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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2010.05.510 Byline: Yanhui Xiang (a)(b), Zhonghua Liu (a), Xun Huang (a) Keywords: npc1; br; Ecdysone; Ring gland; Drosophila Abstract: The growth and metamorphosis of insects are regulated by ecdysteroid hormones produced in the ring gland. Ecdysone biosynthesis-related genes are both highly and specifically expressed in the ring gland. However, the intrinsic regulation of ecdysone biosynthesis has received little attention. Here we used the Drosophila npc1 gene to study the mechanism of ring gland-specific gene expression. npc1 is important for sterol trafficking in the ring gland during ecdysone biosynthesis. We have identified a conserved ring gland-specific cis-regulatory element (RSE) in the npc1 promoter using promoter fusion reporter analysis. Furthermore, genetic loss-of-function analysis and in vitro electrophoretic mobility shift assays revealed that the ecdysone early response gene broad complex (br) is a vital factor in the positive regulation of npc1 ring gland expression. Moreover, br also affects the ring gland expression of many other ecdysone biosynthetic genes as well as torso and InR, two key factors in the regulation of ecdysone biosynthesis. These results imply that ecdysone could potentially act through its early response gene br to achieve positive feedback regulation of ecdysone biosynthesis during development. Author Affiliation: (a) Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (b) Graduate University of Chinese Academy of Sciences, Beijing 100049, China Article History: Received 24 February 2010; Revised 21 May 2010; Accepted 26 May 2010

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2010.05.493 Byline: Jenny M. Rhodes (a), Fiona K. Bentley (a), Cristin G. Print (b), Dale Dorsett (c), Ziva Misulovin (c), Emma J. Dickinson (a), Kathryn E. Crosier (b), Philip S. Crosier (b), Julia A. Horsfield (a) Keywords: Cohesin; Zebrafish; Cornelia de Lange syndrome; Myc Abstract: Contact between sister chromatids from S phase to anaphase depends on cohesin, a large multi-subunit protein complex. Mutations in sister chromatid cohesion proteins underlie the human developmental condition, Cornelia de Lange syndrome. Roles for cohesin in regulating gene expression, sometimes in combination with CCCTC-binding factor (CTCF), have emerged. We analyzed zebrafish embryos null for cohesin subunit rad21 using microarrays to determine global effects of cohesin on gene expression during embryogenesis. This identified Rad21-associated gene networks that included myca (zebrafish c-myc), p53 and mdm2. In zebrafish, cohesin binds to the transcription start sites of p53 and mdm2, and depletion of either Rad21 or CTCF increased their transcription. In contrast, myca expression was strongly downregulated upon loss of Rad21 while depletion of CTCF had little effect. Depletion of Rad21 or the cohesin-loading factor Nipped-B in Drosophila cells also reduced expression of myc and Myc target genes. Cohesin bound the transcription start site plus an upstream predicted CTCF binding site at zebrafish myca. Binding and positive regulation of the c-Myc gene by cohesin is conserved through evolution, indicating that this regulation is likely to be direct. The exact mechanism of regulation is unknown, but local changes in histone modification associated with transcription repression at the myca gene were observed in rad21 mutants. Author Affiliation: (a) Department of Pathology, Dunedin School of Medicine, The University of Otago P.O. Box 913, Dunedin, New Zealand (b) Department of Molecular Medicine and Pathology, School of Medical Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand (c) Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1100 South Grand Boulevard, Saint Louis, MO 63104, USA Article History: Received 29 March 2010; Accepted 15 May 2010

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2010.03.001 Byline: Amanda M. Branam (a), Guy G. Hoffman (b), Francisco Pelegri (c), Daniel S. Greenspan (a)(b)(c)(d) Keywords: Chordin-like; Zebrafish; Maternal factor; BMP1; Tolloid; Dorsoventral patterning; BMP antagonist; Loss-of-function Abstract: Chordin is the prototype of a group of cysteine-rich domain-containing proteins that bind and modulate signaling of various TGF[beta]-like ligands. Chordin-like 1 and 2 (CHL1 and 2) are two members of this group that have been described in human, mouse, and chick. However, in vivo roles for CHL1 and 2 in early development are unknown due to lack of loss-of-function analysis. Here we identify and characterize zebrafish, Danio rerio, CHL (Chl). The chl gene is on a region of chromosome 21 syntenic with the area of murine chromosome 7 bearing the CHL2 gene. Inability to identify a separate zebrafish gene corresponding to the mammalian CHL1 gene suggests that Chl may serve roles in zebrafish distributed between CHL1 and CHL2 in other species. Chl is a maternal factor that is also zygotically expressed later in development and has spatiotemporal expression patterns that differ from but overlap those of zebrafish chordin (Chd), suggesting differences but also possible overlap in developmental roles of the two proteins. Chl, like Chd, dorsalizes embryos upon overexpression and is cleaved by BMP1, which antagonizes this activity. Loss-of-function experiments demonstrate that Chl serves as a BMP antagonist with functions that overlap and are redundant with those of Chd in forming the dorsoventral axis. Author Affiliation: (a) Molecular and Cellular Pharmacology Program, University of Wisconsin, 1300 University Ave, Madison, WI 53706, USA (b) Department of Pathology and Laboratory Medicine, University of Wisconsin, 1300 University Ave, Madison, WI 53706, USA (c) Laboratory of Genetics, University of Wisconsin, 425 Henry Mall, Madison, WI 53706, USA (d) Department of Pharmacology, University of Wisconsin, 1300 University Ave, Madison, WI 53706. USA Article History: Received 2 November 2009; Revised 26 February 2010; Accepted 1 March 2010

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2010.02.013 Byline: Shigehiro Kuraku (a), Yoko Takio (a), Fumiaki Sugahara (a)(b), Masaki Takechi (a), Shigeru Kuratani (a) Keywords: Lamprey; Jaw; Pharyngeal arch; Dlx-code; Endothelin Abstract: In jawed vertebrates, the Dlx code, or nested expression patterns of Dlx genes, specify the dorsoventral polarity of pharyngeal arches, downstream of endothelin-1 (Edn-1) and its effectors, Bapx1 (Nkx3.2) and dHand (Hand2). To elucidate the evolution of the specification mechanism of the oropharyngeal skeletal system, lamprey homologs of Dlx, Edn, endothelin receptor (Ednr), Bapx1, and dHand were identified. Our analysis suggested that the Edn gene family emerged at the advent of vertebrates, and that gene duplications leading to the different Edn gnathostome subtypes (Edn1-3) occurred before the cyclostome-gnathostome split. This timing of gene duplications, giving rise to multiple subtypes, was also implied for Dlx, Ednr, Hand, and Bapx. In lamprey embryos, nested expressions of Dlx genes were not observed in pharyngeal arches, nor was any focal expression of Bapx1, known in gnathostomes to specify the jaw joint. The dHand homolog, however, was expressed more intensively ventrally, as in gnathostomes. Lamprey homologs of Edn-1 and EdnrA were also shown to be expressed as described in mice, indicating involvement of this signaling pathway in the craniofacial patterning early in vertebrate evolution. These results suggest that the last common ancestor of all the extant vertebrates would have possessed basic gene repertoires involved in oropharyngeal patterning in gnathostomes, but the elaborate genetic program leading to the Dlx code is likely to have been acquired uniquely in gnathostomes. Author Affiliation: (a) Laboratory for Evolutionary Morphology, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minami, Chuo-ku, Kobe 650-0047, Japan (b) Graduate School of Science, Kobe University, Kobe 657-8501, Japan Article History: Received 1 January 2010; Revised 5 February 2010; Accepted 6 February 2010

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2010.02.001 Byline: Kristen L. Holler (a), Tyler J. Hendershot (a), Sophia E. Troy (a), Joshua W. Vincentz (b), Anthony B. Firulli (b), Marthe J. Howard (a) Keywords: Hand2(dHand); Neural crest; bHLH; Heart; Outflow tract; Cardiac cushion; Cell type-specific gene regulation; Gene microarray Abstract: The basic helix-loop-helix DNA binding protein Hand2 has critical functions in cardiac development both in neural crest-derived and mesoderm-derived structures. Targeted deletion of Hand2 in the neural crest has allowed us to genetically dissect Hand2-dependent defects specifically in outflow tract and cardiac cushion independent of Hand2 functions in mesoderm-derived structures. Targeted deletion of Hand2 in the neural crest results in misalignment of the aortic arch arteries and outflow tract, contributing to development of double outlet right ventricle (DORV) and ventricular septal defects (VSD). These neural crest-derived developmental anomalies are associated with altered expression of Hand2-target genes we have identified by gene profiling. A number of Hand2 direct target genes have been identified using ChIP and ChIP-on-chip analyses. We have identified and validated a number of genes related to cell migration, proliferation/cell cycle and intracellular signaling whose expression is affected by Hand2 deletion in the neural crest and which are associated with development of VSD and DORV. Our data suggest that Hand2 is a multifunctional DNA binding protein affecting expression of target genes associated with a number of functional interactions in neural crest-derived cells required for proper patterning of the outflow tract, generation of the appropriate number of neural crest-derived cells for elongation of the conotruncus and cardiac cushion organization. Our genetic model has made it possible to investigate the molecular genetics of neural crest contributions to outflow tract morphogenesis and cell differentiation. Author Affiliation: (a) Department of Neurosciences and Program in Neurosciences and Degenerative Disease, Health Sciences Campus, University of Toledo,.sup.1 3000 Arlington Ave., Toledo, OH 43614-1007, USA (b) Riley Heart Research Center, Herman B Webb Center for Pediatric Research, Indiana Medical School, 1044 W. Walnut St., Indianapolis, IN 46202-5225, USA Article History: Received 20 May 2009; Revised 29 January 2010; Accepted 1 February 2010

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2010.01.035 Byline: Beena Punnamoottil (a)(b)(c), Carl Herrmann (d), Juan Pascual-Anaya (e), Salvatore D'Aniello (e), Jordi Garcia-Fernandez (e), Altuna Akalin (b)(f), Thomas S. Becker (b)(c), Silke Rinkwitz (a)(b)(c) Keywords: Hox; Cis-regulation; CNE; Zebrafish; GFP; Transgenesis; Amphioxus; Sequence conservation; Cluster Abstract: Hox genes are key regulators of anterior-posterior axis patterning and have a major role in hindbrain development. The zebrafish Hox4 paralogs have strong overlapping activities in hindbrain rhombomeres 7 and 8, in the spinal cord and in the pharyngeal arches. With the aim to predict enhancers that act on the hoxa4a, hoxb4a, hoxc4a and hoxd4a genes, we used sequence conservation around the Hox4 genes to analyze all fish:human conserved non-coding sequences by reporter assays in stable zebrafish transgenesis. Thirty-four elements were functionally tested in GFP reporter gene constructs and more than 100 F1 lines were analyzed to establish a correlation between sequence conservation and cis-regulatory function, constituting a catalog of Hox4 CNEs. Sixteen tissue-specific enhancers could be identified. Multiple alignments of the CNEs revealed paralogous cis-regulatory sequences, however, the CNE sequence similarities were found not to correlate with tissue specificity. To identify ancestral enhancers that direct Hox4 gene activity, genome sequence alignments of mammals, teleosts, horn shark and the cephalochordate amphioxus, which is the most basal extant chordate possessing a single prototypical Hox cluster, were performed. Three elements were identified and two of them exhibited regulatory activity in transgenic zebrafish, however revealing no specificity. Our data show that the approach to identify cis-regulatory sequences by genome sequence alignments and subsequent testing in zebrafish transgenesis can be used to define enhancers within the Hox clusters and that these have significantly diverged in their function during evolution. Author Affiliation: (a) Carl von Ossietzky University, Oldenburg, Germany (b) Sars International Center, Bergen, Norway (c) Brain and Mind Research Institute, University of Sydney, Sydney, Australia (d) TAGC Inserm U928 and Universite de la Mediterranee, Marseille, France (e) Departament de Genetica, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain (f) Computational Biology Unit, University of Bergen, 5008 Bergen, Norway Article History: Received 7 January 2009; Revised 17 January 2010; Accepted 30 January 2010

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.11.006 Byline: Lionel Christiaen (a)(b), Alberto Stolfi (a), Michael Levine (a) Keywords: TGF-beta; Threshold response; Feedback loop; Ascidian; Kernel Abstract: Gene regulatory networks control the progressive specification of cell types and govern morphogenetic events during development. However, how morphogenetic events reciprocally affect gene expression remains poorly understood. Here, we analyzed the function of BMP signaling and expression of downstream target genes during cell migration of the precardiac mesoderm (trunk ventral cells, TVCs) in ascidian embryos. Our results indicate that migrating TVCs experience increasing BMP signaling as they migrate towards the ventral trunk epidermis, which expresses sustained levels of Bmp2/4. This increasing signaling intensity allows the successive activation of GATAa, Tolloid, Bmp2/4 and NK4. Initial activation of GATAa, Tolloid and Bmp2/4 contribute to a positive feedback loop involving cell migration, chordin inhibition and BMP ligand production. Sustained levels of BMP signaling become sufficient to activate NK4 expression, which in turn contributes to a negative feedback loop inhibiting Bmp2/4 and Tolloid expression. In addition, NK4 appears to inhibit cell migration thus providing a 'transcriptional brake' to stop TVC migration. Our observations led us to propose a model for the coordination of cell migration and gene expression based on the temporal unfolding of a gene regulatory sub-network in a relevant developmental context. Author Affiliation: (a) Center for Integrative Genomics, Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA (b) Department of Biology, New York University, 1009 Silver Building, 100 Washington Square East, New York, NY 10003-6688, USA Article History: Received 7 July 2009; Revised 3 November 2009; Accepted 4 November 2009 Article Note: (footnote) [star] This article was accepted in 2009 to celebrate the 50th anniversary of Developmental Biology.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.10.032 Byline: Bartek Wilczynski (a)(b), Eileen E.M. Furlong (a) Keywords: Transcriptional networks; Gene regulatory networks; Developmental networks; Modeling; Drosophila; CRMs Abstract: Development is regulated by dynamic patterns of gene expression, which are orchestrated through the action of complex gene regulatory networks (GRNs). Substantial progress has been made in modeling transcriptional regulation in recent years, including qualitative 'coarse-grain' models operating at the gene level to very 'fine-grain' quantitative models operating at the biophysical 'transcription factor-DNA level'. Recent advances in genome-wide studies have revealed an enormous increase in the size and complexity or GRNs. Even relatively simple developmental processes can involve hundreds of regulatory molecules, with extensive interconnectivity and cooperative regulation. This leads to an explosion in the number of regulatory functions, effectively impeding Boolean-based qualitative modeling approaches. At the same time, the lack of information on the biophysical properties for the majority of transcription factors within a global network restricts quantitative approaches. In this review, we explore the current challenges in moving from modeling medium scale well-characterized networks to more poorly characterized global networks. We suggest to integrate coarse- and find-grain approaches to model gene regulatory networks in cis. We focus on two very well-studied examples from Drosophila, which likely represent typical developmental regulatory modules across metazoans. Author Affiliation: (a) European Molecular Biology Laboratory (EMBL), D-69117 Heidelberg, Germany (b) Institute of Informatics, University of Warsaw, Warsaw, Poland Article History: Received 21 July 2009; Revised 14 October 2009; Accepted 21 October 2009 Article Note: (footnote) [star] This article was accepted in 2009 to celebrate the 50th anniversary of Developmental Biology.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.12.039 Byline: Julia Zeitlinger (a)(b), Alexander Stark (c) Keywords: Genetics; Genomics; Development; Drosophila; RNA Pol II stalling; Polycomb; Chromatin Abstract: Genetic experiments over the last few decades have identified many developmental control genes critical for pattern formation and cell fate specification during the development of multicellular organisms. A large fraction of these genes encode transcription factors and signaling molecules, show highly dynamic expression patterns during development, and are deeply evolutionarily conserved and deregulated in various human diseases such as cancer. Because of their importance in development, evolution, and disease, a fundamental question in biology is how these developmental control genes are regulated in such an extensive and precise fashion. Using genomics methods, it has become clear that developmental control genes are a distinct group of genes with special regulatory characteristics. However, a systematic analysis of these characteristics has not been presented. Here we review how developmental control genes were discovered, evaluate their genome-wide regulation and gene structure, discuss emerging evidence for their mode of regulation, and estimate their overall abundance in the genome. Understanding the global regulation of developmental control genes may provide a new perspective on development in the era genomics. Author Affiliation: (a) Stowers Institute for Medical Research, Kansas City, MO 64110, USA (b) University of Kansas Medical Center, Kansas City, KS 66103, USA (c) Research Institute of Molecular Pathology (IMP), A-1030 Vienna, Austria Article History: Received 24 July 2009; Revised 4 December 2009; Accepted 23 December 2009

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.10.036 Byline: Maximilian Schuff (a), Doreen Siegel (a), Nabila Bardine (a), Franz Oswald (b), Cornelia Donow (a), Walter Knochel (a) Keywords: Xenopus laevis; FoxO genes; Gastrulation; Posttranslational modification; Akt-1; Antisense morpholino oligonucleotide; Heart; Eye Abstract: Forkhead box (Fox) transcription factors of subclass O are involved in cell survival, proliferation, apoptosis, cell metabolism and prevention of oxidative stress. FoxO genes are highly conserved throughout evolution and their functions were analyzed in several vertebrate and invertebrate organisms. We here report on the identification of FoxO4 and FoxO6 genes in Xenopus laevis and analyze their expression patterns in comparison with the previously described FoxO1 and FoxO3 genes. We demonstrate significant differences in their temporal and spatial expression during embryogenesis and in their relative expression within adult tissues. Overexpression of FoxO1, FoxO4 or FoxO6 results in severe gastrulation defects, while overexpression of FoxO3 reveals this defect only in a constitutively active form containing mutations of Akt-1 target sites. Injections of FoxO antisense morpholino oligonucleotides (MO) did not influence gastrulation, but, later onwards, the embryos showed a delay of development, severe body axis reduction and, finally, a high rate of lethality. Injection of FoxO4MO leads to specific defects in eye formation, neural crest migration and heart development, the latter being accompanied by loss of myocardin expression. Our observations suggest that FoxO genes in X. laevis are dispensable until blastopore closure but are required for tissue differentiation and organogenesis. Author Affiliation: (a) Institute of Biochemistry, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany (b) Department of Internal Medicine I, University of Ulm, Albert-Einstein-Allee 23, 89081 Ulm, Germany Article History: Received 20 May 2009; Revised 27 October 2009; Accepted 27 October 2009

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.08.070 Byline: Florian Raible (a)(b), Kristin Tessmar-Raible (b), Enrique Arboleda (c), Tobias Kaller (b)(d), Peer Bork (a), Detlev Arendt (b), Maria I. Arnone (c) Keywords: Sea urchin; Rhodopsin-type G-protein-coupled receptor (GPCR); Opsin; Surreal-GPCR; Gene cluster; Chemosensation; Olfaction; Pedicellariae, Tube feet Abstract: Rhodopsin-type G-protein-coupled receptors (GPCRs) contribute the majority of sensory receptors in vertebrates. With 979 members, they form the largest GPCR family in the sequenced sea urchin genome, constituting more than 3% of all predicted genes. The sea urchin genome encodes at least six Opsin proteins. Of these, one rhabdomeric, one ciliary and two G.sub.o-type Opsins can be assigned to ancient bilaterian Opsin subfamilies. Moreover, we identified four greatly expanded subfamilies of rhodopsin-type GPCRs that we call sea urchin specific rapidly expanded lineages of GPCRs (surreal-GPCRs). Our analysis of two of these groups revealed genomic clustering and single-exon gene structures similar to the most expanded group of vertebrate rhodopsin-type GPCRs, the olfactory receptors. We hypothesize that these genes arose by rapid duplication in the echinoid lineage and act as chemosensory receptors of the animal. In support of this, group B surreal-GPCRs are most prominently expressed in distinct classes of pedicellariae and tube feet of the adult sea urchin, structures that have previously been shown to react to chemical stimuli and to harbor sensory neurons in echinoderms. Notably, these structures also express different opsins, indicating that sea urchins possess an intricate molecular set-up to sense their environment. Author Affiliation: (a) Computational Unit, EMBL, 69117 Heidelberg, Germany (b) Developmental Unit, EMBL, 69117 Heidelberg, Germany (c) Stazione Zoologica Anton Dohrn, 80121 Napoli, Italy (d) FU Berlin, Institut fur Biologie, Chemie, Pharmazie, Zoologie, Evolution und Systematik der Tiere, 14195 Berlin, Germany Article History: Received 7 June 2006; Revised 28 August 2006; Accepted 30 August 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.08.007 Byline: R.D. Burke (a), L.M. Angerer (b), M.R. Elphick (c), G.W. Humphrey (d), S. Yaguchi (a), T. Kiyama (e), S. Liang (e), X. Mu (e), C. Agca (f), W.H. Klein (e), B.P. Brandhorst (h), M. Rowe (c), K. Wilson (j), A.M. Churcher (g), J.S. Taylor (g), N. Chen (h), G. Murray (h), D. Wang (g), D. Mellott (g), R. Olinski (i), F. Hallbook (i), M.C. Thorndyke (j) Keywords: Genomics; Neurobiology; Neural development; Deuterostome; Evolution; Echinoderm Abstract: The sequencing of the Strongylocentrotus purpuratus genome provides a unique opportunity to investigate the function and evolution of neural genes. The neurobiology of sea urchins is of particular interest because they have a close phylogenetic relationship with chordates, yet a distinctive pentaradiate body plan and unusual neural organization. Orthologues of transcription factors that regulate neurogenesis in other animals have been identified and several are expressed in neurogenic domains before gastrulation indicating that they may operate near the top of a conserved neural gene regulatory network. A family of genes encoding voltage-gated ion channels is present but, surprisingly, genes encoding gap junction proteins (connexins and pannexins) appear to be absent. Genes required for synapse formation and function have been identified and genes for synthesis and transport of neurotransmitters are present. There is a large family of G-protein-coupled receptors, including 874 rhodopsin-type receptors, 28 metabotropic glutamate-like receptors and a remarkably expanded group of 161 secretin receptor-like proteins. Absence of cannabinoid, lysophospholipid and melanocortin receptors indicates that this group may be unique to chordates. There are at least 37 putative G-protein-coupled peptide receptors and precursors for several neuropeptides and peptide hormones have been identified, including SALMFamides, NGFFFamide, a vasotocin-like peptide, glycoprotein hormones and insulin/insulin-like growth factors. Identification of a neurotrophin-like gene and Trk receptor in sea urchin indicates that this neural signaling system is not unique to chordates. Several hundred chemoreceptor genes have been predicted using several approaches, a number similar to that for other animals. Intriguingly, genes encoding homologues of rhodopsin, Pax6 and several other key mammalian retinal transcription factors are expressed in tube feet, suggesting tube feet function as photosensory organs. Analysis of the sea urchin genome presents a unique perspective on the evolutionary history of deuterostome nervous systems and reveals new approaches to investigate the development and neurobiology of sea urchins. Author Affiliation: (a) Department of Biology, Dept of Biochemistry/Microbiology, University of Victoria, Victoria, POB 3020, STN CSC, Victoria, BC, Canada V8W 3N5 (b) National Institute for Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA (c) School of Biological and Chemical Sciences, Queen Mary, University of London, London, UK (d) Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Development, National Institutes of Health, Bethesda, MD, USA (e) University of Texas, M. D. Anderson Cancer Center, Biochemistry and Molecular Biology, Houston, TX, USA (f) Department of Cell Biology and Anatomy, Louisiana State University, Health Sciences Center, New Orleans, LA, USA (g) Department of Biology, University or Victoria, Victoria, BC, Canada (h) Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada (i) Department Neuroscience, Uppsala University, Uppsala, Sweden (j) Royal Swedish Academy of Sciences, Kristineberg Marine Research Station, Fiskebackskil, 450 34, Sweden Article History: Received 1 June 2006; Revised 29 July 2006; Accepted 2 August 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.08.065 Byline: Taku Hibino (a), Mariano Loza-Coll (a), Cynthia Messier (a), Audrey J. Majeske (b), Avis H. Cohen (c), David P. Terwilliger (b), Katherine M. Buckley (b), Virginia Brockton (b), Sham V. Nair (d), Kevin Berney (e), Sebastian D. Fugmann (f), Michele K. Anderson (g), Zeev Pancer (h), R. Andrew Cameron (e), L. Courtney Smith (b), Jonathan P. Rast (a) Keywords: Hematopoiesis; Developmental immunology; Innate immunity; TLR; RAG; NOD; Echinoderm; Scavenger receptor; Complement; Cytokine Abstract: Echinoderms occupy a critical and largely unexplored phylogenetic vantage point from which to infer both the early evolution of bilaterian immunity and the underpinnings of the vertebrate adaptive immune system. Here we present an initial survey of the purple sea urchin genome for genes associated with immunity. An elaborate repertoire of potential immune receptors, regulators and effectors is present, including unprecedented expansions of innate pathogen recognition genes. These include a diverse array of 222 Toll-like receptor (TLR) genes and a coordinate expansion of directly associated signaling adaptors. Notably, a subset of sea urchin TLR genes encodes receptors with structural characteristics previously identified only in protostomes. A similarly expanded set of 203 NOD/NALP-like cytoplasmic recognition proteins is present. These genes have previously been identified only in vertebrates where they are represented in much lower numbers. Genes that mediate the alternative and lectin complement pathways are described, while gene homologues of the terminal pathway are not present. We have also identified several homologues of genes that function in jawed vertebrate adaptive immunity. The most striking of these is a gene cluster with similarity to the jawed vertebrate Recombination Activating Genes 1 and 2 (RAG1/2). Sea urchins are long-lived, complex organisms and these findings reveal an innate immune system of unprecedented complexity. Whether the presumably intense selective processes that molded these gene families also gave rise to novel immune mechanisms akin to adaptive systems remains to be seen. The genome sequence provides immediate opportunities to apply the advantages of the sea urchin model toward problems in developmental and evolutionary immunobiology. Author Affiliation: (a) Sunnybrook Research Institute and Department of Medical Biophysics, University of Toronto, 2075 Bayview Ave., Room S-126b, Toronto, Ontario, Canada M4N 3M5 (b) 340 Lisner Hall, Department of Biological Sciences, George Washington University, 2023 G St. NW, Washington, DC 20052, USA (c) Department of Biology and the Institute of Systems Research, University of Maryland, College Park, MD 20742, USA (d) Department of Biological Sciences, Macquarie University, Sydney, Australia (e) Division of Biology, 156-29 California Institute of Technology, Pasadena, CA 91125, USA (f) Laboratory of Cellular and Molecular Biology, National Institute on Aging, Baltimore, MD 21224, USA (g) Sunnybrook Research Institute and Department of Immunology, University of Toronto. 2075 Bayview Ave., Room A-340, Toronto, Ontario, Canada M4N 3M5 (h) Center of Marine Biotechnology, UMBI, Columbus Center, Suite 236 701 East Pratt St. Baltimore, MD 21202, USA Article History: Received 1 June 2006; Revised 21 August 2006; Accepted 28 August 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.07.036 Byline: Julia Morales (a), Odile Mulner-Lorillon (a), Bertrand Cosson (a), Emmanuelle Morin (b), Robert Belle (a), Cynthia A. Bradham (c), Wendy S. Beane (c), Patrick Cormier (a) Keywords: Sea urchin; Translational control; Cap-dependent initiation; Elongation factors Abstract: Sea urchin eggs and early cleavage stage embryos provide an example of regulated gene expression at the level of translation. The availability of the sea urchin genome offers the opportunity to investigate the 'translational control' toolkit of this model system. The annotation of the genome reveals that most of the factors implicated in translational control are encoded by nonredundant genes in echinoderm, an advantage for future functional studies. In this paper, we focus on translation factors that have been shown or suggested to play crucial role in cell cycle and development of sea urchin embryos. Addressing the cap-binding translational control, three closely related eIF4E genes (class I, II, III) are present, whereas its repressor 4E-BP and its activator eIF4G are both encoded by one gene. Analysis of the class III eIF4E proteins in various phyla shows an echinoderm-specific amino acid substitution. Furthermore, an interaction site between eIF4G and poly(A)-binding protein is uncovered in the sea urchin eIF4G proteins and is conserved in metazoan evolution. In silico screening of the sea urchin genome has uncovered potential new regulators of eIF4E sharing the common eIF4E recognition motif. Taking together, these data provide new insights regarding the strong requirement of cap-dependent translation following fertilization. The genome analysis gives insights on the complexity of eEF1B structure and motifs of functional relevance, involved in the translational control of gene expression at the level of elongation. Finally, because deregulation of translation process can lead to diseases and tumor formation in humans, the sea urchin orthologs of human genes implicated in human diseases and signaling pathways regulating translation were also discussed. Author Affiliation: (a) Equipe Cycle Cellulaire et Developpement, UMR 7150 CNRS/UPMC, Station Biologique 29680 Roscoff, France (b) Service Informatique et Genomique, Station Biologique 29680 Roscoff, France (c) Developmental, Cellular and Molecular Biology Group, Duke University, Durham NC, USA Article History: Received 29 May 2006; Revised 25 July 2006; Accepted 27 July 2006 Article Note: (footnote) [star] Complete names of species listed in the manuscript: Asterina pectinifera, Bombyx mauri, Caenorhabditis elegans, Callinectes sapidus, Ciona intestinalis, Danio rerio, Dictyostelium discoidum, Drosophila melanogaster, Globodera rostochiensis, Homo sapiens, Mus musculus, Nematostella vectensis, Paracentrotus lividus, Saccharomyces cerevisiae, Sphaerechinus granularis, Strongylocentrotus purpuratus, Xenopus laevis.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.07.047 Byline: B.T. Livingston (a), C.E. Killian (b), F. Wilt (b), A. Cameron (c), M.J. Landrum (d), O. Ermolaeva (d), V. Sapojnikov (d), D.R. Maglott (d), A.M. Buchanan (e), C.A. Ettensohn (e) Keywords: Biomineralization; Genome; Echinoderm Abstract: Biomineralization, the biologically controlled formation of mineral deposits, is of widespread importance in biology, medicine, and engineering. Mineralized structures are found in most metazoan phyla and often have supportive, protective, or feeding functions. Among deuterostomes, only echinoderms and vertebrates produce extensive biomineralized structures. Although skeletons appeared independently in these two groups, ancestors of the vertebrates and echinoderms may have utilized similar components of a shared genetic 'toolkit' to carry out biomineralization. The present study had two goals. First, we sought to expand our understanding of the proteins involved in biomineralization in the sea urchin, a powerful model system for analyzing the basic cellular and molecular mechanisms that underlie this process. Second, we sought to shed light on the possible evolutionary relationships between biomineralization in echinoderms and vertebrates. We used several computational methods to survey the genome of the purple sea urchin Strongylocentrotus purpuratus for gene products involved in biomineralization. Our analysis has greatly expanded the collection of biomineralization-related proteins. We have found that these proteins are often members of small families encoded by genes that are clustered in the genome. Most of the proteins are sea urchin-specific; that is, they have no apparent homologues in other invertebrate deuterostomes or vertebrates. Similarly, many of the vertebrate proteins that mediate mineral deposition do not have counterparts in the S. purpuratus genome. Our findings therefore reveal substantial differences in the primary sequences of proteins that mediate biomineral formation in echinoderms and vertebrates, possibly reflecting loose constraints on the primary structures of the proteins involved. On the other hand, certain cellular and molecular processes associated with earlier events in skeletogenesis appear similar in echinoderms and vertebrates, leaving open the possibility of deeper evolutionary relationships. Author Affiliation: (a) Department of Biology, University of South Florida, Tampa, FL 33620, USA (b) Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA (c) Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA (d) National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD 20892, USA (e) Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA Article History: Received 6 June 2006; Revised 26 July 2006; Accepted 31 July 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.09.012 Byline: Antonio Fernandez-Guerra (a), Antoine Aze (a), Julia Morales (b), Odile Mulner-Lorillon (b), Bertrand Cosson (b), Patrick Cormier (b), Cynthia Bradham (c), Nikki Adams (d), Anthony J. Robertson (e), William F. Marzluff (f), James A. Coffman (e), Anne-Marie Geneviere (a) Keywords: Sea urchin; Cell cycle; Replication; Mitosis; Checkpoint; Kinases Abstract: A search of the Strongylocentrotus purpuratus genome for genes associated with cell cycle control and DNA metabolism shows that the known repertoire of these genes is conserved in the sea urchin, although with fewer family members represented than in vertebrates, and with some cases of echinoderm-specific gene diversifications. For example, while homologues of the known cyclins are mostly encoded by single genes in S. purpuratus (unlike vertebrates, which have multiple isoforms), there are additional genes encoding novel cyclins of the B and K/L types. Almost all known cyclin-dependent kinases (CDKs) or CDK-like proteins have an orthologue in S. purpuratus; CDK3 is one exception, whereas CDK4 and 6 are represented by a single homologue, referred to as CDK4. While the complexity of the two families of mitotic kinases, Polo and Aurora, is close to that found in the nematode, the diversity of the NIMA-related kinases (NEK proteins) approaches that of vertebrates. Among the nine NEK proteins found in S. purpuratus, eight could be assigned orthologues in vertebrates, whereas the ninth is unique to sea urchins. Most known DNA replication, DNA repair and mitotic checkpoint genes are also present, as are homologues of the pRB (two) and p53 (one) tumor suppressors. Interestingly, the p21/p27 family of CDK inhibitors is represented by one homologue, whereas the INK4 and ARF families of tumor suppressors appear to be absent, suggesting that these evolved only in vertebrates. Our results suggest that, while the cell cycle control mechanisms known from other animals are generally conserved in sea urchin, parts of the machinery have diversified within the echinoderm lineage. The set of genes uncovered in this analysis of the S. purpuratus genome should enhance future research on cell cycle control and developmental regulation in this model. Author Affiliation: (a) Observatoire Oceanologique de Banyuls-Laboratoire Arago, CNRS-UMR7628/UPMC, 66650 Banyuls-sur-Mer, France (b) Station Biologique de Roscoff, CNRS-UMR7150/UPMC, 29680 Roscoff, France (c) Developmental, Cellular, and Molecular Biology Group, Duke University, Durham, NC, USA (d) Department of Biological Sciences, California Polytechnic State University, San Luis Obispo, CA, USA (e) Mount Desert Island Biological Laboratory, Salisbury Cove, ME, USA (f) Program in Molecular Biology, University of North Carolina, Chapel Hill, NC, USA Article History: Received 30 May 2006; Revised 6 September 2006; Accepted 7 September 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.08.052 Byline: R.L. Morris (a), M.P. Hoffman (b), R.A. Obar (c), S.S. McCafferty (a), I.R. Gibbons (d), A.D. Leone (b), J. Cool (a), E.L. Allgood (a), A.M. Musante (a), K.M. Judkins (a), B.J. Rossetti (a), A.P. Rawson (a), D.R. Burgess (b) Keywords: Sea urchin; Cytoskeleton; Actin; Myosin; Intermediate filament; Tubulin; Microtubule; Microfilament; MAP; Dynein; Kinesin; Motility; Genome Abstract: The sea urchin embryo is a classical model system for studying the role of the cytoskeleton in such events as fertilization, mitosis, cleavage, cell migration and gastrulation. We have conducted an analysis of gene models derived from the Strongylocentrotus purpuratus genome assembly and have gathered strong evidence for the existence of multiple gene families encoding cytoskeletal proteins and their regulators in sea urchin. While many cytoskeletal genes have been cloned from sea urchin with sequences already existing in public databases, genome analysis reveals a significantly higher degree of diversity within certain gene families. Furthermore, genes are described corresponding to homologs of cytoskeletal proteins not previously documented in sea urchins. To illustrate the varying degree of sequence diversity that exists within cytoskeletal gene families, we conducted an analysis of genes encoding actins, specific actin-binding proteins, myosins, tubulins, kinesins, dyneins, specific microtubule-associated proteins, and intermediate filaments. We conducted ontological analysis of select genes to better understand the relatedness of urchin cytoskeletal genes to those of other deuterostomes. We analyzed developmental expression (EST) data to confirm the existence of select gene models and to understand their differential expression during various stages of early development. Author Affiliation: (a) Department of Biology, Wheaton College, Norton, MA 02766, USA (b) Department of Biology, Boston College, Chestnut Hill, MA 0246, USA (c) Tethys Research, LLC, 53 Downing Road, Bangor, ME 04401, USA (d) Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA Article History: Received 6 June 2006; Revised 18 August 2006; Accepted 22 August 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.08.074 Byline: Cynthia A. Bradham (a), Kathy R. Foltz (b), Wendy S. Beane (a), Maria I. Arnone (c), Francesca Rizzo (c), James A. Coffman (d), Arcady Mushegian (e), Manisha Goel (e), Julia Morales (f), Anne-Marie Geneviere (g), Francois Lapraz (h), Anthony J. Robertson (d), Hemant Kelkar (i), Mariano Loza-Coll (j), Ian K. Townley (b), Michael Raisch (b), Michelle M. Roux (b), Thierry Lepage (h), Christian Gache (h), David R. McClay (a), Gerard Manning (k) Keywords: Kinase; Signal transduction; Genome; Kinome Abstract: This paper reports a preliminary in silico analysis of the sea urchin kinome. The predicted protein kinases in the sea urchin genome were identified, annotated and classified, according to both function and kinase domain taxonomy. The results show that the sea urchin kinome, consisting of 353 protein kinases, is closer to the Drosophila kinome (239) than the human kinome (518) with respect to total kinase number. However, the diversity of sea urchin kinases is surprisingly similar to humans, since the urchin kinome is missing only 4 of 186 human subfamilies, while Drosophila lacks 24. Thus, the sea urchin kinome combines the simplicity of a non-duplicated genome with the diversity of function and signaling previously considered to be vertebrate-specific. More than half of the sea urchin kinases are involved with signal transduction, and approximately 88% of the signaling kinases are expressed in the developing embryo. These results support the strength of this nonchordate deuterostome as a pivotal developmental and evolutionary model organism. Author Affiliation: (a) DCMB Group, Duke University, Box 91000, Durham, NC 27708, USA (b) Department Molecular, Cellular and Developmental Biology, UCSB, Santa Barbara, CA, USA (c) Stazione Zoologica A, Dohrn, Napoli, Italy (d) Mount Desert Island Biological Laboratory, Salisbury Cove, ME, USA (e) Stowers Institute for Medical Research, Kansas City MO and Dept. of Microbiology, Kansas University Medical Center, Kansas City, KS, USA (f) Equipe Cycle cellulaire et Developpement, UMR 7150 CNRS/UPMC, Station Biologique BP74, Roscoff, France (g) Observatoire Oceanologique de Banyuls, Laboratoire Arago, CNRS-UMR7628/Universite Pierre et Marie Curie (Paris 6), Banyuls-sur-Mer, France (h) Laboratory of Developmental Biology, UMR 7009, CNRS and Universite Pierre and Marie Curie (Paris 6), Observatoire Oceanologique, Villefranche-sur-Mer, France (i) Center for Bioinformatics, Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, NC, USA (j) Sunnybrook Research Institute and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada (k) Razavi-Newman Center for Bioinformatics, Salk Institute for Biological Studies, La Jolla, CA, USA Article History: Received 31 May 2006; Revised 19 August 2006; Accepted 22 August 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.08.046 Byline: Wendy S. Beane (a), Ekaterina Voronina (b), Gary M. Wessel (b), David R. McClay (a) Keywords: Ras GTPase; Rho; G protein; Galpha; Dynamin; SRP; Signal recognition particle receptor; Translation; Gene duplication; Phylogenomics; Echinoderm Abstract: In every organism, GTP-binding proteins control many aspects of cell signaling. Here, we examine in silico several GTPase families from the Strongylocentrotus purpuratus genome: the monomeric Ras superfamily, the heterotrimeric G proteins, the dynamin superfamily, the SRP/SR family, and the 'protein biosynthesis' translational GTPases. Identified were 174 GTPases, of which over 90% are expressed in the embryo as shown by tiling array and expressed sequence tag data. Phylogenomic comparisons restricted to Drosophila, Ciona, and humans (protostomes, urochordates, and vertebrates, respectively) revealed both common and unique elements in the expected composition of these families. G[alpha] and dynamin families contain vertebrate expansions, consistent with whole genome duplications, whereas SRP/SR and translational GTPases are highly conserved. Unexpectedly, Ras superfamily analyses revealed several large (5+) lineage-specific expansions in the sea urchin. For Rho, Rab, Arf, and Ras subfamilies, comparing total human gene numbers to the number of sea urchin genes with vertebrate orthologs suggests reduced genomic complexity in the sea urchin. However, gene duplications in the sea urchin increase overall numbers such that total sea urchin gene numbers approximate vertebrate gene numbers for each monomeric GTPase family. These findings suggest that lineage-specific expansions may be an important component of genomic evolution in signal transduction. Author Affiliation: (a) Department of Biology, Developmental, Cell and Molecular Group, Duke University, Box 91000, Durham, NC 27708, USA (b) Department of Molecular and Cell Biology and Biochemistry, Brown University, 69 Brown Street, Providence, RI 02912, USA Article History: Received 11 May 2006; Revised 18 August 2006; Accepted 19 August 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.08.048 Byline: Francois Lapraz (a), Eric Rottinger (a), Veronique Duboc (a), Ryan Range (a), Louise Duloquin (a), Katherine Walton (b), Shu-Yu Wu (b), Cynthia Bradham (b), Mariano A. Loza (c), Taku Hibino (c), Karen Wilson (d), Albert Poustka (e), Dave McClay (b), Lynne Angerer (f), Christian Gache (a), Thierry Lepage (a) Keywords: Receptor tyrosine kinase; Signaling; TGF-[beta]; Sea urchin; Genome; Deuterostome; FGFR; Nodal; TGF-[beta] receptors; Smad Abstract: The Receptor Tyrosine kinase (RTK) and TGF-[beta] signaling pathways play essential roles during development in many organisms and regulate a plethora of cellular responses. From the genome sequence of Strongylocentrotus purpuratus, we have made an inventory of the genes encoding receptor tyrosine kinases and their ligands, and of the genes encoding cytokines of the TGF-[beta] superfamily and their downstream components. The sea urchin genome contains at least 20 genes coding for canonical receptor tyrosine kinases. Seventeen of the nineteen vertebrate RTK families are represented in the sea urchin. Fourteen of these RTK among which ALK, CCK4/PTK7, DDR, EGFR, EPH, LMR, MET/RON, MUSK, RET, ROR, ROS, RYK, TIE and TRK are present as single copy genes while pairs of related genes are present for VEGFR, FGFR and INSR. Similarly, nearly all the subfamilies of TGF-[beta] ligands identified in vertebrates are present in the sea urchin genome including the BMP, ADMP, GDF, Activin, Myostatin, Nodal and Lefty, as well as the TGF-[beta] sensu stricto that had not been characterized in invertebrates so far. Expression analysis indicates that the early expression of nodal, BMP2/4 and lefty is restricted to the oral ectoderm reflecting their role in providing positional information along the oral-aboral axis of the embryo. The coincidence between the emergence of TGF-[beta]-related factors such as Nodal and Lefty and the emergence of the deuterostome lineage strongly suggests that the ancestral function of Nodal could have been related to the secondary opening of the mouth which characterizes this clade, a hypothesis supported by functional data in the extant species. The sea urchin genome contains 6 genes encoding TGF-[beta] receptors and 4 genes encoding prototypical Smad proteins. Furthermore, most of the transcriptional activators and repressors shown to interact with Smads in vertebrates have orthologues in echinoderms. Finally, the sea urchin genome contains an almost complete repertoire of genes encoding extracellular modulators of BMP signaling including Chordin, Noggin, Sclerotin, SFRP, Gremlin, DAN and Twisted gastrulation. Taken together, these findings indicate that the sea urchin complement of genes of the RTK and TGF-[beta] signaling pathways is qualitatively very similar to the repertoire present in vertebrates, and that these genes are part of the common genetool kit for intercellular signaling of deuterostomes. Author Affiliation: (a) UMR 7009 CNRS, Universite Pierre et Marie Curie-Paris 6, Observatoire Oceanologique, 06230 Villefranche-sur-Mer, France (b) Developmental, Molecular, and Cellular Biology Group, Duke University, Durham, NC, USA (c) Sunnybrook and Women's Research Institute and Department of Medical Biophysics, University of Toronto. 2075 Bayview Ave., Toronto, Ontario, Canada M4N 3M5 (d) Kristineberg Marine Research Station 45034 Fiskebackskil, Sweden (e) Evolution and Development Group Max-Planck Institut fuer Molekulare Genetik Ihnestrasse 7314195, Berlin (f) National Institute for Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA Article History: Received 25 May 2006; Revised 16 August 2006; Accepted 16 August 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.08.020 Byline: Laurence Covassin (a), Julio D. Amigo (a), Kana Suzuki (b), Viktor Teplyuk (b), Juerg Straubhaar (b), Nathan D. Lawson (a) Keywords: Zebrafish; Hematopoietic; Vascular; Endothelial; Microarray Abstract: In this study, we utilize fluorescent activated cell sorting (FACS) of cells from transgenic zebrafish coupled with microarray analysis to globally analyze expression of cell type specific genes. We find that it is possible to isolate cell populations from Tg(fli1:egfp).sup.y1 zebrafish embryos that are enriched in vascular, hematopoietic and pharyngeal arch cell types. Microarray analysis of GFP.sup.+ versus GFP.sup.- cells isolated from Tg(fli1:egfp).sup.y1 embryos identifies genes expressed in hematopoietic, vascular and pharyngeal arch tissue, consistent with the expression of the fli1:egfp transgene in these cell types. Comparison of expression profiles from GFP.sup.+ cells isolated from embryos at two different time points reveals that genes expressed in different fli1.sup.+ cell types display distinct temporal expression profiles. We also demonstrate the utility of this approach for gene discovery by identifying numerous previously uncharacterized genes that we find are expressed in fli1:egfp-positive cells, including new markers of blood, endothelial and pharyngeal arch cell types. In parallel, we have developed a database to allow easy access to both our microarray and in situ results. Our results demonstrate that this is a robust approach for identification of cell type specific genes as well as for global analysis of cell type specific gene expression in zebrafish embryos. Author Affiliation: (a) Program in Gene Function and Expression, University of Massachusetts Medical School, Lazare Research Building, Room 617, 364 Plantation Street, Worcester, MA 01605, USA (b) Diabetes and Endocrinology Research Center, University of Massachusetts Medical School, Biotech 2, Suite 218, 373 Plantation Street, Worcester, MA 01605, USA Article History: Received 27 April 2006; Revised 28 July 2006; Accepted 6 August 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.08.040 Byline: Iain D. Keenan (a)(c), R. Michael Sharrard (b), Harry V. Isaacs (a) Keywords: Xenopus; FGF; Cdx; ERK; PI-3 kinase; Mesoderm Abstract: Cdx homeodomain transcription factors play important roles in the development of the vertebrate body axis and gut epithelium. Signaling involving FGF, wnt and retinoic acid ligands has been implicated in the regulation of individual Cdx genes. In this study we examine the requirement for FGF-dependent signal transduction pathways in the regulation of Cdx gene expression. In the amphibian Xenopus laevis the earliest expression of Cdx1, Cdx2 and Cdx4 is within the developing mesoderm. We show that a functional FGF signaling pathway is required for the normal expression of all three amphibian Cdx genes during gastrula stages. We show that FGF stimulation activates signaling through both the MAP kinase pathway and the PI-3 kinase pathway in Xenopus tissue explants. However, our analysis of these pathways in gastrula stage embryos indicates that the MAP kinase pathway is required for Cdx gene expression, whereas the PI-3 kinase pathway is not. We show that FGF and wnt signaling can interact in the regulation of Cdx genes and during gastrula stages the normal expression of the Cdx genes requires the activity of both pathways. Furthermore, we show that wnt mediated Cdx regulation is independent of the MAP kinase pathway. Author Affiliation: (a) Area 11, Department of Biology, University of York, York, YO10 5YW, UK (b) YCR Cancer Research Unit, Department of Biology, University of York, York, YO10 5YW, UK (c) Molecular Pharmacology Unit, Cancer Research UK, Biomedical Research Centre, Ninewells Hospital, Dundee, DD1 5SY, UK Article History: Received 19 September 2005; Revised 7 August 2006; Accepted 21 August 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.07.023 Byline: Hajime Ono (a)(b), Kim F. Rewitz (f), Tetsuro Shinoda (d), Kyo Itoyama (e), Anna Petryk (a)(c), Robert Rybczynski (f), Michael Jarcho (c), James T. Warren (f), Guillermo Marques (a)(b), Mary Jane Shimell (a), Lawrence I. Gilbert (f), Michael B. O'Connor (a)(b) Keywords: Ecdysone; Molting; CYP; p450 Abstract: Ecdysteroids regulate many key developmental events in arthropods including molting and metamorphosis. Recently, members of the Drosophila Halloween group of genes, that are required for embryonic viability and cuticle deposition, have been shown to code for several cytochrome P450 enzymes that catalyze the terminal hydroxylation steps in the conversion of cholesterol to the molting hormone 20-hydroxyecdysone. These P450s are conserved in other insects and each is thought to function throughout development as the sole mediator of a particular biosynthetic step since, where analyzed, each is expressed at all stages of development and shows no closely related homolog in their respective genomes. In contrast, we show here that several dipteran genomes encode two novel, highly related, microsomal P450 enzymes, Cyp307A1 and Cyp307A2, that likely participate as stage-specific components of the ecdysone biosynthetic machinery. This hypothesis comes from the observation that Cyp307A1 is encoded by the Halloween gene spook (spo), but unlike other Halloween class genes, Dmspo is not expressed during the larval stages. In contrast, Cyp307a2, dubbed spookier (spok), is expressed primarily during larval stages within the prothoracic gland cells of the ring gland. RNAi mediated reduction in the expression of this heterochromatin localized gene leads to arrest at the first instar stage which can be rescued by feeding the larva 20E, E or ketodiol but not 7dC. In addition, spok expression is eliminated in larvae carrying mutations in molting defective (mld), a gene encoding a nuclear zinc finger protein that is required for production of ecdysone during Drosophila larval development. Intriguingly, mld is not present in the Bombyx mori genome, and we have identified only one spook homolog in both Bombyx and Manduca that is expressed in both embryos and larva. These studies suggest an evolutionary split between Diptera and Lepidoptera in how the ecdysone biosynthetic pathway is regulated during development. Author Affiliation: (a) The Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 55455, USA (b) Howard Hughes Medical Institute, University of Minnesota, Minneapolis, MN, 55455, USA (c) Department of Pediatrics, University of Minnesota, Minneapolis, MN, 55455, USA (d) National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, 305-8634 Japan (e) Akita Prefectural Agricultural Experiment Station, 34-1 Genpachisawa, Aikawa, Yuwa, Kawabe, Akita 010-1231, Japan (f) Department of Biology, University of North Carolina, Chapel Hill, NC 27599-3280, USA Article History: Received 30 March 2006; Revised 7 July 2006; Accepted 9 July 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.04.446 Byline: Thorsten Hadrys (a)(b), Beena Punnamoottil (b), Mareike Pieper (b), Hiroshi Kikuta (c), Guillaume Pezeron (d), Thomas S. Becker (c), Victoria Prince (e), Robert Baker (a), Silke Rinkwitz (b) Keywords: Hox; Transcription; cis-regulation; Exon sharing; Conserved non-coding sequences; Genome; Transient transgenics; Enhancer detection; Hindbrain; Micro-RNA; miR-10b Abstract: The expression of zebrafish hoxb3a and hoxb4a has been found to be mediated through five transcripts, hoxb3a transcripts I-III and hoxb4a transcripts I-II, driven by four promoters. A 'master' promoter, located about 2 kb downstream of hoxb5a, controls transcription of a pre-mRNA comprising exon sequences of both genes. This unique gene structure is proposed to provide a novel mechanism to ensure overlapping, tissue-specific expression of both genes in the posterior hindbrain and spinal cord. Transgenic approaches were used to analyze the functions of zebrafish hoxb3a/hoxb4a promoters and enhancer sequences containing regions of homology that were previously identified by comparative genomics. Two neural enhancers were shown to establish specific anterior expression borders within the hindbrain and mediate expression in defined neuronal populations derived from hindbrain rhombomeres (r) 5 to 8, suggesting a late role of the genes in neuronal cell lineage specification. Species comparison showed that the zebrafish hoxb3a r5 and r6 enhancer corresponded to a sequence within the mouse HoxA cluster controlling activity of Hoxa3 in r5 and r6, whereas a homologous region within the HoxB cluster activated Hoxb3 expression but limited to r5. We conclude that the similarity of hoxb3a/Hoxa3 regulatory mechanisms reflect the shared descent of both genes from a single ancestral paralog group 3 gene. Author Affiliation: (a) Department of Physiology and Neuroscience, NYU Medical School, New York, NY 10016, USA (b) Institute of Biology, University of Oldenburg, 26111 Oldenburg, Germany (c) Sars Centre for Marine Molecular Biology, University of Bergen, ThormA[cedilla]hlensgt. 55, 5008 Bergen, Norway (d) Biologie Moleculaire du Developpement INSERM U368 Ecole Normale Superieure 46 Rue d'Ulm, 75230 Paris Cedex 05, France (e) Organismal Biology and Anatomy, University of Chicago, 1027 E 57th Street, Chicago, IL 60637, USA Article History: Received 12 September 2005; Revised 16 March 2006; Accepted 12 April 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.04.455 Byline: Selen Muratoglu (a), Betsy Garratt (a), Kristy Hyman (b), Kathleen Gajewski (b), Robert A. Schulz (b), Nancy Fossett (a)(b) Keywords: Drosophila; Friend of GATA; GATA; Gene regulation; Hematopoiesis; RUNX Abstract: Friend of GATA proteins interact with GATA factors to regulate development in a variety of tissues. We analyzed cis- and trans-regulation of the Drosophila gene, u-shaped, to better understand the transcriptional control of this important gene family during hematopoiesis. Using overlapping genomic fragments driving tissue-specific reporter-gene (lacZ) expression, we identified two minimal hematopoietic enhancers within the 7.4 kb region upstream of the transcription start site. One enhancer was active in all classes of hemocytes, whereas the other was active in hemocyte precursors and plasmatocytes only. The GATA factor, Serpent, directly regulated the activity of both enhancers. However, activity in the crystal cell lineage not only required Serpent but also the RUNX homologue, Lozenge. This is the first demonstration of GATA and RUNX direct regulation of Friend of GATA gene expression and provides additional evidence for the combinatorial control of crystal cell lineage commitment by Serpent, Lozenge, and U-shaped. In addition, we analyzed cis-regulation of ush expression in the lymph gland and identified similarities and differences between regulatory strategies used during embryonic and lymph gland hematopoiesis. The results of these studies provide information to analyze further the regulation of this conserved gene family and its role during hematopoietic lineage commitment. Author Affiliation: (a) Center for Vascular and Inflammatory Diseases and the Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201, USA (b) Department of Biochemistry and Molecular Biology, Graduate Program in Genes and Development, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA Article History: Received 10 February 2006; Revised 12 April 2006; Accepted 19 April 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.04.460 Byline: Benjamin Altenhein (a), Angela Becker (a), Christian Busold (b), Boris Beckmann (b), Jorg D. Hoheisel (b), Gerhard M. Technau (a) Keywords: Glial development; gcm; Glial genes; Microarrays; Drosophila embryogenesis Abstract: In the central nervous system of Drosophila, the induction of the glial cell fate is dependent on the transcription factor glial cells missing (gcm). Though a considerable number of other genes have been shown to be expressed in all or in subsets of glial cells, the course of glial cell differentiation and subtype specification is only poorly understood. This prompted us to design a whole genome microarray approach comparing gcm gain-of-function and, for the first time, gcm loss-of-function genetics to wildtype in time course experiments along embryogenesis. The microarray data were analyzed with special emphasis on the temporal profile of differential regulation. A comparison of both experiments enabled us to identify more than 300 potential gcm target genes. Validation by in situ hybridization revealed expression in glial cells, macrophages, and tendon cells (all three cell types depend on gcm) for 70 genes, of which more than 50 had been unknown to be under gcm control. Eighteen genes are exclusively expressed in glial cells, and their dependence on gcm was confirmed in situ. Initial considerations regarding the role of the newly discovered glial genes are discussed based on gene ontology and the temporal profile and subtype specificity of their expression. This collection of glial genes provides an important basis for the clarification of the genetic network controlling various aspects of glial development and function. Author Affiliation: (a) Institute of Genetics, University of Mainz, Germany (b) Division of Functional Genome Analysis, Deutsches Krebsforschungszentrum Heidelberg, Germany Article History: Received 22 March 2006; Revised 19 April 2006; Accepted 21 April 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.03.036 Byline: Jan Schlueter (a), Jorg Manner (b), Thomas Brand (a) Keywords: Wt1; Tbx18; Cfc; Bmp2; Bmp4; Popdc2; Nkx2.5; Proepicardium; Pericardium; Cardiac myocytes; Chick embryo Abstract: The proepicardium (PE) is a transient structure formed by pericardial coelomic mesothelium at the venous pole of the embryonic heart and gives rise to several cell types of the mature heart. In order to study PE development in chick embryos, we have analyzed the expression pattern of the marker genes Tbx18, Wt1, and Cfc. During PE induction, the three marker genes displayed a left-right asymmetric expression pattern. In each case, expression on the right side was stronger than on the left side. The left-right asymmetric gene expression observed here is in accord with the asymmetric formation of the proepicardium in the chick embryo. While initially the marker genes were expressed in the primitive sinus horn, subsequently, expression became confined to the PE mesothelium. In order to search for signaling factors involved in PE development, we studied Bmp2 and Bmp4 expression. Bmp2 was bilaterally expressed in the sinus venosus. In contrast, Bmp4 expression was initially expressed unilaterally in the right sinus horn and subsequently in the PE. In order to assess its functional role, BMP signaling was experimentally modulated by supplying exogenous BMP2 and by inhibiting endogenous BMP signaling through the addition of Noggin. Both supplying BMP and blocking BMP signaling resulted in a loss of PE marker gene expression. Surprisingly, both experimental situations lead to cardiac myocyte formation in the PE cultures. Careful titration experiments with exogenously added BMP2 or Noggin revealed that PE-specific marker gene expression depends on a low level of BMP signaling. Implantation of BMP2-secreting cells or beads filled with Noggin protein into the right sinus horn of HH stage 11 embryos resulted in downregulation of Tbx18 expression, corresponding to the results of the explant assay. Thus, a distinct level of BMP signaling is required for PE formation in the chick embryo. Author Affiliation: (a) Cell and Developmental Biology, Theodor-Boveri-Institute, University of Wurzburg, Am Hubland, D-97074 Wurzburg, Germany (b) Department of Anatomy and Embryology, University of Gottingen, Germany Article History: Received 22 August 2005; Revised 16 March 2006; Accepted 28 March 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.03.028 Byline: Macie B. Walker (a), Craig T. Miller (a), Jared Coffin Talbot (a), David W. Stock (b), Charles B. Kimmel (a) Keywords: Sturgeon; Furin; Endothelin1; Dlx; Barx1; Sox9a; Runx2b Abstract: Endothelin1 (Edn1) signaling promotes ventral character to the facial skeleton. In zebrafish edn1 mutants, the ventral jaw structures are severely reduced and fused to their dorsal counterparts, with a loss of joints that normally form at an intermediate dorsal-ventral position. Loss of function at another locus, sturgeon, also yields joint losses, but only mild reductions in the ventral jaw structures. We show that sturgeon encodes one of two orthologs of Furin present in zebrafish, and that both furin genes may function partially redundantly to activate Edn1 signaling. Supporting this hypothesis, early expression of edn1-dependent genes is downregulated in sturgeon (furinA) mutants. Later in development, expression of most of these genes recovers to near wild-type levels in furinA mutants but not in edn1 mutants. The recovery explains the less severe furinA mutant skeletal phenotype and suggests that late gene expression is dependent on a critical level of Edn1 signaling not present in the more severe edn1 mutants. However, expression defects in the intermediate joint-forming domains in both mutants persist, explaining the joint losses observed later in both mutants. We further show that in both mutants the arches fail to correctly undergo ventral elongation before skeletogenesis begins and propose a model in which this failure is largely responsible for the loss of an Edn1-dependent compartmentation of the arch into the intermediate and ventral domains. Author Affiliation: (a) Institute of Neuroscience, 1254 University of Oregon, Eugene, OR 97403, USA (b) Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, CO 80309-0334, USA Article History: Received 14 October 2005; Revised 13 March 2006; Accepted 20 March 2006 Article Note: (footnote) [star] The zebrafish gene zgc:110654 has been renamed barx1 on April 19, 2006.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.02.020 Byline: Raman M. Das (a), Nick J. Van Hateren (a), Gareth R. Howell (a), Elizabeth R. Farrell (b), Fiona K. Bangs (b), Victoria C. Porteous (a), Elizabeth M. Manning (c), Michael J. McGrew (d), Kyoji Ohyama (c), Melanie A. Sacco (e), Pam A. Halley (b), Helen M. Sang (d), Kate G. Storey (b), Marysia Placzek (c), Cheryll Tickle (b), Venugopal K. Nair (e), Stuart A. Wilson (a) Keywords: Chicken; Embryo; RNA; Interference; microRNA; RNAi; siRNA Abstract: RNA interference (RNAi) provides an effective method to silence gene expression and investigate gene function. However, RNAi tools for the chicken embryo have largely been adapted from vectors designed for mammalian cells. Here we present plasmid and retroviral RNAi vectors specifically designed for optimal gene silencing in chicken cells. The vectors use a chicken U6 promoter to express RNAs modelled on microRNA30, which are embedded within chicken microRNA operon sequences to ensure optimal Drosha and Dicer processing of transcripts. The chicken U6 promoter works significantly better than promoters of mammalian origin and in combination with a microRNA operon expression cassette (MOEC), achieves up to 90% silencing of target genes. By using a MOEC, we show that it is also possible to simultaneously silence two genes with a single vector. The vectors express either RFP or GFP markers, allowing simple in vivo tracking of vector delivery. Using these plasmids, we demonstrate effective silencing of Pax3, Pax6, Nkx2.1, Nkx2.2, Notch1 and Shh in discrete regions of the chicken embryonic nervous system. The efficiency and ease of use of this RNAi system paves the way for large-scale genetic screens in the chicken embryo. Author Affiliation: (a) Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK (b) Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee, Dundee DD15 5EH, UK (c) Centre for Developmental and Biomedical Genetics, Department of Biomedical Science, The University of Sheffield, Sheffield S10 2TN, UK (d) Roslin Institute, Roslin, Midlothian EH25 9PS, UK (e) Viral Oncogenesis Group, Institute for Animal Health, Compton, Berkshire RG20 7NN, UK Article History: Received 7 October 2005; Revised 13 February 2006; Accepted 15 February 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.02.023 Byline: Kristopher Schwab (a), Heather A. Hartman (b), Hung-Chi Liang (a), Bruce J. Aronow (c), Larry T. Patterson (b), S. Steven Potter (a) Keywords: Kidney development; Microarrays; Hox genes; Laser capture microdissection; Metanephric mesenchyme; Ureteric bud; Morphogenesis Abstract: The Hox11 paralogous genes play critical roles in kidney development. They are expressed in the early metanephric mesenchyme and are required for the induction of ureteric bud formation and its subsequent branching morphogenesis. They are also required for the normal nephrogenesis response of the metanephric mesenchyme to inductive signals from the ureteric bud. In this report, we use microarrays to perform a comprehensive gene expression analysis of the Hoxa11/Hoxd11 mutant kidney phenotype. We examined E11.5, E12.5, E13.5 and E16.5 developmental time points. A novel high throughput strategy for validation of microarray data is described, using additional biological replicates and an independent microarray platform. The results identified 13 genes with greater than 3-fold change in expression in early mutant kidneys, including Hoxa11s, GATA6, TGFbeta2, chemokine ligand 12, angiotensin receptor like 1, cytochrome P450, cadherin5, and Lymphocyte antigen 6 complex, Iroquois 3, EST A930038C07Rik, Meox2, Prkcn, and Slc40a1. Of interest, many of these genes, and others showing lower fold expression changes, have been connected to processes that make sense in terms of the mutant phenotype, including TGFbeta signaling, iron transport, protein kinase C function, growth arrest and GDNF regulation. These results identify the multiple molecular pathways downstream of Hox11 function in the developing kidney. Author Affiliation: (a) Division of Developmental Biology, Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA (b) Division of Nephrology, Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA (c) Division of Bioinformatics, Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA Article History: Received 13 December 2005; Revised 2 February 2006; Accepted 16 February 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.12.015 Byline: Dan Zou (a), Derek Silvius (a), Julie Davenport (a), Raphaelle Grifone (b), Pascal Maire (b), Pin-Xian Xu (a) Keywords: Six1; Six4; Third pharyngeal pouch; Patterning; Thymus; Parathyroid; Eya1; Pax; Hoxa3; Fox; Tbx1; Fgf8; Wnt5b Abstract: Previous studies have suggested a role of the homeodomain Six family proteins in patterning the developing vertebrate head that involves appropriate segmentation of three tissue layers, the endoderm, the paraxial mesoderm and the neural crest cells; however, the developmental programs and mechanisms by which the Six genes act in the pharyngeal endoderm remain largely unknown. Here, we examined their roles in pharyngeal pouch development. Six1.sup.-/- mice lack thymus and parathyroid and analysis of Six1.sup.-/- third pouch endoderm demonstrated that the patterning of the third pouch into thymus/parathyroid primordia is initiated. However, the endodermal cells of the thymus/parathyroid rudiments fail to maintain the expression of the parathyroid-specific gene Gcm2 and the thymus-specific gene Foxn1 and subsequently undergo abnormal apoptosis, leading to a complete disappearance of organ primordia by E12.5. This thus defines the thymus/parathyroid defects present in the Six1 mutant. Analyses of the thymus/parathyroid development in Six1.sup.-/- ;Six4.sup.-/- double mutant show that both Six1 and Six4 act synergistically to control morphogenetic movements of early thymus/parathyroid tissues, and the threshold of Six1/Six4 appears to be crucial for the regulation of the organ primordia-specific gene expression. Previous studies in flies and mice suggested that Eya and Six genes may function downstream of Pax genes. Our data clearly show that Eya1 and Six1 expression in the pouches does not require Pax1/Pax9 function, suggesting that they may function independently from Pax1/Pax9. In contrast, Pax1 expression in all pharyngeal pouches requires both Eya1 and Six1 function. Moreover, we show that the expression of Tbx1, Fgf8 and Wnt5b in the pouch endoderm was normal in Six1.sup.-/- embryos and slightly reduced in Six1.sup.-/- ;Six4.sup.-/- double mutant, but was largely reduced in Eya1.sup.-/- embryos. These results indicate that Eya1 appears to be upstream of very early events in the initiation of thymus/parathyroid organogenesis, while Six genes appear to act in an early differentiation step during thymus/parathyroid morphogenesis. Together, these analyses establish an essential role for Eya1 and Six genes in patterning the third pouch into organ-specific primordia. Author Affiliation: (a) McLaughlin Research Institute for Biomedical Sciences, Great Falls, MT 59405, USA (b) Institut Cochin-INSERM 567, CNRS UMR 8104, Universite Paris V, 24 Rue du Faubourg Saint Jacques, 75014 Paris, France Article History: Received 27 May 2005; Revised 17 November 2005; Accepted 6 December 2005

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.01.023 Byline: Claire E. Hirst (a), Elizabeth S. Ng (a), Lisa Azzola (a), Anne K. Voss (b), Tim Thomas (b), Edouard G. Stanley (a), Andrew G. Elefanty (a) Keywords: Embryonic stem cell; Cell differentiation; Transcriptional profiling; SLAIN1 Abstract: We analyzed the transcriptional profiles of differentiating mouse embryonic stem cells (mESCs) and show that embryoid bodies (EBs) sequentially expressed genes associated with the epiblast, primitive streak, mesoderm and endoderm of the developing embryo, validating ESCs as a model system for identifying cohorts of genes marking specific stages of embryogenesis. By comparing the transcriptional profiles of undifferentiated ESCs to those of their differentiated progeny, we identified 503 mESC and 983 hESC genes selectively expressed in undifferentiated ES cells. Over 75% of the mESC genes were expressed in hESC and vice versa, attesting to the underlying similarity of mESCs and hESCs. The expression of a cohort of 68 genes decreased greater than 2-fold during differentiation in both mESCs and hESCs. As well as containing many validated ESC genes such as Oct4 [Pou5f1], Nanog and Nodal, this cohort included an uncharacterised gene (FLJ30046), which we designated SLAIN1/Slain1. Slain1 was expressed at the stem cell and epiblast stages of ESC differentiation and in the epiblast, nervous system, tailbud and somites of the developing mouse embryo. SLAIN1 and its more widely expressed homologue SLAIN2 comprise a new family of structurally unique genes conserved throughout vertebrate evolution. Author Affiliation: (a) Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, VIC 3800, Australia (b) The Walter and Eliza Hall Institute of Medical Research, Royal Parade, Parkville, VIC 3050, Australia Article History: Received 16 December 2005; Accepted 20 January 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.02.003 Byline: Dirk Sieger (a), Bastian Ackermann (a), Christoph Winkler (b), Diethard Tautz (a), Martin Gajewski (a) Keywords: Somitogenesis; bHLH transcription factor; her genes; Anterior somites; Morpholino-oligonucleotide mediated knockdown Abstract: Delta-Notch and FGF signaling are involved in the control of somitogenesis in zebrafish. her genes are generally known as downstream targets of Delta-Notch signaling, but the her13.2 gene from zebrafish has recently been shown to depend on FGF signaling only. We have here studied the functional role of her13.2 in conjunction with her genes that are under Delta-Notch control. We show that joint inactivation of her1 and her13.2 leads to a complete loss of all somitic borders, including the most anterior ones. This somitic phenotype is much stronger than would be expected from the effects of the inactivation of either gene alone. A joint inactivation of her13.2 and her7, which is a paralogue of her1, does not show this enhanced effect. Thus, our results confirm inferences from in vitro studies that her1 and her13.2 form specific heterodimers, which may directly be required for regulating further target genes. These two her genes thus constitute the link between Delta-Notch pathway and FGF signaling during entire somitogenesis. We show that this interaction is conserved in the rice fish medaka, as a joint inactivation of the respective orthologues leads also to the same phenotype as in zebrafish. In addition, our results suggest that the mechanisms for anterior and posterior somite formation are not principally different, although the anterior somites often seem more refractory to genetic perturbations. Author Affiliation: (a) Universitat zu Koln, Institut fur Genetik, Zuelpicherstr. 47, 50674 Koln, Germany (b) Universitat Wurzburg, Physiologische Chemie I, Biozentrum, Am Hubland, 97074 Wurzburg, Germany Article History: Received 18 November 2005; Revised 27 January 2006; Accepted 3 February 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.12.016 Byline: Atsushi Takebe (a)(c), Takumi Era (a), Mitsuhiro Okada (a)(b), Lars Martin Jakt (a)(b), Yoshikazu Kuroda (c), Shin-Ichi Nishikawa (a) Keywords: ES cell; In vitro differentiation; DNA microarray; PDGFR[alpha]; ARID3b Abstract: An inherent difficulty in using DNA microarray technology on the early mouse embryo is its relatively small size. In this study, we investigated whether use of ES cell differentiation culture, which has no theoretical limit in the number of cells that can be generated, can improve this situation. Seven distinct ES-cell-derived populations were analyzed by DNA microarray and examined for genes whose distribution patterns are similar to those of PDGFR[alpha], a gene implicated in differentiation of mesoderm/mesenchymal lineages. Using software developed in our laboratory, we formed a group of 30 genes which showed the highest similarity to PDGFR[alpha], 18 of these genes were shown to be involved in development of either mesodermal, mesenchymal or neural crest cells. This list also contains several genes whose role in embryogenesis has not yet been fully identified. One such molecule is mARID3b. The mARID3b expression is found in the paraxial mesoderm and cranial mesenchyme. mARID3b-null mouse showed early embryonic lethality, and most phenotypes of this mutant appear to develop from a failure to generate a sufficient number of cranial mesenchymal cells. These results demonstrate the potential use of ES cell differentiation culture in identifying novel genes playing an indispensable role in embryogenesis. Author Affiliation: (a) Laboratory for Stem Cell Biology, RIKEN Center for Development Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan (b) The Foundation for Biomedical Research and Innovation, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan (c) Division of Gastroenterological Surgery, Graduate School of Medicine, Kobe University, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan Article History: Received 27 June 2005; Revised 18 November 2005; Accepted 6 December 2005

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.12.054 Byline: Jonathan R. Chubb (a)(h)(i), Gareth Bloomfield (b)(c), Qikai Xu (d)(e), Markus Kaller (f), Al Ivens (b)(c), Jason Skelton (b)(c), Bryan M. Turner (g), Wolfgang Nellen (f), Gad Shaulsky (d)(e), Robert R. Kay (b)(c), Wendy A. Bickmore (h), Robert H. Singer (a) Keywords: Chromatin; Dictyostelium; Set1; Lysine 4 methylation; Gene cluster; Clock Abstract: Histone-modifying enzymes have enormous potential as regulators of the large-scale changes in gene expression occurring during differentiation. It is unclear how different combinations of histone modification coordinate regimes of transcription during development. We show that different methylation states of lysine 4 of histone H3 (H3K4) mark distinct developmental phases of the simple eukaryote, Dictyostelium. We demonstrate that the enzyme responsible for all mono, di and tri-methylation of H3K4 is the Dictyostelium homolog of the Set1 histone methyltransferase. In the absence of Set1, cells display unusually rapid development, characterized by precocious aggregation of amoebae into multicellular aggregates. Early differentiation markers are abundantly expressed in growing set1 cells, indicating the differentiation program is ectopically activated during growth. This phenotype is caused specifically by the loss of Set1 catalytic activity. Set1 mutants induce premature differentiation in wild-type cells, indicating Set1 regulates production of an extra-cellular factor required for the correct perception of growth conditions. Microarray analysis of the set1 mutants reveals genomic clustering of mis-expressed genes, suggesting a requirement for Set1 in the regulation of chromatin-mediated events at gene clusters. Author Affiliation: (a) Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, The Bronx, NY 10461, USA (b) MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK (c) The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK (d) Graduate Program in Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, TX 77030, USA (e) Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA (f) Abt. Genetik, Universitat Kassel, Heinrich-Plett-Str. 40, D-34132 Kassel, Germany (g) Chromatin and Gene Expression Group, Anatomy Department, University of Birmingham Medical School, Birmingham B15 2TT, UK (h) MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK (i) Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK Article History: Received 3 November 2005; Accepted 30 December 2005

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.11.028 Byline: Limin Hao (a), Gudrun Aspock (b), Thomas R. Burglin (a)(c) Keywords: Caenorhabditis elegans; Embryonic lethal; warthog (wrt) genes; wrt-5; Seam cells; Cold sensitive Abstract: The Caenorhabditis elegans genome encodes a series of hedgehog-related genes, which are thought to have evolved and diverged from an ancestral Hh gene. They are classified into several families based on their N-terminal domains. Here, we analyze the expression and function of a member of the warthog gene family, wrt-5, that lacks the Hint/Hog domain. wrt-5 is expressed in seam cells, the pharynx, pharyngeal-intestinal valve cells, neurons, neuronal support cells, the excretory cell, and the reproductive system. WRT-5 protein is secreted into the extracelluar space during embryogenesis. Furthermore, during larval development, WRT-5 protein is secreted into the pharyngeal lumen and the pharyngeal expression changes in a cyclical manner in phase with the molting cycle. Deletion mutations in wrt-5 cause embryonic lethality, which are temperature sensitive and more severe at 15[degrees]C than at 25[degrees]C. Animals that hatch exhibit variable abnormal morphology, for example, bagging worms, blistering, molting defects, or Roller phenotypes. We examined hypodermal cell junctions using the AJM-1a*GFP marker in the wrt-5 mutant background and observed cell boundary abnormalities in the arrested embryos. AJM-1a*GFP protein is also misplaced in pharyngeal muscle cells in the absence of WRT-5. In conclusion, we show that wrt-5 is an essential gene that - despite its lack of a Hint domain - has multiple functions in C. elegans and is implicated in cell shape integrity. Author Affiliation: (a) Department of Biosciences at Novum, and Center for Genomics and Bioinformatics, Karolinska Institutet, Halsovagen 7, SE-141 57 Huddinge, Sweden (b) Biozentrum, University of Basel, 4056 Basel, Switzerland (c) Life Sciences, Sodertorns Hogskola, Alfred Nobels Alle 7, SE-141 89 Huddinge, Sweden Article History: Received 26 May 2005; Revised 17 October 2005; Accepted 14 November 2005

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.11.009 Byline: Xi-De Wang (a), Ching Ching Leow (a), Jiping Zha (b), Zhijun Tang (c), Zora Modrusan (a), Freddy Radtke (d), Michel Aguet (e), Frederic J. de Sauvage (a), Wei-Qiang Gao (a) Keywords: Notch; Prostate development; Cell proliferation; Cell differentiation; Mouse models; Prostate cancer Abbreviations: BPH, benign prostatic hyperplasia; CK, cytokeratin; Cre, Cre-recombinase; DAPI, 4,6-diamidino-2-phenylindole Abstract: Notch pathway is crucial for stem/progenitor cell maintenance, growth and differentiation in a variety of tissues. Using a transgenic cell ablation approach, we found in our previous study that cells expressing Notch1 are crucial for prostate early development and re-growth. Here, we further define the role of Notch signaling in regulating prostatic epithelial cell growth and differentiation using biochemical and genetic approaches in ex vivo or in vivo systems. Treatment of developing prostate grown in culture with inhibitors of gamma-secretase/presenilin, which is required for Notch cleavage and activation, caused a robust increase in proliferation of epithelial cells co-expressing cytokeratin 8 and 14, lack of luminal/basal layer segregation and dramatically reduced branching morphogenesis. Using conditional Notch1 gene deletion mouse models, we found that inactivation of Notch1 signaling resulted in profound prostatic alterations, including increased tufting, bridging and enhanced epithelial proliferation. Cells within these lesions co-expressed both luminal and basal cell markers, a feature of prostatic epithelial cells in predifferentiation developmental stages. Microarray analysis revealed that the gene expression in a number of genetic networks was altered following Notch1 gene deletion in prostate. Furthermore, expression of Notch1 and its effector Hey-1 gene in human prostate adenocarcinomas were found significantly down-regulated compared to normal control tissues. Taken together, these data suggest that Notch signaling is critical for normal cell proliferation and differentiation in the prostate, and deregulation of this pathway may facilitate prostatic tumorigenesis. Author Affiliation: (a) Department of Molecular Biology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA (b) Department of Pathology, Genentech, Inc., South San Francisco, CA 94080, USA (c) Department of Bioinformatics, Genentech, Inc., South San Francisco, CA 94080, USA (d) Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, 1066 Epalinges, Switzerland (e) Swiss Institute of Experimental Cancer Research, 1066 Epalinges, Switzerland Article History: Received 21 September 2005; Revised 5 November 2005; Accepted 7 November 2005

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.09.047 Byline: Takuya Minokawa (a), Athula H. Wikramanayake (b), Eric H. Davidson (a) Keywords: wnt8; cis-regulation; Gene network; Sea urchin embryo Abstract: Expression of the wnt8 gene is the key transcriptional motivator of an intercellular signaling loop which drives endomesoderm specification forward early in sea urchin embryogenesis. This gene was predicted by network perturbation analysis to be activated by inputs from the blimp1/krox gene, itself expressed zygotically in the endomesoderm during cleavage; and by a Tcf1/[beta]-catenin input. The implication is that zygotic expression of wnt8 is stimulated in neighboring cells by its own gene product, since reception of the Wnt8 ligand causes [beta]-catenin nuclearization. Here, the modular cis-regulatory system of the wnt8 gene of Strongylocentrotus purpuratus was characterized functionally, and shown to respond to blockade of both Blimp1/Krox and Tcf1/[beta]-catenin inputs just as does the endogenous gene. The genomic target sites for these factors were demonstrated by mutation in one of the cis-regulatory modules. The Tcf1/[beta]-catenin and Blimp1/Krox inputs are both necessary for normal endomesodermal expression mediated by this cis-regulatory module; thus, the genomic regulatory code underlying the predicted signaling loop thus resides in the wnt8 cis-regulatory sequence. In a second regulatory region, which initiates expression in micromere and macromere descendant cells early in cleavage, Tcf1 sites act to repress ectopic transcription in prospective ectoderm cells. Author Affiliation: (a) Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA (b) Department of Zoology, University of Hawaii at Manoa, Honolulu, HI 96822, USA Article History: Received 30 August 2005; Revised 21 September 2005; Accepted 23 September 2005

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.08.023 Byline: Hua Pan (a), Marilyn J. O'Brien (b), Karen Wigglesworth (b), John J. Eppig (b), Richard M. Schultz (a) Keywords: Mouse oocyte development; Gene expression; Microarrays; Meiotic competence; Developmental competence; Development in vitro; Gonadotropin priming Abstract: The molecular basis for acquisition of meiotic and developmental competence, the two main outcomes of oocyte development and essential for producing an egg capable of being fertilized and supporting development to term, is largely unknown. Using microarrays, we characterized global changes in gene expression in oocytes derived from primordial, primary, secondary, small antral, and large antral follicles and used Expression Analysis Systematic Explorer (EASE) to identify biological and molecular processes that accompany these transitions and likely underpin acquisition of meiotic and developmental competence. The greatest degree of change in gene expression occurs during the primordial to primary follicle transition. Of particular interest is that specific chromosomes display significant changes in their overall transcriptional activity and that in some cases these changes are largely confined to specific regions on these chromosomes. We also examined the transcript profile of oocytes that developed in vitro, as well as following eCG priming. Remarkably, the expression profiles only differed by 4% and 2% from oocytes that developed in vivo when compared to oocytes that developed in vitro from either primordial or secondary follicles, respectively. About 1% of the genes were commonly mis-expressed, and EASE analysis revealed there is an over-representation of genes involved in transcription. Developmental competence of oocytes obtained from eCG-primed mice was substantially improved when compared to oocytes obtained from unprimed mice, and this correlated with decreased expression of genes implicated in basal transcription. Author Affiliation: (a) Department of Biology, University of Pennsylvania, 415 South University Avenue, Philadelphia, PA 19104-6018, USA (b) The Jackson Laboratory, Bar Harbor, ME 04609, USA Article History: Received 3 May 2005; Revised 9 August 2005; Accepted 11 August 2005

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.07.002 Byline: Rebecca L. Bradford (a), Chenwei Wang (a), Donald J. Zack (a)(b)(c), Ruben Adler (a)(b) Keywords: Photoreceptor; Differentiation; Chick embryo; Visual pigments; Cell culture; Growth factors Abstract: Photoreceptor differentiation requires the coordinated expression of numerous genes. It is unknown whether those genes share common regulatory mechanisms or are independently regulated by distinct mechanisms. To distinguish between these scenarios, we have used in situ hybridization, RT-PCR, and real-time PCR to analyze the expression of visual pigments and other photoreceptor-specific genes during chick embryo retinal development in ovo, as well as in retinal cell cultures treated with molecules that regulate the expression of particular visual pigments. In ovo, onset of gene expression was asynchronous, becoming detectable at the time of photoreceptor generation (ED 5-8) for some photoreceptor genes, but only around the time of outer segment formation (ED 14-16) for others. Treatment of retinal cell cultures with activin, staurosporine, or CNTF selectively induced or down-regulated specific visual pigment genes, but many cognate rod- or cone-specific genes were not affected by the treatments. These results indicate that many photoreceptor genes are independently regulated during development, are consistent with the existence of at least two distinct stages of gene expression during photoreceptor differentiation, suggest that intrinsic, coordinated regulation of a cascade of gene expression triggered by a commitment to the photoreceptor fate is not a general mechanism of photoreceptor differentiation, and imply that using a single photoreceptor-specific 'marker' as a proxy to identify photoreceptor cell fate is problematic. Author Affiliation: (a) The Department of Ophthalmology, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA (b) The Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA (c) The Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA Article History: Received 2 June 2005; Revised 29 June 2005; Accepted 6 July 2005

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.06.026 Byline: Sarah Ivins (a), Kelly Lammerts van Beuren (a), Catherine Roberts (a), Chela James (a), Elizabeth Lindsay (b), Antonio Baldini (b), Paris Ataliotis (a), Peter J. Scambler (a) Keywords: Expression microarray; DiGeorge syndrome; 22q11 deletion; Pharyngeal development Tbx1 Abstract: 22q11-deletion (DiGeorge/velocardiofacial) syndrome (22q11DS) is modeled by mutation of murine transcription factor Tbx1. As part of efforts to identify transcriptional targets of Tbx1, we analyzed the transcriptome of the pharyngeal region of Df1/+;Tbx1.sup.+/- embryos at 9.5 days of embryonic development using two independent microarray platforms. In this model, embryos are null for Tbx1, with hemizygosity of genes in cis with Tbx1 on one chromosome providing a positive control for array sensitivity. Reduced mRNA levels of genes deleted from Df1 were detected on both platforms. Expression level filtering and statistical analysis identified several genes that were consistently differentially expressed between mutant and wild type embryos. Real-time quantitative PCR and in situ hybridization validated diminished expression of Pax9 and Gcm2, genes known to be required for normal thymus and parathyroid gland morphogenesis, whereas Pax1, Hoxa3, Eya1, and Foxn1, which are similarly required, were not down-regulated. Gbx2, a gene required for normal arch artery development, was down-regulated specifically in the pharyngeal endoderm and the posterior part of pharyngeal arch 1, and is a potential point of cross talk between the Tbx1 and Fgf8 controlled pathways. These experiments highlight which genes and pathways potentially affected by lack of Tbx1, and whose role may be explored further by testing for epistasis using mouse mutants. Author Affiliation: (a) Molecular Medicine Unit, Institute of Child Health, London WC1N 1EH, UK (b) Department of Pediatrics (Cardiology), Baylor College of Medicine, 6621 Fannin St., FC430.09, Houston, TX 77030, USA Article History: Received 6 April 2005; Revised 20 June 2005; Accepted 21 June 2005

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.06.014 Byline: Gregory K. Davis (a), Joseph A. D'Alessio (b), Nipam H. Patel (b)(c)(d) Keywords: Pax; Arthropod; Crustacean; Centipede; Spider; Paired; Gooseberry; Segmentation; Pair-rule; Pattern formation; Evolution Abstract: Several features of Pax3/7 gene expression are shared among distantly related insects, including pair-rule, segment polarity, and neural patterns. Recent data from arachnids imply that roles in segmentation and neurogenesis are likely to be played by Pax3/7 genes in all arthropods. To further investigate Pax3/7 genes in non-insect arthropods, we isolated two monoclonal antibodies that recognize the products of Pax3/7 genes in a wide range of taxa, allowing us to quickly survey Pax3/7 expression in all four major arthropod groups. Epitope analysis reveals that these antibodies react to a small subset of Paired-class homeodomains, which includes the products of all known Pax3/7 genes. Using these antibodies, we find that Pax3/7 genes in crustaceans are expressed in an early broad and, in one case, dynamic domain followed by segmental stripes, while myriapods and chelicerates exhibit segmental stripes that form early in the posterior-most part of the germ band. This suggests that Pax3/7 genes acquired their role in segmentation deep within, or perhaps prior to, the arthropod lineage. However, we do not detect evidence of pair-rule patterning in either myriapods or chelicerates, suggesting that the early pair-rule expression pattern of Pax3/7 genes in insects may have been acquired within the crustacean-hexapod lineage. Author Affiliation: (a) Committee on Developmental Biology, University of Chicago, Chicago, IL 60637, USA (b) Department of Molecular Cell Biology, University of California, Berkeley, CA 94720-3140, USA (c) Department of Integrative Biology, University of California, Berkeley, CA 94720-3140, USA (d) Howard Hughes Medical Institute, University of California, Berkeley, CA 94720-3140, USA Article History: Received 6 May 2005; Revised 6 June 2005; Accepted 13 June 2005

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.06.004 Byline: Diana L. Walker (a), Scott J. Vacha (b), Margaret L. Kirby (c), Cecilia W. Lo (a) Keywords: Coronary vessels; Connexin43; Knockout; VEGF; Vascular smooth muscle; Endothelial cells; Heart development Abstract: The connexin43 knockout (Cx43[alpha]1 KO) mouse dies at birth from outflow obstruction associated with infundibular pouches. To elucidate the origin of the infundibular pouches, we used microarray analysis to investigate gene expression changes in the pouch tissue. We found elevated expression of many genes encoding markers for vascular smooth muscle (VSM), endothelial cells, and fibroblasts, cell types that are epicardially derived and essential for coronary vasculogenesis. This was accompanied by increased expression of VEGF and genes in the TGF[beta] and VEGF/Notch/Eph cell-signaling pathways known to regulate vasculogenesis/angiogenesis. Using immunohistochemistry and a VSM lacZ reporter gene, we confirmed an abundance of ectopic VSM and endothelial cells in the infundibular pouch and in some regions of the right ventricle forming secondary pouches. This was associated with distinct thinning of the compact myocardium. TUNEL labeling showed increased apoptosis in the pouch tissue, in agreement with the finding of altered expression of many apoptotic genes. Defects in vascular remodeling were indicated by a marked reduction in the branching complexity of the distal coronary arteries. In the near term KO mouse, we also observed a profusion of large coronary vascular plexuses subepicardially. This was associated with elevated epicardial expression of VEGF and abnormal epicardial cell morphology. Together, these observations indicate that dysregulated coronary vasculogenesis plays a pivotal role in formation of the infundibular pouches and suggests an essential role for Cx43[alpha]1 gap junctions in coronary vasculogenesis and vascular remodeling. Author Affiliation: (a) Laboratory of Developmental Biology, National Heart, Lung and Blood Institute, National Institutes of Health, Building 50/Room 4537, 9000 Rockville Pike, Bethesda, MD 20892-8019, USA (b) Agilent Technologies, 3500 Deer Creek Road, Palo Alto, CA 94304, USA (c) Neonatal Perinatal Research Institute, Division of Neonatology, Duke University Medical Center, Durham, NC 27710, USA Article History: Received 4 January 2005; Revised 3 June 2005; Accepted 8 June 2005

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.05.017 Byline: Kara Thoemke (a), Woelsung Yi (a), Jennifer M. Ross (a), Shinseog Kim (a), Valerie Reinke (b), David Zarkower (a) Keywords: Sex determination; Sex-enriched; Sexual dimorphism; tra-1; C. elegans Abstract: Sex determination in C. elegans is controlled by the TRA-1 zinc finger protein, a Ci/GLI homolog that promotes female cell fates throughout the body. The regulatory hierarchy that controls TRA-1 is well established, but the downstream effectors that establish sexual dimorphism during larval development remain largely unknown. Here, we describe the use of cDNA microarrays to identify sex-enriched transcripts expressed during three stages of C. elegans larval development. By excluding previously identified germline-enriched transcripts, we focused on somatic sexual development. This approach identified a large number of sex-enriched transcripts that are good candidates to encode regulators of somatic sexual development. We found little overlap between genes with sex-enriched expression in early versus late larval development, indicating that distinct sexual regulatory programs operate at these times. Genes with sex-enriched expression are found throughout the genome, with no strong bias between autosomes and X chromosomes. Reporter gene analysis revealed that these genes are expressed in highly specific patterns in a variety of sexually dimorphic cells. We searched for TRA-1 consensus DNA binding sites near genes with sex-enriched expression, and found that most strongly sex-enriched mRNAs are likely to be regulated indirectly by TRA-1. These results suggest that TRA-1 controls sexual dimorphism through a small number of intermediary regulators rather than by acting directly on the full constellation of genes involved in sex-specific differentiation. Author Affiliation: (a) Department of Genetics, Cell Biology, and Development, and Developmental Biology Center, University of Minnesota, Minneapolis, MN 55455, USA (b) Department of Genetics, Yale University School of Medicine, New Haven, CT 06520, USA Article History: Received 2 February 2005; Revised 16 May 2005; Accepted 16 May 2005

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/S0012-1606(02)00032-5 Byline: Thomas M Brodigan (a), J.i Liu (b), Morgan Park (a), Edward T Kipreos (b), Michael Krause (a) Keywords: Cyclin; cye-1; Cyclin-dependent kinase; Expression Abstract: Our interest in the coordination of cell cycle control and differentiation has led us to investigate the Caenorhabditis elegans cye-1 gene encoding the G.sub.1 cell cycle regulator cyclin E. We have studied the expression and function of cye-1 by using monoclonal antibodies directed against CYE-1 protein, cye-1::GFP reporter genes, and a cye-1 chromosomal deletion mutation. We show that a ubiquitous embryonic pattern of expression becomes restricted and dynamic during postembryonic development. Promoter analysis reveals a relatively small region of cis-acting sequences that are necessary for the complex pattern of expression of this gene. Our studies demonstrate that two other G.sub.1 cell cycle genes, encoding cyclin D and CDK4/6, have similarly compact promoter requirements. This suggests that a relatively simple mechanism of regulation may underlie the dynamic developmental patterns of expression exhibited by these three G.sub.1 cell cycle genes. Our analysis of a new cye-1 deletion allele confirms and extends previous studies of two point mutations in the gene. Author Affiliation: (a) Laboratory of Molecular Biology, NIDDK, NIH, Bethesda, MD 20892, USA (b) Department of Cellular Biology, University of Georgia, Athens, GA 30602, USA Article History: Received 1 August 2002; Revised 23 October 2002; Accepted 30 October 2002