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Background Deciphering how ectodermal tissues form, and how they maintain their integrity, is crucial for understanding epidermal development and pathogenesis. However, lack of simple and rapid gene manipulation techniques limits genetic studies to elucidate mechanisms underlying these events. Results Here we describe an easy method for electroporation of chick limb bud ectoderm enabling gene manipulation during ectoderm development and wound healing. Taking advantage of a small parafilm well that constrains DNA plasmids locally and the fact that the limb ectoderm arises from a defined site, we target the limb ectoderm forming region by in ovo electroporation. This approach results in focal and efficient transgenesis of the limb ectodermal cells. Further, using a previously described Msx2 promoter, gene manipulation can be specifically targeted to the apical ectodermal ridge (AER), a signaling center regulating limb development. Using the electroporation technique to deliver a fluorescent marker into the embryonic limb ectoderm, we show its utility in performing time-lapse imaging during wound healing. This analysis revealed previously unrecognized dynamic remodeling of the actin cytoskeleton and lamellipodia formation at the edges of the wound. We find that the lamellipodia formation requires activity of Rac1 GTPase, suggesting its necessity for wound closure. Conclusion Our method is simple and easy. Thus, it would permit high throughput tests for gene function during limb ectodermal development and wound healing.

Background Interdigits (IDs) determine digit identity in chick limbs. They are located between the digital rays and act as secondary signaling centers downstream of sonic hedgehog to provide positional information for determining digit identity in the phalanx-forming region (PFR). We examined the dynamic developmental mechanism by which PFR cells obtain positional information from IDs to determine the identity of individual digits in the chick hindlimb. Results We identified the specific region of the IDs responsible for determining digit identity and showed that PFR cells actively receive positional information only from the posteriorly, and not the anteriorly, located IDs. We also demonstrated that digits 1, 2, and 3 are interchangeable with each other, but not with digit 4. Finally, we found that both ID4 and digital ray 4 are necessary for determining digit 4 identity. Conclusions The digital rays are naive during the initial stages of their development, at which time digit identity is not determined. To determine digit identity, each PFR cell shows a unidirectional response to obtain positional information specifically from the IDs located posterior to the PFR, regardless of the signal strength from the anteriorly located IDs.

Before limbs or fins, can be patterned and grow they must be initiated. Initiation of the limb first involves designating a portion of lateral plate mesoderm along the flank as the site of the future limb. Following specification, a myriad of cellular and molecular events interact to generate a bud that will grow and form the limb. The past three decades has provided a wealth of understanding on how those events generate the limb bud and how variations in them result in different limb forms. Comparatively, much less attention has been given to the earliest steps of limb formation and what impacts altering the position and initiation of the limb have had on evolution. Here, we first review the processes and pathways involved in these two phases of limb initiation, as determined from amniote model systems. We then broaden our scope to examine how variation in the limb initiation module has contributed to biological diversity in amniotes. Finally, we review what is known about limb initiation in fish and amphibians, and consider what mechanisms are conserved across vertebrates.

BACKGROUND Xenopus embryos and tadpoles are versatile models for embryological, cell biological, and regenerative studies. Genomic and transcriptomic approaches have been increasingly employed in these frogs. Most of these genome-wide analyses have profiled tissues in bulk, but there are many scenarios where isolation of single cells may be advantageous, including isolation of a preferred cell type, or generation of a single-cell suspension for applications such as scRNA-Seq. RESULTS Here we present a protocol for the disaggregation of complex tail and limb bud tissue, and use cell type-specific fluorescence in transgenic X. tropicalis appendages to isolate specific cell populations using fluorescence activated cell sorting (FACS). Our protocol addresses a specific challenge in Xenopus embryos and tadpoles: the storage of maternal yolk platelets in each cell, which can introduce light scatter and thereby false positives into FACS analysis. CONCLUSIONS Here we gate against both nontransgenic and ubiquitously transgenic animals to reduce both false positives and false negatives. We use the Xtr.Tg(pax6:GFP;cryga:RFP;actc1:RFP)Papal transgenic line as a test case to demonstrate that nucleic acid preparations made from sorted cells are high quality and specific. We anticipate this method will be adaptable to study various cell types that have transgenic reporter lines to better profile cell types of interest.

Background Fgf10 is expressed in various tissues and organs, such as the limb bud, heart, inner ear, and head mesenchyme. Previous studies identified Fgf10 enhancers for the inner ear and heart. However, Fgf10 enhancers for other tissues have not been identified. Results By using primary culture chick embryo lateral plate mesoderm cells, we compared activities of deletion constructs of the Fgf10 promoter region, cloned into a promoter-less luciferase reporter vector. We identified a 0.34-kb proximal promoter that can activate luciferase expression. Then, we cloned 11 evolutionarily conserved sequences located within or outside of the Fgf10 gene into the 0.34-kb promoter-luciferase vector, and tested their activities in vitro using primary cultured cells. Two sequences showed the highest activities. By using the Tol2 system and electroporation into chick embryos, activities of the 0.34-kb promoter with and without the two sequences were tested in vivo. No activities were detected in limb buds. However, the 0.34-kb promoter exhibited activities in the dorsal midline of the brain, while Fgf10 is detected in broader region in the brain. The two noncoding sequences negatively acted on the 0.34-kb promoter in the brain. Conclusions The proximal 0.34-kb promoter has activities to drive expression in restricted areas of the brain. Developmental Dynamics 247:1253-1263, 2018. © 2018 Wiley Periodicals, Inc.

Background The organizing center, which serves as a morphogen source, has crucial functions in morphogenesis in animal development. The center is necessarily located in a certain restricted area in the morphogenetic field, and there are several ways in which an organizing center can be restricted. The organizing center for limb morphogenesis, the ZPA (zone of polarizing activity), specifically expresses the Shh gene and is restricted to the posterior region of the developing limb bud. Results The pre-pattern along the limb anteroposterior axis, provided by anterior Gli3 expression and posterior Hand2 expression, seems insufficient for the initiation of Shh expression restricted to a narrow, small spot in the posterior limb field. Comparison of the spatiotemporal patterns of gene expression between Shh and some candidate genes (Fgf8, Hoxd10, Hoxd11, Tbx2, and Alx4) upstream of Shh expression suggested that a combination of these genes' expression provides the restricted initiation of Shh expression. Conclusions Taken together with results of functional assays, we propose a model in which positive and negative transcriptional regulatory networks accumulate their functions in the intersection area of their expression regions to provide a restricted spot for the ZPA, the source of morphogen, Shh. Developmental Dynamics 246:417-430, 2017. © 2017 Wiley Periodicals, Inc.

Background: Fras1 encodes an extracellular matrix protein that is critical for the establishment of the epidermal basement membrane during gestation. In humans, mutations in FRAS1 cause Fraser Syndrome (FS), a pleiotropic condition with many clinical presentations such as limb, eye, kidney, and craniofacial deformations. Many of these defects are mimicked by loss of Fras1 in mice, and are preceded by the formation of epidermal blisters in utero. Results: In this study, we identified a novel ENU-derived rounded foot (rdf) mouse mutant with highly penetrant hindlimb soft-tissue syndactyly, among other structural defects. Mapping and sequencing revealed that rdf is a novel loss-of-function nonsense allele of Fras1 (Fras1rdf). Focusing on the limb, we found that the Fras1rdf syndactyly phenotype originates from loss of interdigital cell death (ICD). Despite normal expression of bone morphogenetic protein (BMP) ligands and their receptors, the BMP downstream target gene Msx2, which is also necessary and sufficient to promote ICD, was down-regulated in the interdigital regions of Fras1rdf hindlimb buds. Conclusions: The close correlation between limb bud epidermal blistering, decreased Msx2 expression, and reduced ICD in the Fras1rdf hindlimb buds suggests that epithelium detachment from the mesenchyme may create a physical gap that interrupts the transmission of BMP, among other signals, resulting in soft tissue syndactyly. Developmental Dynamics 245:497–507, 2016. © 2015 Wiley Periodicals, Inc.

Background: GLI2, a zinc finger transcription factor, mediates Sonic hedgehog signaling, a critical pathway in vertebrate embryogenesis. GLI2 has been implicated in diverse set of embryonic developmental processes, including patterning of central nervous system and limbs. In humans, mutations in GLI2 are associated with several developmental defects, including holoprosencephaly and polydactyly. Results: Here, we demonstrate in transient transgenic zebrafish assays, the potential of a subset of tetrapod-teleost conserved non-coding elements (CNEs) residing within human GLI2 intronic intervals to induce reporter gene expression at known regions of endogenous GLI2 transcription. The regulatory activities of these elements are observed in several embryonic domains, including neural tube and pectoral fin. Moreover, our data reveal an overlapping expression profile of duplicated copies of an enhancer during zebrafish evolution. Conclusions: Our data suggest that during vertebrate history GLI2 acquired a high level of complexity in the genetic mechanisms regulating its expression during spatiotemporal patterning of the central nervous system (CNS) and limbs. Developmental Dynamics 244:681–692, 2015. © 2015 Wiley Periodicals, Inc.

Background: The vertebrate limb bud is a well-established system for studying the mechanisms driving growth and patterning of an embryonic tissue. However, approaches for manipulating gene expression are currently limited to time-consuming methods. Culturing primary limb bud cells could potentially be used as a quicker assay. However, limb cells in culture quickly differentiate into cartilage under normal conditions, and approaches delivering DNA and siRNA into primary limb cells in culture are limited. These technical limitations have restricted the utility of limb buds for investigating problems that require higher-throughput approaches. Results: In this report, we describe adaptations to a method for culturing primary limb bud cells in a pre-chondrogenic state, and generate a population of mouse primary limb cells that are responsive to Hedgehog (Hh) signaling. Hh-stimulated cells upregulate Hh target genes as well as an exogenous Hh-responsive reporter. We then describe a method for highly efficient delivery of plasmids and siRNAs into cultured primary limb bud cells in a 96-well format. Conclusions: Cultures of primary limb bud cells are amenable to gene manipulation under conditions that maintain the limb cells in an Hh-responsive, undifferentiated state. This approach provides a medium-throughput system to manipulate gene expression, and test DNA regulatory elements. Developmental Dynamics 243:928–936, 2014. © 2014 Wiley Periodicals, Inc.

Background: Avian forelimb (wing) contains only three digits, and the three-digit formation in the bird forelimb is one of the avian-specific limb characteristics that have been evolutionarily inherited from the common ancestral form in dinosaurs. Despite many studies on digit formation in the chick limb bud, the developmental mechanisms giving rise to the three-digit forelimb in birds have not been completely clarified. Results: To identify which cell populations of the early limb bud contribute to digit formation in the late limb bud, fate maps of the early fore- and hindlimb buds were prepared. Based on these fate maps, we found that the digit-forming region in the forelimb bud is narrower than that in the hindlimb bud, suggesting that some developmental mechanisms on the anterior-most region lead to a reduced number of digits in the forelimb. We also found temporal differences in the onset of appearance of the ANZ (anterior necrotic zone) as well as differences in the position of the anterior edge of the AER. Conclusions: Forelimb-specific events in the anterior limb bud are possible developmental mechanisms that might generate the different cell fates in the fore- and hindlimb buds, regulating the number of digits in birds. Developmental Dynamics 243:741–752, 2014. © 2014 Wiley Periodicals, Inc.

Background: ATIPs (Angiotensin receptor type 2 [AT2] interacting proteins) are described as AT2 interacting protein variants, whereas their expression and functions during development are not known yet. Results: Here, we provide a detailed expression pattern of ATIP variants during mouse development by visualizing Mtus1 promotor activity, Mtus1 RNA, and subsequent ATIP protein expression. ATIPs are strongly expressed in the developing cardiovascular system, including the vascular plexus of the yolk sac and the fetal vascular part of the placenta. Moreover, ATIP is expressed spatially distinct during eye and limb bud development, and in later stages in lung and nervous system. The three murine ATIP isoforms are expressed in a distinct manner, whereupon isoform 1 and 4 are correlated to cardiovascular, lung, and limb bud development and isoform 3 is restricted to brain and eye development. Interestingly, Mtus1 expression is not necessarily correlated to Agtr2 expression, suggesting novel but yet unknown functions for ATIP, independent of AT2 signaling. Conclusions: ATIPs seem to be mainly involved in the developmental regulation of the cardiovascular system and may act in different AT2-dependent and -independent manners. Hence, these results deliver valuable information to further elucidate the different functions of ATIPs in the processes of mammalian development. Developmental Dynamics 243:699–711, 2014. © 2013 Wiley Periodicals, Inc.

Background: Vertebrate muscles are defined and patterned at the stage of primary myotube formation, but there is no clear description of how these cells form in vivo. Of particular interest is whether primary myotubes are “seeded” by a unique myoblast population that differentiates as mononucleated myocytes, similar to the founder myoblasts of insects. Results: We analyzed the cell populations and processes leading to initiation of primary myogenesis in limb buds of rats and mice. Pax3+ve myogenic precursors migrate into the limb bud and initially consolidate into dorsal and ventral muscle masses in the absence of Pax7 expression. Approximately a day later, Pax7+ve cells appear in the central aspect of the limb base and subsequently throughout the limb muscle masses. Primary myogenesis is initiated within each muscle mass at a time when only Pax3, and not Pax7, protein can be detected. Primary myotubes form initially as elongate mononucleated myocytes, well before cleavage of the muscle masses has occurred. Multinucleate myotubes appear approximately a day later. A similar process is seen during initiation of chick limb primary myogenesis. Conclusions: Primary myotubes of vertebrate limb muscles are initiated by mononucleated myocytes, that appear structurally analogous to the founder myoblasts of insects. Developmental Dynamics 242:1043–1055, 2013. © 2013 Wiley Periodicals, Inc.

Background: Whole-mount in situ hybridization (ISH) is a prevalent tool to examine the spatial distribution of gene transcripts in intact embryos. Chromogenic-based methods of signal development are commonly used in mouse embryos because of their high sensitivity. Fluorescence techniques, however, offer several advantages over chromogenic methods including the ability to visualize multiple signals in a specimen at once. Results: We describe a procedure for fluorescence in situ hybridization (FISH) for whole mouse embryos up to embryonic day 13.5. We show that this approach successfully produces a bright expression signal for several genes, validating the procedure in multiple tissues. Further, we show that double FISH can be used to visualize the expression of two genes in a single embryo by determining that Hoxd13 and Shh are co-expressed in both the limb bud and the hindgut. Finally, we demonstrate that FISH can be paired with confocal microscopy to take optical sections of interior regions of the embryo. Conclusions: FISH is a valid alternative to chromogenic-based ISH for visualizing gene expression in whole mouse embryos. This work provides a framework to add additional fluorescence signals in the mouse such as visualizing both mRNA and protein by pairing the procedure with immunofluorescence. Developmental Dynamics 242:1094–1100, 2013. © 2013 Wiley Periodicals, Inc.

Background: Dact gene family encodes multifunctional proteins that are important modulators of Wnt and TGF-β signaling pathways. Given that these pathways coordinate multiple steps of limb development, we investigated the expression pattern of the two chicken Dact genes (Dact1 and Dact2) from early limb bud up to stages when several tissues are differentiating. Results: During early limb development (HH24-HH30) Dact1 and Dact2 were mainly expressed in the cartilaginous rudiments of the appendicular skeleton and perichondrium, presenting expression profiles related, but distinct. At later stages of development (HH31–HH35), the main sites of Dact1 and Dact2 expression were the developing synovial joints. In this context, Dact1 expression was shown to co-localize with regions enriched in the nuclear β-catenin protein, such as developing joint capsule and interzone. In contrast, Dact2 expression was restricted to the interzone surrounding the domains of bmpR-1b expression, a TGF-β receptor with crucial roles during digit morphogenesis. Additional sites of Dact expression were the developing tendons and digit blastemas. Conclusions: Our data indicate that Dact genes are good candidates to modulate and, possibly, integrate Wnt and TGF-β signaling during limb development, bringing new and interesting perspectives about the roles of Dact molecules in limb birth defects and human diseases. Developmental Dynamics 243:428–439, 2014. © 2013 Wiley Periodicals, Inc.

Background: Digit patterning integrates signaling by the Sonic Hedgehog (SHH), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) pathways. GLI3, a component of the SHH pathway, is a major regulator of digit number and identity. Neogenin (encoded by Neo1) is a cell surface protein that serves to transduce signals from several ligands, including BMPs, in various developmental contexts. Although neogenin is implicated in BMP signaling, it has not been linked to SHH signaling and its role in digit patterning is unknown. Results: We report that Neo1 mutant mice have preaxial polydactyly with low penetrance. Expression of SHH target genes, but not BMP target genes, is altered in Neo1 mutant limb buds. Analysis of mice carrying mutations in both Neo1 and Gli3 reveals that, although neogenin plays a role in constraint of digit numbers, suppressing polydactyly, it is also required for the severe polydactyly caused by loss of GLI3. Furthermore, embryo fibroblasts from Neo1 mutant mice are sensitized to SHH pathway activation in vitro. Conclusions: Our findings indicate that neogenin regulates SHH signaling in the limb bud to achieve proper digit patterning. Developmental Dynamics 241:627–637, 2012. © 2012 Wiley Periodicals Inc.

We have used the chick limb as a model to gain insight into the longstanding question of regulative vs. mosaic development. To test the influence of signals on limb proximodistal development, distal limb bud tips of several stages were grafted to regions of the embryo known to provide different signaling environments. Of interest, thin grafts (100-micron thick) formed elements more proximal in character when grafted to the proximal limb region than when grafted to other regions. The extra elements were derived from host tissue, presumably distalized and recruited by the graft's apical ectodermal ridge signals. The results of classic and recent experiments have been reinterpreted in light of our conclusions.

Over the past decade, the genetics of external genital development have begun to be understood. Male and female external genitalia develop from the genital tubercle. The early tubercle has a superficial resemblance to the limb bud, but an important distinction is that the limb consists of only mesoderm and ectoderm, whereas the genital tubercle also has an endodermal component, the urethral epithelium. Urethral epithelium, which expresses Sonic hedgehog, acts as a signaling region that controls outgrowth and pattern formation, and ultimately differentiates into the urethral tube. While there are intriguing parallels between limb and genital development, recent studies have identified some key differences, including the role of Fgf signaling. Our understanding of the mechanisms of genital development still lags far behind the limb, and major questions remain to be answered, including the molecular nature of the signals that initiate genital budding, sustain outgrowth, induce tissue polarity and orchestrate urethral tubulogenesis.

Questions regarding morphogenesis have played second fiddle to those pertaining to pattern formation among the limb development set for some time. A recent series of publications has reinvigorated the search for mechanisms by which the limb bud arises, elongates and acquires its peculiar shape. While there are stage-specific variations, the theme that resonates across these studies is that mesoderm and cartilage cells in the limb bud exhibit polarity that drives directional movement and oriented division. Noncanonical Wnt signalling is important for these cell behaviors at all stages of limb development. While the emerging morphogenetic mechanisms underlying limb bud outgrowth are partly analogous to those of other developing structures, insights from the limb have the potential to reveal intriguing new mechanisms by which three dimensional mesoderm changes shape.

The vertebrate limb is an excellent model organ system to investigate how signaling pathways interact. Over the last half-century, experiments investigating patterning in the vertebrate limb have led directly to the discovery of many of the molecules and molecular pathways that are not only responsible for limb patterning but the patterning of many different organ systems. In the limb bud, the zone of polarizing activity (ZPA) has long been known to produce factor(s) that are essential for normal limb formation. Recently, one of these factors, Sonic Hedgehog (SHH) was shown to function not only in the limb bud mesenchyme but also in the ectoderm overlying the ZPA. This review discusses the role and potential implications hedgehog signaling in the limb bud ectoderm plays in patterning the vertebrate limb. Developmental Dynamics 240:915–919, 2011. © 2011 Wiley-Liss, Inc.

During vertebrate limb development, various molecules are expressed in the presumptive limb field or the limb bud in a spatiotemporal-specific manner. The combination of these molecules regulates cellular properties that affect limb initiation and its morphogenesis, especially cartilage formation. Cell adhesiveness of the limb mesenchyme is a key factor in the regulation of cell distribution. Differential adhesiveness of mesenchymal cells is first observed between cells in the presumptive limb field and flank region, and the adhesiveness of the cells in the limb field is higher than that of cells in the flank region. In the limb bud, the adhesiveness of mesenchymal cells shows spatiotemporal difference, which reflects the positional identity of the cells. Position-dependent cell adhesiveness is also observed in blastema cells of the regenerating limb. Therefore, local changes in cell adhesiveness are observed during limb development and regeneration, suggesting significant roles for cell adhesiveness in limb morphogenesis.

Lx mutation in SHR.Lx rat manifests in homozygotes as hindlimb preaxial polydactyly. It was previously mapped to a chromosome 8 segment containing the Plzf gene. Plzf (promyelocytic leukemia zinc finger protein) influences limb development as a direct repressor of posterior HoxD genes. However, the Plzf coding sequence is intact in the Lx mutants. Using linkage mapping in F2 hybrids, we downsized the segment containing Lx to 155 kb and sequenced conserved noncoding elements (CNEs) inside. A 2,964-bp deletion in Plzf intron 2, never detected in control animals, is the only candidate for Lx. The deletion removes the most deeply conserved CNE in the 155-kb segment, suggesting a regulatory influence on Plzf expression. Correspondingly, using in situ hybridization and quantitative real-time polymerase chain reaction, we found a decrease of Plzf expression in Lx/Lx limb buds with concomitant anterior expansion of expression domains of its targets, Hoxd10–13 genes, in the absence of ectopic Sonic hedgehog expression. Upstream regulation of Plzf in limb buds is currently unknown. We present here the first candidate Plzf cis-regulatory sequence. Developmental Dynamics 238:673–684, 2009. © 2009 Wiley-Liss, Inc.

RCAS is a replication-competent retroviral vector system that allows sustained misexpression of a gene of interest in avian cells. This tool has been used to gain fundamental insights into skeletal development in the chick embryo, and consequently into broader principles of morphogenesis. In this review, we discuss a range of RCAS-based strategies that have been employed to examine gene function during early patterning of the limb bud, later skeletal differentiation, and craniofacial morphogenesis. The potential for the RCAS system as a tool for loss-of-function studies is also discussed.

Twist genes code for regulatory bHLH proteins essential for embryonic development and conserved across the metazoa. There are four genes that constitute the zebrafish twist family: twist1a, twist1b, twist2--orthologs of the mammalian twist1 and twist2 genes; and twist3--a gene from a new clade that does not exist in mammals. Presented here are their embryonic mRNA expression profiles. The study extends the known conservation of twist developmental patterns in tetrapods to the fish, e.g., expression in cephalic neural crest, sclerotome and lateral plate mesoderm. Some other expression domains are unique, like hypochord and dorsal aorta; some, like the notochord, may be ancestral patterns retained from protochordates; and the expression in invaginating/migrating cells may have been retained from the jellyfish. Perhaps this is one of the more ancient functions of twist--conserved from diploblasts to humans--to facilitate cell movement.

Vasohibin is an angiogenesis inhibitor that is induced in endothelial cells in an autocrine manner. In this study, we cloned a 500-bp fragment of chick Vasohibin cDNA and analyzed its expression pattern by in situ hybridization during chick development. From HH-stage 3, expression of Vasohibin is observed in the area opaca and it is expressed throughout the primitive streak during later stages. At HH-stage 11, Vasohibin is expressed in head paraxial mesoderm, in the vitelline vein, dorsal neural tube, intermediate and lateral plate mesoderm, Wolffian duct, and blood islands at the caudal part of the embryo. In epithelial somites, expression is seen in the region around the somitocoel, and after somite maturation, expression is observed in the myotome, which becomes stronger with development. Expression is detected in fore and hind brain, also in the retina and lens vesicle of the developing eye. In the early limb bud, expression is initiated in the mesenchyme and becomes stronger during later stages. Expression in the limb mesoderm remains strong at the margins but decreases in the central mesenchyme. At day 7, expression is seen in interdigital grooves of the digits and digit-demarcating regions. During organogenesis, expression is seen in the anlagen of the esophagus, trachea, duodenum, lungs, liver, heart, and gut. Our analysis shows that Vasohibin is expressed in a wide range of tissues and organs suggesting that Vasohibin acts as a physiological regulator of vascular development during chick embryogenesis.

Newborn neurons elaborate an axon that undertakes a complicated journey to find its ultimate target in the brain or periphery. Although major progress in the study of this process has been made by analysis of dissociated neurons in vitro, one would like to observe and manipulate axonal outgrowth and pathfinding as it occurs in situ, as fasciculated nerves growing within the tissue itself. Here, we present a simple technique to do this, through cultivation of embryonic mouse slices expressing enhanced green fluorescent protein (EGFP) specifically in newborn neurons. This system allows for imaging of outgrowth of peripheral nerves into structures such as the developing limb. We demonstrate a reproduction of normal innervation patterns by spinal nerves derived from spinal cord motor neurons and sensory neurons of the dorsal root ganglia. The slices can be manipulated pharmacologically as well as genetically, by crossing the EGFP-expressing line with lines containing targeted mutations in genes of interest. Developmental Dynamics 236:3514–3523, 2007. © 2007 Wiley-Liss, Inc.

In the developing atrioventricular (AV) valve, limb bud, and somites, cartilage cell lineage differentiation is regulated by bone morphogenetic protein (BMP), while fibroblast growth factor (FGF) controls tendon cell fate. We observed aggrecan and sox9, characteristic of cartilage cell types, and scleraxis and tenascin, characteristic of tendon cell types, in developing avian semilunar valves. Addition of BMP4 to outflow tract (OFT) precursor cells of young (E4.5) but not older (E6) chick embryos activated Smad1/5/8 and induced sox9 and aggrecan expression, while FGF4 treatment increased phosphorylated MAPK (dpERK) signaling and promoted expression of scleraxis and tenascin. These results identify BMP and FGF pathways that promote expression of cartilage- or tendon-like characteristics in semilunar valve precursor cells. In contrast to AV valve precursor cells, which diversify into leaflets (cartilage-like) or chordae tendineae (tendon-like), semilunar valve cells exhibit both cartilage- and tendon-like characteristics in the developing and mature valve cusp. Developmental Dynamics 236:971–980, 2007. © 2007 Wiley-Liss, Inc.

We have performed in situ hybridization to study the expression of Wise in early chick embryos. Wise expression is first detectable in the ectoderm at posterior levels of late neurula. As development proceeds, Wise expression is seen in specific patterns in the ectoderm of the trunk region, pharyngeal arches, limb buds, and feather buds. In addition to these areas, particular cartilages such as the ones in the maxillary process and limbs start to express Wise at the late pharyngula stage, and the expression in these cartilages becomes stronger than that in epidermal components at later stages. Importantly, Wise is expressed in regions where other signaling molecules such as Wnt, Bmp, and Shh are known to function in morphogenesis and differentiation. Direct comparisons of the expression of Wise and these genes are also demonstrated. Developmental Dynamics 236:2277–2284, 2007. Published 2007 Wiley-Liss, Inc.

C-terminal binding proteins (CtBPs) are transcriptional corepressors of mediators of Notch, Wnt, and other signalling pathways. Thus, they are potential players in the control of several developmentally important processes, including segmentation, somitogenesis, and neural tube and limb patterning. We have cloned the avian orthologues of Ctbp1 and Ctbp2 and examined their expression pattern by whole-mount in situ hybridization between Hamburger and Hamilton (HH) stages 3 and 24. Both Ctbp genes show similar expression patterns during embryonic development, and both are detected from HH stage 3 in the developing central nervous system, by HH stage 7 in the paraxial mesoderm and later in the limb bud. In most places, Ctbp1 and Ctbp2 are expressed in overlapping domains. However, there are interesting domains and/or temporal expression patterns that are specific to each Ctbp gene. For instance, Ctbp1 is predominantly expressed in the epiblast, whereas Ctbp2 is in the primitive streak at HH stage 3. However, by HH stage 4, both genes are found in the primitive streak and in the ectoderm. Similarly, although both genes display similar expression patterns in early somitogenesis, in mature somites, Ctbp1 transcripts are located in myotomal cells, whereas Ctbp2 transcripts are observed in dermomyotomal cells. Finally, we found that emigrating neural crest cells express Ctbp2, whereas dorsal root ganglia express Ctbp1. These data suggest that Ctbp1 and Ctbp2 may be functionally redundant in some tissues and have unique functions in other tissues. Developmental Dynamics 235:490–495, 2005. © 2005 Wiley-Liss, Inc.

Members of the Dact protein family initially were identified through binding to Dishevelled (Dvl), a cytoplasmic protein central to Wnt signaling. During mouse development, Dact1 is detected in the presomitic mesoderm and somites during segmentation, in the limb bud mesenchyme and other mesoderm-derived tissues, and in the central nervous system (CNS). Dact2 expression is most prominent during organogenesis of the thymus, kidneys, and salivary glands, with much lower levels in the somites and in the developing CNS. Dact3, not previously described in any organism, is expressed in the ventral region of maturing somites, limb bud and branchial arch mesenchyme, and in the embryonic CNS; of the three paralogs, it is the most highly expressed in the adult cerebral cortex. These data are consistent with studies in other vertebrates showing that Dact paralogs have distinct signaling and developmental roles and suggest they may differentially contribute to postnatal brain physiology.

Cell death and cell proliferation are basic cellular processes that need to be precisely controlled during embryonic development. The developing vertebrate limb illustrates particularly well how correct morphogenesis depends on the appropriate spatial and temporal balance between cell death and cell proliferation. Precise knowledge of the patterns of cell proliferation and cell death during limb development is required to understand how their modifications may contribute to the generation of the great diversity of limb phenotypes that result from spontaneous mutations or induced genetic manipulations. We have performed a comprehensive analysis of the patterns of cell death, assayed by terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL), and cell proliferation, assayed by anti-phosphorylated histone H3 immunohistochemistry, in consecutive sections of forelimbs and hindlimbs covering an extensive period of chick and mouse limb development. Our results confirm and expand previous reports and show common and specific areas of cell death for each species. Mitotic cells were found scattered in a uniform distribution across the early limb bud, with the exception of the areas of cell death in which mitotic cells were scarce. At later stages, mitotic cells were seen more abundantly in the digital tips. The aim of the present study was to satisfy the need for organized data sets describing these processes, which will allow the side-by-side comparison between the two major model organisms of limb development, i.e., the mouse and the chick.

The Notch family of receptors is involved in a wide variety of developmental processes, including cell fate specification, cell proliferation, and cell survival decisions during cell differentiation and tissue morphogenesis. Notch1 and Notch ligands are expressed in the developing limbs, and Notch signalling has been implicated in the formation of a variety of tissues that comprise the limb, such as the skeleton, musculature, and vasculature. Notch signalling has also been implicated in regulating overall limb size. We have used a conditional allele of Notch1 in combination with two different Cre transgenic lines to delete Notch1 function either in the limb mesenchyme or in the apical ectodermal ridge (AER) and limb ectoderm. We demonstrate that Notch signalling, involving Notch1 and Jagged2, is required to regulate the number of Fgf8-expressing cells that comprise the AER and that regulation of the levels of fibroblast growth factor signalling is important for the freeing of the digits during normal limb formation. Regulation of the extent of the AER is achieved by Notch signalling positively regulating apoptosis in the AER. We also demonstrate that Notch1 is not required for proper formation of all the derivatives of the limb mesenchyme.

The apical ectodermal ridge (AER) is indispensable for vertebrate limb development and requires Wnt/beta-catenin signaling for induction and maintenance. We report identification and involvement of Wnt10a in AER formation during chick limb development. Chicken Wnt10a has 82% identity with mouse Wnt10a in the amino acid sequence. The Wnt10a gene was expressed broadly in the surface ectoderm from as early as stage 10. By stage 15, the expression was restricted to the surface ectoderm overlying the lateral plate mesoderm. Wnt10a expression became intensified in the presumptive limb ectoderm during AER formation, and subsequently intense expression signals persisted in the AER. Wnt10a misexpression led to ectopic Fgf8 expression in the developing limb ectoderm and induced translocation of beta-catenin in chick embryo fibroblasts. These results suggest that Wnt10a is involved in AER formation in the chick limb bud through the Wnt/beta-catenin signaling pathway.

A variety of teratogens induce the loss of postaxial forelimb structures when administered during mid-gestation to the mouse. Previous studies demonstrated that teratogen exposure is associated with a reduction in zone of polarizing activity (ZPA) -related polarizing activity without a noticeable loss of Shh expression. Herein, we quantitatively confirm that expression of Shh, Ptch1, and Gli3 are unaltered by teratogen exposure and demonstrate that sonic hedgehog (Shh) translation is unaffected. Examination of the polarizing response of host chick wings to teratogen-exposed ZPA tissue revealed an induced growth response and ectopic induction of Fgf4, Bmp2, Ptch1, and Gli1 expression similar to control ZPA tissue. Control ZPA tissue altered the fate of cells destined to die in the anterior necrotic zone, whereas cell death ensued in hosts receiving teratogen-exposed grafts. Immunohistochemical studies localized Shh protein in the mouse limb to the posterior mesoderm and overlying ectoderm. We postulate that teratogen exposure alters the ability of Shh to signal to the ectoderm and present microarray and reverse transcriptase-polymerase chain reaction data, indicating that Shh signaling could occur in the limb bud ectoderm.

Removal of the posterior wing bud leads to massive apoptosis of the remaining anterior wing bud mesoderm. We show here that this finding correlates with an increase in the level of the repressor form of the Gli3 protein, due to the absence of the Sonic hedgehog (Shh) protein signaling. Therefore, we used the anterior wing bud mesoderm as a model system to analyze the relationship between the repressor form of Gli3 and apoptosis in the developing limb. With increased Gli3R levels, we demonstrate a concomitant increase in Bmp4 expression and signaling in the anterior mesoderm deprived of Shh signaling. Several experimental approaches show that the apoptosis can be prevented by exogenous Noggin, indicating that Bmp signaling mediates it. The analysis of Bmp4 expression in several mouse and chick mutations with defects in either expression or processing of Gli3 indicates a correlation between the level of the repressor form of Gli3 and Bmp4 expression in the distal mesoderm. Our analysis adds new insights into the way Shh differentially controls the processing of Gli3 and how, subsequently, BMP4 expression may mediate cell survival or cell death in the developing limb bud in a position-dependent manner.

The vertebrate hindbrain develops from a series of segments (rhombomeres) distributed along the anteroposterior axis. We are studying the roles of Wnt and Delta-Notch signaling in maintaining rhombomere boundaries as organizing centers in the zebrafish hindbrain. Several wnt genes (wnt1, wnt3a, wnt8b, and wnt10b) show elevated expression at rhombomere boundaries, whereas several delta genes (dlA, dlB, and dlD) are expressed in transverse stripes flanking rhombomere boundaries. Partial disruption of Wnt signaling by knockdown of multiple wnt genes, or the Wnt mediator tcf3b, ablates boundaries and associated cell types. Expression of dlA is chaotic, and cell types associated with rhombomere centers are disorganized. Similar patterning defects are observed in segmentation mutants spiel-ohne-grenzen (spg) and valentino (val), which fail to form rhombomere boundaries due to faulty interactions between adjacent rhombomeres. Stripes of wnt expression are variably disrupted, with corresponding disturbances in metameric patterning. Mutations in dlA or mind bomb (mib) disrupt Delta-Notch signaling and cause a wide range of patterning defects in the hindbrain. Stripes of wnt1 are initially normal but subsequently dissipate, and metameric patterning becomes increasingly disorganized. Driving wnt1 expression using a heat-shock construct partially rescues metameric patterning in mib mutants. Thus, rhombomere boundaries act as Wnt signaling centers required for precise metameric patterning, and Delta signals from flanking cells provide feedback to maintain wnt expression at boundaries. Similar feedback mechanisms operate in the Drosophila wing disc and vertebrate limb bud, suggesting coaptation of a conserved signaling module that spatially organizes cells in complex organ systems.

Mouse tooth germ development is currently studied by three main approaches: in wild-type and mutant mouse lines, after transplantation of tooth germs to ectopic sites, and in organ culture. The in vivo approaches are the most physiological but do not provide accessibility to tooth germs for further experimental manipulation. Organ cultures, although readily accessible, do not sustain full tooth germ development and are appropriate for short-term analysis. Thus, we sought to establish a new approach that would combine experimental accessibility with sustained development. We implanted fragments of embryonic day 12 mouse embryo first branchial arch containing early bud stage tooth germs into the lateral mesenchyme of day 4-5 chick embryo wing buds in ovo. Eggs were reincubated, and implanted tissues were examined by histochemistry and in situ hybridization over time. The tooth germs underwent seemingly normal growth, differentiation, and morphogenesis. They reached the cap, bell, and crown stages in approximately 3, 6, and 10 days, respectively, mimicking in a striking manner native temporal patterns. To examine mechanisms regulating tooth germ development, we first implanted tooth germ fragments, microinjected them with neutralizing antibodies to the key signaling molecule Sonic hedgehog (Shh), and examined them over time. Tooth germ development was markedly delayed, as revealed by poor morphogenesis and lack of mature ameloblasts and odontoblasts displaying characteristic traits such as an elongated cell shape, nuclear relocalization, and amelogenin gene expression. These phenotypic changes began to be reversed upon further incubation. The data show that the limb bud represents an effective, experimentally accessible as well as economical system for growth and analysis of developing tooth germs. The inhibitory effects of Shh neutralizing antibody treatment are discussed in relation to roles of this signaling pathway proposed by this and other groups previously.

Fgf-8 is one of the key signaling molecules implicated in the initiation, outgrowth, and patterning of vertebrate limbs. However, it is not clear whether FGF-8 plays similar role in development and regeneration of urodele limbs. We isolated a Fgf-8 cDNA from the Mexican axolotl (Ambystoma mexicanum) through the screening of an embryo cDNA library. The cloned 1.26-kb cDNA contained an open reading frame encoding 212 amino acid residues with 84%, 86%, and 80% amino acid identities to those of Xenopus, chick, and mouse, respectively. By using the above clone as a probe, we examined the temporal and spatial expression patterns of Fgf-8 in developing embryos and in regenerating larval limbs. In developing embryos, Fgf-8 was expressed in the neural fold, midbrain-hindbrain junction, tail and limb buds, pharyngeal clefts, and primordia of maxilla and mandible. In the developing axolotl limb, Fgf-8 began to be expressed in the prospective forelimb region at pre–limb-bud and limb bud stages. Interestingly, strong expression was detected in the mesenchymal tissue of the limb bud before digit forming stages. In the regenerating limb, Fgf-8 expression was noted in the basal layer of the apical epithelial cap (AEC) and the underlying thin layer of mesenchymal tissue during blastema formation stages. These data suggest that Fgf-8 is involved in the organogenesis of various craniofacial structures, the initiation and outgrowth of limb development, and the blastema formation and outgrowth of regenerating limbs. In the developing limb of axolotl, unlike in Xenopus or in amniotes such as chick and mouse, the Fgf-8 expression domain was localized mainly in the mesenchyme rather than epidermis. The unique expression pattern of Fgf-8 in axolotl suggests that the regulatory mechanism of Fgf-8 expression is different between urodeles and other higher species. The expression of Fgf-8 in the deep layer of the AEC and the thin layer of underlying mesenchymal tissue in the regenerating limbs support the previous notion that the amphibian AEC is a functional equivalent of the AER in amniotes. © 2001 Wiley-Liss, Inc.

Teneurin-2 is a member of a novel family of transmembrane proteins characterized to date in fish, birds, mammals, and Drosophila (e.g., the pair-rule gene product Ten-m). We have shown that teneurin-2 is expressed by neurons in the developing avian visual system in a pattern complementary to the expression of teneurin-1 and that recombinant teneurin-2 induces morphologic changes in neuronal cells in culture (Rubin et al., 1999). Here we have used cRNA probes to two newly identified splice variants and a teneurin-2-specific antibody to determine whether teneurin-2 is also expressed outside the nervous system. Both reverse transcriptase-polymerase chain reaction and in situ hybridization indicate that the three splice variants known so far are coexpressed at sites of pattern formation during development. Teneurin-2 mRNAs and protein are found in the developing limbs, somites, and craniofacial mesenchyme. In addition to expression of teneurin-2 by the apical ectodermal ridge, teneurin-2 transcripts also appear transiently at sites of tendon development. Teneurin-2 expression patterns were strikingly similar to those of fibroblast growth factor 8 (FGF8). In agreement with the overlapping expression pattern, FGF8-coated beads implanted into chicken limb buds induced the ectopic expression of teneurin-2 and soluble FGF8 induced teneurin-2 in limb explant cultures. Thus, teneurin-2 could act downstream of FGF8 during morphogenesis. © 2001 Wiley-Liss, Inc.

A steady ionic current is driven out of both developing and regenerating amphibian limbs. In the developing limbs of anurans and urodeles, focal outwardly directed current (0.5-2 microA/cm(2)) predicts the location of mesenchyme accumulations producing the early bud. Here, we report measurements of a similar outwardly directed ionic current associated with the development of the limb bud in the mouse and chick embryo by using a noninvasive, self-referencing electrode for the measurement of extracellular current. In both the mouse and chick embryo, flank currents were usually inwardly directed - the direction of Na(+) uptake by ectoderm. Outward currents associated with the mouse limb bud ranged from 0.04-10.8 microA/cm(2). Mouse limb bud and flank currents were similar to those measured in amphibian larvae, because they were reversibly collapsed and/or reversed by application of 30 microM amiloride, a Na(+) channel blocker. Unlike the amphibian embryos, flank ectoderm adjacent to the mouse limb bud in the anterior/posterior axis was usually associated with outwardly directed ionic current. This raises the possibility of a different, or changing, gradient of extracellular voltage experienced by mesenchyme cells in this plane of development than that observed in other regions of the limb bud. In the chick flank caudal to the somites, a striking reversal of the inwardly directed flank currents to very large ( approximately 100 microA/cm(2)) outwardly directed currents occurred three developmental stages before limb bud formation. We tested the relevance of this outwardly directed ionic current to limb formation in the chick embryo by reversing it by using an artificially applied "countercurrent" pulled through a microelectrode inserted just beneath the caudal ectoderm of the embryo. This application was performed for approximately 6 hr 2.5-3 developmental stages before hindlimb bud formation. This method resulted in abnormal limb formation by the tenth day of gestation in some embryos, whereas all control embryos developed normally. These data suggest an early physiological control of limb development.

We tested a diffusion gradient model for setting up overlapping domains of Hoxa gene expression in the chick limb bud. The model is based on morphogen production at the limb bud tip where the apical ridge is located and assumes that cells respond to a series of concentration thresholds. Consistent with the model, Hoxa13 gene expression rapidly switches off when the ridge is removed from stage 21/22 buds, while Hoxa11 and Hoxa10 expression is stable; Hoxa13 expression can be initiated and maintained in absence of the ridge by FGF soaked beads; the Hoxa13 domain first expands quickly and then slows up and the size is related to the dose of FGF4. Contrary to the model, addition of FGF4 to early limb buds does not activate Hoxa13 prematurely nor extend the Hoxa13 expression domain proximally. Therefore FGF4 signalling is necessary but not sufficient for Hoxa gene expression in the limb bud.

Urodele amphibians (salaman- ders) are unique among adult vertebrates in their ability to regenerate limbs. The regener- ated structure is often indistinguishable from the developmentally produced original. Thus, the two processes by which the limb is produced — development and regeneration — are likely to use many conserved biochemical and develop- mental pathways. Some of these limb features are also likely to be conserved across vertebrate families. The apical ectodermal ridge (AER) of the developing amniote limb and the larger api- cal epithelial cap (AEC) of the regenerating urodele limb are both found at the limb's distal- most tip and have been suggested to be function- ally similar even though their morphology is quite different. Both structures are necessary for limb outgrowth. However, the AEC is uniformly smooth and thickly covers the entire limb-tip, unlike the AER, which is a protruding ridge cov- ering only the dorsoventral boundary. Previous data from our laboratory suggest the multilay- ered AEC may be subdivided into separate func- tional compartments. We used hematoxylin and eosin (H1E) staining as well as in situ hybridiza- tion to examine the basal layer of the AEC, the layer that lies immediately over the distal limb mesenchyme. In late-stage regenerates, this basal layer expresses fibronectin (FN) message very strongly in a stripe of cells along the dorso- ventral boundary. H1E staining also reveals the unique shape of basal cells in this area. The stripe of cells in the basal AEC also contains the notch/groove structure previously seen in avian and reptilian AERs. In addition, AEC expression of FN message in the cells around the groove correlates with previous amniote AER localiza- tion of FN protein inside the groove. The struc- tural and biochemical analyses presented here suggest that there is a specialized ridge-like com- partment in the basal AEC in late-stage regener- ates. The data also suggest that this compart- ment may be homologous to the AER of the developing amniote limb. Thus, the external dif- ferences between amniote limb development and urodele limb regeneration may be outweighed by internal similarities, which enable both pro- cesses to produce morphologically complete limbs. In addition, we propose that this basal layer of the AEC is uniquely responsible for AEC functions in regeneration, such as secreting mol- ecules to promote mesenchymal cell cycling and dictating the direction of limb outgrowth. Fi- nally, we include here a clarification of existing nomenclature to facilitate further discussion of the AEC and its basal layer. Dev Dyn 2000;217: 216 -224. © 2000 Wiley-Liss, Inc.

Dlx3 is a homeodomain transcription factor in vertebrates, related to Distal-less in Drosophila, that is expressed in differentiating epidermal cells, in neural crest, hair follicles, dental epithelium and mesenchyme, the otic and olfactory placodes, limb bud, placenta, and in the cement gland, which is located in the extreme anterior neural plate in Xenopus embryos. This factor behaves as a transcriptional activator, and positively regulates gene expression in the skin, and negatively regulates central nervous system markers in Xenopus epidermis and anterior neural plate. A mutation in the DLX3 gene is associated with a hereditary syndrome in humans, and loss of Dlx3 function is a developmental lethal in gene-targeted mice, where it is essential for proper modeling of the labyrinthine layer of the placenta. In this review, we discuss the evolution, expression, regulation, and function of Dlx3 in mouse, amphibians, and zebrafish. Published 2000 Wiley-Liss, Inc.

While all cells in eukaryotic organisms probably express the gene encoding NADPH cytochrome P450 reductase, we identified two novel sites which have the highest local concentrations of P450 reductase transcripts during murine embryogenesis. One site is in developing limbs, including lateral limb bud mesenchyme and condensing mesenchyme in the footplate which will form precartilage. A second site is in primitive neuroepithelia, including future olfactory epithelia and olfactory lobes of the brain. These high, local concentrations of P450 reductase transcripts revealed by in situ hybridization were transient and most prominent between embryonic (E) days 12.5–15.5. They cannot be explained by the known functions for P450 reductase. The precursor nature of the highest reductase-expressing cells suggests that differentiation-specific mechanisms regulate P450 reductase gene transcription during organogenesis. The data suggest this multifunctional protein might serve an important role in the formation of precartilage models from condensing limb mesenchyme and in the early development of joints that will form at apposed surfaces of these models. Dev Dyn 1999;216:511–517. ©1999 Wiley-Liss, Inc.

Retinoids long have been implicated in limb development and their endogenous contributions to this process are finally being elucidated. Here we use an established model of retinoid depletion during specific gestational windows to investigate the role of endogenous retinoic acid (RA) in supporting limb outgrowth. Rat embryos were deprived of RA starting at days-postcoitum (dpc) 3.0, 5.5, or 7.0 and harvested at the 35-somite stage (dpc 12–12.5). Although embryos from all these windows possessed many characteristics of gestational retinoid deficiency (frontonasal hypoplasia, straight tail, reduced CRBPI and RARβ), their limb buds emerged with only modest size reductions. Molecular analysis of RA-deficient limb buds revealed enhanced gli-3 and reduced hoxd-12, hoxd-13, shh, and fgf-4, while fgf-8, en-1, and wnt-7a expression remained unaltered. Occasional posterior truncations were observed at low incidence in the longest deficiency window; otherwise, the deficiency window length had no discernable impact on the severity of these changes. At the 45-somite stage, RA-deficient limbs had additional losses of hoxd-13 and fgf-8, accompanied by a flattened AER, suggestive of an ultimate failure in limb bud outgrowth. Results could not confirm a function for endogenous retionids in limb initiation, but show they are required to maintain the signaling loops between the developing mesenchyme and AER that govern limb outgrowth after the initial emergence of limb bud. Dev Dyn 1999;216:469–480. ©1999 Wiley-Liss, Inc.

Fibroblast growth factor 9 (FGF9), originally cloned as glial-activating factor from human glioma cells, is expressed in adult rat brain and kidney. Here we report the chromosomal localization, genomic organization, and embryonic expression pattern of the mouse Fgf9 gene. Fgf9 maps to chromosome 14 near the Ctla6 locus. The gene spans more than 34 kb and contains three exons and two introns. Translation initiation occurs in exon 1, and translation termination occurs in exon 3. Fgf9 RNA was detected during mouse embryogenesis in several tissues in which Fgf gene expression has not been previously described, including intermediate mesoderm of late-stage gastrulation, ventricular myocardium, lung pleura, skeletal myoblasts in the early limb bud, spinal cord motor neurons, olfactory bulb, and gut lumenal epithelium. Fgf9 is coexpressed with other Fgf genes in some skeletal myoblasts, in limb apical ectoderm, in craniofacial ectoderm, and in the retina, inner ear, and tooth bud. Dev Dyn 1999;216:72-88.