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BackgroundPbx genes encode TALE class homeodomain transcription factors that pattern the developing neural tube, pancreas, and blood. Within the hindbrain, Pbx cooperates with Hox proteins to regulate rhombomere segment identity. Pbx cooperates with Eng to regulate midbrain-hindbrain boundary maintenance, and with MyoD to control fast muscle cell differentiation. Although previous results have demonstrated that Pbx is required for proper eye size, functions in regulating retinal cell identity and patterning have not yet been examined.ResultsAnalysis of retinal ganglion cell axon pathfinding and outgrowth in pbx2/4 null embryos demonstrated a key role for pbx genes in regulating neural cell behavior. To identify Pbx-dependent genes involved in regulating retino-tectal pathfinding, we conducted a microarray screen for Pbx-dependent transcripts in zebrafish, and detected genes that are specifically expressed in the eye and tectum. A subset of Pbx-dependent retinal transcripts delineate specific domains in the dorso-temporal lobe of the developing retina. Furthermore, we determined that some Pbx-dependent transcripts also require Meis1 and Gdf6a function. Since gdf6a expression is also dependent on Pbx, we propose a model in which Pbx proteins regulate expression of the growth factor gdf6a, which in turn regulates patterning of the dorso-temporal lobe of the retina. This, in concert with aberrant tectal patterning in pbx2/4 null embryos, may lead to the observed defects in RGC outgrowth.ConclusionThese data define a novel role for Pbx in patterning the vertebrate retina and tectum in a manner required for proper retinal ganglion cell axon outgrowth.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.07.004 Byline: Lisa Maves (a), Ashlee Tyler (a), Cecilia B. Moens (b), Stephen J. Tapscott (a) Keywords: pbx; hand2; Zebrafish; Heart development; Muscle differentiation Abstract: Transcription factors of the basic helix-loop-helix (bHLH) family are critical regulators of muscle cell differentiation. For example, Myod drives skeletal muscle differentiation, and Hand2 potentiates cardiac muscle differentiation. Understanding how these bHLH factors regulate distinct transcriptional targets in a temporally and spatially controlled manner is critical for understanding their activity in cellular differentiation. We previously showed that Pbx homeodomain proteins modulate the activity of Myod to promote the differentiation of fast-twitch skeletal muscle. Here, we test the hypothesis that Pbx proteins are also necessary for cardiac muscle differentiation through interacting with Hand2. We show that Pbx proteins are required for the activation of cardiac muscle differentiation in zebrafish embryos. Loss of Pbx activity leads to delay of myocardial differentiation and subsequent defective cardiac morphogenesis, similar to reduced Hand2 activity. Genetic interaction experiments support the hypothesis that Pbx proteins modulate the activity of Hand2 in myocardial differentiation. Furthermore, we show that Pbx proteins directly bind the promoter of the myocardial differentiation gene myl7 in vitro, supporting a direct role for Pbx proteins in promoting cardiac muscle differentiation. Our findings demonstrate new roles for Pbx proteins in vertebrate cardiac development and also provide new insight into connections between the transcriptional regulation of skeletal and cardiac muscle differentiation programs. Author Affiliation: (a) Division of Human Biology, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, C3-168, Seattle, WA 98109, USA (b) Howard Hughes Medical Institute and Division of Basic Science, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA Article History: Received 9 April 2009; Revised 9 June 2009; Accepted 6 July 2009

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.12.010 Byline: Hilary A. Kemp, Julie E. Cooke, Cecilia B. Moens Keywords: Eph; Ephrin; Efn; Hindbrain; Boundary; Zebrafish; Rhombomere; Neuroepithelium; Cell affinity; Cell sorting Abstract: During vertebrate development, the hindbrain is transiently segmented into 7 distinct rhombomeres (r). Hindbrain segmentation takes place within the context of the complex morphogenesis required for neurulation, which in zebrafish involves a characteristic cross-midline division that distributes progenitor cells bilaterally in the forming neural tube. The Eph receptor tyrosine kinase EphA4 and the membrane-bound Ephrin (Efn) ligand EfnB2a, which are expressed in complementary segments in the early hindbrain, are required for rhombomere boundary formation. We showed previously that EphA4 promotes cell-cell affinity within r3 and r5, and proposed that preferential adhesion within rhombomeres contributes to boundary formation. Here we show that EfnB2a is similarly required in r4 for normal cell affinity and that EphA4 and EfnB2a regulate cell affinity independently within their respective rhombomeres. Live imaging of cell sorting in mosaic embryos shows that both proteins function during cross-midline cell divisions in the hindbrain neural keel. Consistent with this, mosaic EfnB2a over-expression causes widespread cell sorting and disrupts hindbrain organization, but only if induced at or before neural keel stage. We propose a model in which Eph and Efn-dependent cell affinity within rhombomeres serve to maintain rhombomere organization during the potentially disruptive process of teleost neurulation. Author Affiliation: Howard Hughes Medical Institute and Division of Basic Science, Fred Hutchinson Cancer Research Center, B2-152, 1100 Fairview Ave. N., Seattle WA 98109, P.O. Box 19024, USA Article History: Received 14 August 2008; Revised 6 December 2008; Accepted 10 December 2008

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.03.007 Byline: Bruce W. Draper (a)(c), Claire M. McCallum (b), Cecilia B. Moens (a) Keywords: Development; Zebrafish; Nanos; Vasa; Ziwi; Germline stem cells; Primordial germ cells; Ovary; Oogenesis; TILLING Abstract: Development of the germline requires the specification and survival of primordial germ cells (PGCs) in the embryo as well as the maintenance of gamete production during the reproductive life of the adult. These processes appear to be fundamental to all Metazoans, and some components of the genetic pathway regulating germ cell development and function are evolutionarily conserved. In both vertebrates and invertebrates, nanos-related genes, which encode RNA-binding zinc finger proteins, have been shown to play essential and conserved roles during germ cell formation. In Drosophila, maternally supplied nanos is required for survival of PGCs in the embryo, while in adults, nanos is required for the continued production of oocytes by maintaining germline stem cells self-renewal. In mice and zebrafish, nanos orthologs are required for PGC survival during embryogenesis, but a role in adults has not been explored. We show here that nanos1 in zebrafish is expressed in early stage oocytes in the adult female germline. We have identified a mutation in nanos1 using a reverse genetics method and show that young female nanos mutants contain oocytes, but fail to maintain oocyte production. This progressive loss of fertility in homozygous females is not a phenotype that has been described previously in the zebrafish and underlines the value of a reverse genetics approach in this model system. Author Affiliation: (a) Howard Hughes Medical Institute and Fred Hutchinson Cancer Research Center, P.O. Box 19024, Seattle, WA 98109, USA (b) Arcadia Biosciences, 1102 Columbia St., Suite 600, Seattle, WA 98104, USA (c) Section of Molecular and Cellular Biology, University of California, Davis One Shields Ave., Davis, CA 95616, USA Article History: Received 29 April 2006; Revised 6 March 2007; Accepted 6 March 2007

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.08.022 Byline: Timothy Erickson (a), Steffen Scholpp (b)(c), Michael Brand (c), Cecilia B. Moens (d), Andrew Jan Waskiewicz (a) Keywords: Zebrafish; Engrailed; Pbx; Midbrain; Midbrain-hindbrain boundary; Diencephalic-mesencephalic boundary; Hexapeptide; Lineage restriction; Neural patterning Abstract: Pbx proteins are a family of TALE-class transcription factors that are well characterized as Hox co-factors acting to impart segmental identity to the hindbrain rhombomeres. However, no role for Pbx in establishing more anterior neural compartments has been demonstrated. Studies done in Drosophila show that Engrailed requires Exd (Pbx orthologue) for its biological activity. Here, we present evidence that zebrafish Pbx proteins cooperate with Engrailed to compartmentalize the midbrain by regulating the maintenance of the midbrain-hindbrain boundary (MHB) and the diencephalic-mesencephalic boundary (DMB). Embryos lacking Pbx function correctly initiate midbrain patterning, but fail to maintain eng2a, pax2a, fgf8, gbx2, and wnt1 expression at the MHB. Formation of the DMB is also defective as shown by a caudal expansion of diencephalic epha4a and pax6a expression into midbrain territory. These phenotypes are similar to the phenotype of an Engrailed loss-of-function embryo, supporting the hypothesis that Pbx and Engrailed act together on a common genetic pathway. Consistent with this model, we demonstrate that zebrafish Engrailed and Pbx interact in vitro and that this interaction is required for both the eng2a overexpression phenotype and Engrailed's role in patterning the MHB. Our data support a novel model of midbrain development in which Pbx and Engrailed proteins cooperatively pattern the mesencephalic region of the neural tube. Author Affiliation: (a) Department of Biological Sciences, CW405, Biological Sciences Building, University of Alberta, Edmonton AB, Canada T6G2E9 (b) MRC Centre for Developmental Neurobiology, King's College London, New Hunt's House, Guy's Campus London, SE1 1UL, UK (c) Biotechnology Center, University of Technology Dresden, Tatzberg 47-51, 01307 Dresden, Germany (d) Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA Article History: Received 19 May 2006; Revised 26 July 2006; Accepted 7 August 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.10.032 Byline: Cecilia B. Moens (a), Licia Selleri (b) Keywords: Pbx; Hox; Hox cofactors; Hexapeptide motif; Fly; Zebrafish; Mouse; Vertebrate development Abstract: Hox genes encode homeodomain-containing transcription factors that pattern the body axes of animal embryos. It is well established that the exquisite DNA-binding specificity that allows different Hox proteins to specify distinct structures along the body axis is frequently dependent on interactions with other DNA-binding proteins which act as Hox cofactors. These include the PBC and MEIS classes of TALE (Three Amino acid Loop Extension) homeodomain proteins. The PBC class comprises fly Extradenticle (Exd) and vertebrate Pbx homeoproteins, whereas the MEIS class includes fly Homothorax (Hth) and vertebrate Meis and Prep homeoproteins. Exd was first implicated as a Hox cofactor based on mutant phenotypes in the fly. In vertebrates, PBC and MEIS homeobox proteins play important roles in development and disease. In this review, we describe the evidence that these functions reflect a requirement for Pbx and Meis/Prep proteins as Hox cofactors. However, there is mounting evidence that, like in the fly, Pbx and Meis/Prep proteins function more broadly, and we also discuss how 'Hox cofactors' function as partners for other, non-Hox transcription factors during development. Conversely, we review the evidence that Hox proteins have functions that are independent of Pbx and Meis/Prep cofactors and discuss the possibility that other proteins may participate in the DNA-bound Hox complex, contributing to DNA-binding specificity in the absence of, or in addition to, Pbx and Meis/Prep. Author Affiliation: (a) Division of Basic Science and HHMI, Fred Hutchinson Cancer Research Center, B2-152, 1100 Fairview Ave. N., Seattle, WA 98115, USA (b) Department of Cell and Developmental Biology of Cornell University Medical School, and Molecular Biology Program of Cornell University/Sloan Kettering Institute, 1300 York Avenue, New York, NY 1002, USA Article History: Received 10 July 2005; Revised 17 October 2005; Accepted 24 October 2005

Cranial neural crest cells (NCCs) migrate into the pharyngeal arches in three primary streams separated by two cranial neural crest (NC)-free zones. Multiple tissues have been implicated in the guidance of cranial NCC migration; however, the signals provided by these tissues have remained elusive. We investigate the function of semaphorins (semas) and their receptors, neuropilins (nrps), in cranial NCC migration in zebrafish. We fred that genes of the sema3F and sema3G class are expressed in the cranial NC-free zones, while nrp2a and nrp2b are expressed in the migrating NCCs. sema3F/3G expression is expanded homogeneously in the head periphery through which the cranial NCCs migrate in lzr/pbx4 mutants, in which the cranial NC streams are fused. Antisense morpholino knockdown of Sema3F/3G or Nrp2 suppresses the abnormal cranial NC phenotype of lzr/pbx4 mutants, demonstrating that aberrant Sema3F/3G-Nrp2 signaling is responsible for this phenotype and suggesting that repulsive Sema3F/3G-Npn2 signaling normally contributes to the guidance of migrating cranial NCCs. Furthermore, global over-expression of sema3Gb phenocopies the aberrant cranial NC phenotype of lzr/pbx4 mutants when endogenous Sema3 ligands are knocked down, consistent with a model in which the patterned expression of Sema3 ligands in the head periphery coordinates the migration of Nrp-expressing cranial NCCs. Keywords: Cranial neural crest migration; Pbx; Neuropilin; Semaphorin

The vertebrate branchiomotor neurons are organized in a pattern that corresponds with the segments, or rhombomeres, of the developing hindbrain and have identities and behaviors associated with their position along the anterior/posterior axis. These neurons undergo characteristic migrations in the hindbrain and project from stereotyped exit points. We show that lazarus/pbx4, which encodes an essential Hox DNA-binding partner in zebrafish, is required for facial (VIIth cranial nerve) motor neuron migration and for axon pathfinding of trigeminal (Vth cranial nerve) motor axons. We show that lzr/pbx4 is required for Hox paralog group 1 and 2 function, suggesting that Pbx interacts with these proteins. Consistent with this, lzr/pbx4 interacts genetically with hoxbla to control facial motor neuron migration. Using genetic mosaic analysis, we show that lzr/pbx4 and hoxbla are primarily required cell-autonomously within the facial motor neurons; however, analysis of a subtle non-cell-autonomous effect indicates that facial motor neuron migration is promoted by interactions amongst the migrating neurons. At the same time, lzr/pbx4 is required non-cell-autonomously to control the pathfinding of trigeminal motor axons. Thus, Pbx/Hox can function both cell-autonomously and non-cell-autonomously to direct different aspects of hindbrain motor neuron behavior. Keywords: Pbx; Hox; Branchiomotor neurons; Neuronal migration; Zebrafish

Segmentation of the vertebrate hindbrain into rhombomeres is a key step in the development of a complex pattern of differentiated neurons from a homogeneous neuroepithelium. Many of the transcription factors important for establishing the segmental plan and assigning rhombomere identity are now known. However, the downstream effectors that bring about the formation of rhombomere boundaries are only just being characterized. Here we discuss molecules that could be responsible for segregating populations of cells from different rhombomeres. We focus on recent work demonstrating that the Eph family of receptor tyrosine kinases and their ligands, the ephrins, function in rhombomere-specific cell sorting and initiation of a structural boundary. We discuss the contributions of two mechanisms -- cell sorting and plasticity -- to the formation of rhombomere boundaries.

Abstract Background The facial branchiomotor neurons of cranial nerve VII undergo a stereotyped tangential migration in the zebrafish hindbrain that provides an ideal system for examining the complex interactions between neurons and their environment that result in directed migration. Several studies have shown the importance of the planar cell polarity pathway in facial branchiomotor neuron migration but the role of apical-basal polarity has not been determined. Here we examine the role of the PAR-aPKC complex in forming the basal structures that guide facial branchiomotor neurons on an appropriate migratory path. Results High resolution timelapse imaging reveals that facial branchiomotor neurons begin their migration by moving slowly ventrally and posteriorly with their centrosomes oriented medially and then, upon contact with the Laminin-containing basement membrane at the rhombomere 4-rhombomere 5 boundary, speed up and reorient their centrosomes on the anterior-posterior axis. Disruption of the PAR-aPKC complex members aPKCλ, aPKCζ, and Pard6gb results in an ectopic ventral migration in which facial branchiomotor neurons escape from the hindbrain through holes in the Laminin-containing basement membrane. Mosaic analysis reveals that the requirement for aPKC is cell-nonautonomous, indicating that it is likely required in the surrounding polarized neuroepithelium rather than in facial motor neurons themselves. Ventral facial motor neuron ectopia can be phenocopied by mutation of lamininα1, suggesting that it is defects in maintenance of the laminin-containing basement membrane that are the likely cause of ventral mismigration in aPKCλ+ζ double morphants. Conclusions Our results suggest that the laminin-containing ventral basement membrane, dependent on the activity of the PAR-aPKC complex in the hindbrain neuroepithelium, is both a substrate for migration and a boundary that constrains facial branchiomotor neurons to the appropriate migratory path.