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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.

Polydactyly is a common congenital anomaly of the hand and foot. Postaxial polydactyly (PAP) is characterized by one or more posterior or postaxial digits. In a Pakistani family with autosomal recessive nonsyndromic postaxial polydactyly type A (PAPA), we performed genomewide genotyping, linkage analysis, and exome and Sanger sequencing. Exome sequencing revealed a homozygous nonsense variant (c.478C>T, p.[Arg160*]) in the FAM92A gene within the mapped region on 8q21.13-q24.12 that segregated with the PAPA phenotype. We found that FAM92A is expressed in the developing mouse limb and E11.5 limb bud including the progress zone and the apical ectodermal ridge, where it strongly localizes at the cilia level, suggesting an important role in limb patterning. The identified variant leads to a loss of the FAM92A/Chibby1 complex that is crucial for ciliogenesis and impairs the recruitment and the colocalization of FAM92A with Chibby1 at the base of the cilia. In addition, we show that Fam92a-/- homozygous mice also exhibit an abnormal digit morphology, including metatarsal osteomas and polysyndactyly, in addition to distinct abnormalities on the deltoid tuberosity of their humeri. In conclusion, we present a new nonsyndromic PAPA ciliopathy due to a loss-of-function variant in FAM92A. © 2018 American Society for Bone and Mineral Research.

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 Retinoic acid (RA) is a vitamin A derivative. Exposure to exogenous RA generates congenital limb malformations (CLMs) in species from frogs to humans. These CLMs include but are not limited to oligodactyly and long-bone hypoplasia. The processes by which exogenous RA induces CLMs in mammals have been best studied in mouse, but as of yet remain unresolved. Methods We investigated the impact of exogenous RA on the cellular and molecular development of the limbs of a nonrodent model mammal, the opossum Monodelphis domestica. Opossums exposed to exogenous retinoic acid display CLMs including oligodactly, and results are consistent with opossum development being more susceptible to RA-induced disruptions than mouse development. Results Exposure of developing opossums to exogenous RA leads to an increase in cell death in the limb mesenchyme that is most pronounced in the zone of polarizing activity, and a reduction in cell proliferation throughout the limb mesenchyme. Exogenous RA also disrupts the expression of Shh in the zone of polarizing activity, and Fgf8 in the apical ectodermal ridge, and other genes with roles in the regulation of limb development and cell death. Conclusion Results are consistent with RA inducing CLMs in opossum limbs by disrupting the functions of the apical ectodermal ridge and zone of polarizing activity, and driving an increase in cell death and reduction of cell proliferation in the mesenchyme of the developing limb. Birth Defects Research (Part A) 103:225–234, 2015. © 2015 Wiley Periodicals, Inc.

Background: Tooth development is highly regulated in mammals and it is regulated by networks of signaling pathways (e. g. Tnf, Wnt, Shh, Fgf and Bmp) whose activities are controlled by the balance between ligands, activators, inhibitors and receptors. The members of the R-spondin family are known as activators of Wnt signaling, and Lgr4, Lgr5, and Lgr6 have been identified as receptors for R-spondins. The role of R-spondin/Lgr signaling in tooth development, however, remains unclear. Results: We first carried out comparative in situ hybridization analysis of R-spondins and Lgrs, and identified their dynamic spatio-temporal expression in murine odontogenesis. R-spondin2 expression was found both in tooth germs and the tooth-less region, the diastema. We further examined tooth development in R-spondin2 mutant mice, and although molars and incisors exhibited no significant abnormalities, supernumerary teeth were observed in the diastema. Conclusions: R-spondin/Lgr signaling is thus involved in tooth development. Developmental Dynamics 243:844–851, 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: At birth, marsupial neonates have precociously developed forelimbs. The development of the tammar wallaby (Macropus eugenii) hindlimbs lags significantly behind that of the forelimbs. This differs from the grey short-tailed opossum, Monodelphis domestica, which has relatively similar fore- and hindlimbs at birth. This study examines the expression of the key patterning genes TBX4, TBX5, PITX1, FGF8, and SHH in developing limb buds in the tammar wallaby. Results: All genes examined were highly conserved with orthologues from opossum and mouse. TBX4 expression appeared earlier in development than in the mouse, but later than in the opossum. SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. Conclusions: The most marked divergence in limb size in marsupials occurs in the kangaroos and wallabies. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs. Developmental Dynamics 243:324–338, 2014. © 2013 Wiley Periodicals, Inc.

BMP signaling is pivotal for normal limb bud development in vertebrate embryos and genetic analysis of receptors and ligands in the mouse revealed their requirement in both mesenchymal and ectodermal limb bud compartments. In this study, we genetically assessed the potential essential functions of SMAD4, a mediator of canonical BMP/TGFs signal transduction, in the mouse limb bud ectoderm. Msx2-Cre was used to conditionally inactivate Smad4 in the ectoderm of fore- and hindlimb buds. In hindlimb buds, the Smad4 inactivation disrupts the establishment and signaling by the apical ectodermal ridge (AER) from early limb bud stages onwards, which results in severe hypoplasia and/or aplasia of zeugo- and autopodal skeletal elements. In contrast, the developmentally later inactivation of Smad4 in forelimb buds does not alter AER formation and signaling, but prolongs epithelial-mesenchymal feedback signaling in advanced limb buds. The late termination of SHH and AER-FGF signaling delays distal progression of digit ray formation and inhibits interdigit apoptosis. In summary, our genetic analysis reveals the temporally and functionally distinct dual requirement of ectodermal Smad4 during initiation and termination of AER signaling.

We conducted a population-based case-control study of single nucleotide polymorphisms (SNPs) in selected genes to find common variants that play a role in the etiology of limb deficiencies (LDs). Included in the study were 389 infants with LDs of unknown cause and 980 unaffected controls selected from all births in New York State (NYS) for the years 1998-2005. We used cases identified from the NYS Department of Health (DOH) Congenital Malformations Registry. Genotypes were obtained for 132 SNPs in genes involved in limb development (SHH, WNT7A, FGF4, FGF8, FGF10, TBX3, TBX5, SALL4, GREM1, GDF5, CTNNB1, EN1, CYP26A1, CYP26B1), angiogenesis (VEGFA, HIF1A, NOS3), and coagulation (F2, F5, MTHFR). Genotype call rates were97% and SNPs were tested for departure from Hardy-Weinberg expectations by race/ethnic subgroups. For each SNP, odds ratios (OR)s and confidence intervals (CI)s were estimated and corrected for multiple comparisons for all LDs combined and for LD subtypes. Among non-Hispanic white infants, associations between FGF10 SNPs rs10805683 and rs13170645 and all LDs combined were statistically significant following correction for multiple testing (OR = 1.99; 95% CI = 1.43-2.77; uncorrected P = 0.000043 for rs10805683 heterozygous genotype, and OR = 2.37; 95% CI = 1.48-3.78; uncorrected P = 0.00032 for rs13170645 homozygous minor genotype). We also observed suggestive evidence for associations with SNPs in other genes including CYP26B1 and WNT7A. Animal studies have shown that FGF10 induces formation of the apical ectodermal ridge and is necessary for limb development. Our data suggest that common variants in FGF10 increase the risk for a wide range of non-syndromic limb deficiencies.

SUMMARY The vertebrate limb demonstrates remark- able similarity in basic organization across phylogenetically disparate groups. To gain further insight into how this mor- phological similarity is maintained in different developmental contexts, we explored the molecular anatomy of size-reduced embryos of the Puerto Rican coqufrog, Eleutherodactylus coqui. This animal demonstrates direct development, a life- history strategy marked by rapid progression from egg to adult and absence of a free-living, aquatic larva. Nonethe- less, coqu´o exhibits a basal anuran limb structure, with four toes on the forelimb and five toes on the hind limb. We in- vestigated the extent to which coqulimb bud development conforms to the model of limb development derived from am- niote studies. Toward this end, we characterized dynamic pat- terns of expression for 13 critical patterning genes across three principle stages of limb development. As expected, most genes demonstrate expression patterns that are es- sentially unchanged compared to amniote species. For ex- ample, we identified an EcFgf8-expression domain within the apical ectodermal ridge (AER). This expression pattern de- fines a putatively functional AER signaling domain, despite the absence of a morphological ridge in coqu´o embryos. However, two genes, EcMeis2 and EcAlx4, demonstrate al- tered domains of expression, which imply a potential shift in gene function between coqufrogs and amniote model sys- tems. Unexpectedly, several genes thought to be critical for limb patterning in other systems, including EcFgf4, EcWnt3a, EcWnt7a, and EcGremlin, demonstrated no evident expres- sion pattern in the limb at the three stages we analyzed. The absence of EcFgf4 and EcWnt3a expression during limb patterning is perhaps not surprising, given that neither gene is critical for proper limb development in the mouse, based on knockout and expression analyses. In contrast, absence of EcWnt7a and EcGremlin is surprising, given that expres- sion of these molecules appears to be absolutely essential in all other model systems so far examined. Although this analysis substantiates the existence of a core set of an- cient limb-patterning molecules, which likely mediate iden- tical functions across highly diverse vertebrate forms, it also reveals remarkable evolutionary flexibility in the genetic con- trol of a conserved morphological pattern across evolutionary time.

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.

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.

Anatomical history over centuries includes description of a wide variety of malformations involving the lower limbs. This article offers an organized review of these diverse abnormalities, including new understanding of mechanisms through recent discoveries in genetics and molecular biology. In 19th century Europe, a number of unique anomalies were reported, as well as evidence of foot amputations occurring in ancient Peruvian culture. Embryologically, the limbs develop early, with the lower limb being recognizable for the first time at stage 13 of development. By stage 23, the toes are clearly defined and by birth, although the legs appear bowed, the tibia and fibula are straight. Removal of the apical ectodermal ridge results in cessation of limb development, conversely, a second apical ectodermal ridge results in duplication of distal structures. Supernumerary limbs have been documented to occur as part of a teratoma with unique morphology and accompanying blood supply. Additionally, many examples of polydactyly occur in the foot postulating that deletion of chromosome 22q11 is involved in postaxial polydactyly. Such deletions occur near the middle of the chromosome at a location designated q11.2 (i.e., on the long arm of one of the pair of chromosomes 22) and this syndrome is also referred to as DiGeorge syndrome, which has a prevalence estimated at 1:4,000. Absence of the lower limbs has also been noted, with hypoplasia of the fibula being the most common manifestation of congenital bone absences in the lower limb. In addition to fibular aplasia, cases of tibial aplasia have been reported. This article is important for surgeons attempting correctional repair of lower limb anomalies, as well as providing analysis of the historical, anatomical and clinical aspects of supernumerary and absent limbs and digits for the lower limb.

Because all-trans retinoic acid (atRA) is teratogenic in all species tested and many of the specific defects induced are common across the phylogenetic spectrum, it would be logical to predict that murine strain differences in teratology to this agent are minimal. However, for specific defects, strain susceptibilities are vastly different. Studies with atRA have shown stark differences between C57BL/6 and SWV mouse strains in susceptibility to postaxial forelimb ectrodactyly and ectopic hindlimb formation, with the C57 strain being more susceptible for both defects. Various approaches were used to determine why these strains differ in susceptibility, but the mechanisms remain unknown. Hindlimb duplications were hypothesized to be caused by the formation of ectopic posterior body axes. For forelimb ectrodactyly, a locus on chromosome 11, Rafar, has linkage to the strain difference, and mRNA localization has shown that specific genes (Fgf8, Dlx3, Bmp4, and Sp8) in the postaxial preAER (prior to formation of the apical ectodermal ridge) of the developing limb bud (the site of the defect) were downregulated hours after atRA administration more in the susceptible C57 than in the SWV strain. Because both atRA and divalent cadmium induce postaxial forelimb ectrodactyly (right-sided predominance) at a high rate in C57BL/6 and low in the SWV strain, there is debate as to whether they share a common mechanism. These teratogens cause a greater-than-additive level of forelimb ectrodactyly when coadministered at low doses, but cadmium does not induce ectopic hindlimb formation. The hypothesis is that these agents have separate molecular pathologic pathways that converge to perturb a common anatomic structure.

Wnt signalling regulates cell proliferation and cell fate determination during embryogenesis. However, little is known about the developmental role of one Wnt family member, Wnt-3, during avian development. To investigate the possible functions of Wnt-3, its expression pattern was determined using whole-mount in situ hybridization. Wnt-3 is expressed in important signalling centres, including the dorsal neural tube, Hensen's node and the AER (apical ectodermal ridge). Most interestingly, Wnt-3 is expressed in the dorsal neural tube as a gradient, with the strongest expression anterior in the trunk. Furthermore, this study showed that Wnt-3 and Wnt-3a play a different role in neural crest lineages derived from different axial level of neural tube. Wnt-3 might be involved in proliferation of neural crest lineages, whereas Wnt-3a plays an important role in melanogenesis in vagal. However, both Wnt-3 and Wnt-3a cause a significant increase in melanogenesis in the trunk neural crest lineage.

Marsupials give birth after short gestation times to neonates that have an intriguing combination of precocial and altricial features, based on their functional necessity after birth. Perhaps most noticeably, marsupial newborns have highly developed forelimbs, which provide the propulsion necessary for the newborn's crawl to the teat. To achieve their advanced state at birth, the development of marsupial forelimbs is accelerated. The development of the newborn's hind limb, which plays no part in the crawl, is not accelerated, and is likely even delayed. Given the large differences in the rate of limb outgrowth among marsupials and placentals, we hypothesize that the pathways underlying the early development and outgrowth of marsupial limbs, especially that of their forelimbs, will also be divergent. As a first step toward testing this, we examine the development of one of the two major signaling centers of the developing limb, the apical ectodermal ridge (AER), in a marsupial, Monodelphis domestica. We found that, while both opossum limbs have reduced physical AER's, in the opossum forelimb this reduction has been taken to the extreme. Where the M. domestica forelimb should have an AER, it instead has only a few patches of disorganized cells. These results make the marsupial, M. domestica, the only known amniote (without reduced limbs) to exhibit no morphological AER. However, both M. domestica limbs normally express Fgf8, a molecular marker of the AER.

Heparan sulfate (HS) interacts with numerous growth factors, morphogens, receptors, and extracellular matrix proteins. Disruption of HS synthetic enzymes causes perturbation of growth factor signaling and malformation in vertebrate and invertebrate development. Our previous studies show that the O-sulfation patterns of HS are essential for the specific binding of growth factors to HS chains, and that depletion of O-sulfotransferases results in remarkable developmental defects in Drosophila, zebrafish, chick, and mouse. Here, we show that inhibition of chick HS-6-O-sulfotransferases (HS6ST-1 and HS6ST-2) in the prospective limb region by RNA interference (RNAi) resulted in the truncation of limb buds and reduced Fgf-8 and Fgf-10 expressions in the apical ectodermal ridge and in the underlying mesenchyme, respectively. HS6ST-2 RNAi resulted in a higher frequency of limb truncation and a more marked change in both Fgf-8 and Fgf-10 expressions than that achieved with HS6ST-1 RNAi. HS6ST-1 RNAi and HS6ST-2 RNAi caused a significant but distinct reduction in the levels of different 6-O-sulfation in HS, possibly as a result of their different substrate specificities. Our data support a model where proper levels and patterns of 6-O-sulfation of HS play essential roles in chick limb bud development.

The rhombomere 4(r4)-restricted expression of the mouse Hoxb2 gene is regulated by a 1.4-kb enhancer-containing fragment. Here, we showthat transgenic mouse lines expressing cre driven by this fragment (B2-r4-Cre), activated the R26R Cre reporter in rhombomere 4 and the second branchial arch, the epithelium of the first branchial arch, apical ectodermal ridge of the limb buds and the tail region. Of particular interest is Cre activity in the developing inner ear. Cre activity was found in the preotic field and otic placode at E8.5 and otocyst at E9.5-E12.5, in the cochleovestibular and facio-acoustic ganglia at E10.5 and the vestibular and spiral ganglia and all the otic epithelia derived from the otocyst at E15.5 and P0. Our data suggest that the B2-r4-Cre transgenic mice provide an important tool for conditional gene manipulation and lineage tracing in the inner ear. In combination with other transgenic lines expressing cre exclusively in the otic vesicle, the relative contributions of the hindbrain, periotic mesenchyme and otic epithelium in otic development can be dissected.

The apical ectodermal ridge (AER) in Neoceratodus develops after an initial period of mesenchymal proliferation and outgrowth of the fin bud and persists until chondrogenesis of the stylopod and zeugopod is initiated. At this time, the lateral margins of the AER convert to the fin fold leading to subsequent development of the dermal fin skeleton. Thorogood's (1991) fin fold model predicts that the AER should persist longer in Neoceratodus than it does in actinopterygians because of the comparatively extensive endochondral skeleton in lungfish. While the AER does persist into early chondrogenesis and is extended compared to actinopterygians (lost before fin radial chondrogenesis) it does not persist into further stages of chondrogenesis, providing partial support for Thorogood's model. Fgf8 appears in the lungfish fin epithelium during the initial period of fin outgrowth before a physical AER forms, when Fgf8 is restricted to the AER plus the preaxial and postaxial epithelium immediately adjacent to the AER. Fgf8 is no longer detected after the AER is replaced by a fin fold. Neoceratodus appears to provide a halfway point between ray fins and limbs during very early development as Thorogood proposed, but not precisely for the reasons his model suggests.

Patterning of the developing vertebrate limb along the anterior-posterior axis is controlled by the zone of polarizing activity (ZPA) via the expression of Sonic hedgehog (Shh) and along the proximal-distal axis by the apical ectodermal ridge (AER) through the production of fibroblast growth factors (FGFs). ZPA grafting, as well as ectopic application of SHH to the anterior chick limb bud, demonstrate that digit patterning is largely influenced by these secreted factors. Although signal transduction pathways have been well characterized for SHH and for FGFs, little is known of how these signals are regulated extracellularly in the limb. The present study shows that alteration of the extracellular environment through trypsin treatment can have profound effects on digit patterning. These effects appear to be mediated by the induction of Shh in host tissues and by ectopic AER formation, implicating the extracellular matrix in regulating the signaling activities of key patterning genes in the limb.

Btg/Tob genes encode for a new family of proteins with antiproliferative functions, which are also able to stimulate cell differentiation. Btg1 and Btg2 are the most closely related members in terms of gene sequence. We analyzed their expression patterns in avian embryos by in situ hybridization, from embryonic day 1 to 3. Btg1 was distinctively expressed in the Hensen's node, the notochord, the cardiogenic mesoderm, the lens vesicle, and in the apical ectodermal ridge and mesenchyme of the limb buds. On the other hand, Btg2 expression domains included the neural plate border, presomitic mesoderm, trigeminal placode, and mesonephros. Both genes were commonly expressed in the myotome, epibranchial placodes, and dorsal neural tube. The results suggest that Btg1 and Btg2 are involved in multiple developmental processes. Overlapping expression of Btg1 and Btg2 may imply redundant functions, but unique expression patterns suggest also differential regulation and function.

alphaVbeta3 is a receptor for vitronectin and other extracellular matrix ligands, and it has been implicated in angiogenesis and osteoclast function in mammals. We have cloned full-length cDNAs of zebrafish integrin alphaV (itgalphaV), and two paralogous zebrafish beta3 integrins (itgbeta3.1 and itgbeta3.2). Whole-mount in situ hybridization analysis revealed that alphaV and beta3.1 share overlapping expression domains in apical ectodermal ridge, ventricular myocardium, hypothalamus, posterior tuberculum, medial tectal proliferation zone, and in the odontogenic field of the bilateral pharyngeal dentitions. In contrast to beta3.1, beta3.2 is transiently expressed throughout the developing embryo. In situ hybridization profiles and heterologous expression of proteins in tissue culture cells suggest that beta3.1 is the major beta3 paralog that associates with alphaV in zebrafish. Furthermore, when beta3.1 expression profiles are compared to those of other potential alphaV partners (beta1, beta5, and beta8), pharyngeal dentitions appear to represent a unique expression field for alphaV and beta3.1.

Early in vertebrate limb development, a program initiates that polarizes the limb along the antero-posterior axis. The mesenchyme at the posterior margin is ultimately responsible for the asymmetry due to a region called the zone of polarizing activity (ZPA). The ZPA produces and secretes the molecule SHH, which coordinates the patterning of the resulting digits. Preaxial polydactyly (PPD) is a commonly occurring limb abnormality; investigating the genetic basis of this defect has provided insights into our understanding of digit patterning. PPD disrupts limb asymmetry by producing an ectopic ZPA at the opposite margin of the limb bud. Mutations in the long-range, limb-specific regulatory element of the Shh gene are responsible for the defect. Genetic analysis of this limb abnormality provides an important approach in understanding the mechanisms that control digit patterning.

The prevalence of clinical phenotypes that exhibit combinations of central polydactyly, syndactyly, or cleft hand or foot is higher than would be expected for random independent mutations. We have previously demonstrated that maternal ingestion of a chemotherapeutic agent, busulfan, at embryonic day 11 (E11) induces these defects in various combinations in rat embryo limbs. In an effort to determine the mechanism by which busulfan disrupts digital development, we examined cell death by Nile Blue staining and TdT-mediated dUTP nick end labeling (TUNEL) assays; we also carried out whole mount in situ hybridization for fibroblast growth factor-8 (Fgf8), bone morphogenetic protein-4 (Bmp4), and sonic hedgehog (Shh) to examine developmental pathways linked to these defects. In busulfan-treated embryos, diffuse cell death was evident in both ectoderm and mesoderm, peaking at E13. The increased cell death leads to regression of Fgf8 in the apical ectodermal ridge (AER) and Bmp4 and Shh in the underlying mesoderm. The subsequent pattern of interdigital apoptosis and cartilage condensation was variably disrupted. These results suggest that busulfan manifests its teratogenic effects by inducing cell death of both ectoderm and mesoderm, with an associated reduction in tissue and a disruption in the generation of patterning molecules during critical periods of digit specification.

Digit formation is the last step in the skeletal patterning of developing limbs. This process involves important aspects such as determination of chondrogenic versus interdigital areas; growth of digital rays with periodic segmentation to form joints and thus phalanges, and finally tip formation. Traditionally it was believed that the properties of digital rays were fixed at earlier stages, but recently a surprising plasticity of digit primordia at the time of condensation has been demonstrated. This implies the presence of local interactions that are able to modulate the particular programs that make a given digit, but we don't fully understand how they operate. An involvement of signaling from the interdigital spaces and from the apical ectodermal ridge has been proposed. Another interesting question is the formation of the last limb structure, digit tips, which may involve a specific molecular and cellular program. Indeed, the expression of several developmentally important genes is restricted to digit tips at late stages of limb development. Understanding the molecular and cellular interactions that lead to digit morphogenesis has important implications not only in the context of embryonic development (for example, how early cues received by cells are translated into anatomy or what are the mechanisms that control the cease of activity of signaling regions) but also in terms of limb diversification during evolution.

BACKGROUND: Defects in digit number or fusion as a teratogenic response are well documented in humans and intensively studied in various mouse models. Maternal exposure to excess levels of all-trans-retinoic acid (RA) at gestational day 9.5 induces postaxial ectrodactyly (digit loss) in the murine C57BL/6N strain but not in the SWV/Fnn strain. METHODS: Whole-mount in situ hybridization was used to examine the differential expression of limb patterning genes at the transcriptional level between the two mouse strains following the maternal exposure to a teratogenic level of RA. The detection of a gene with altered expression was followed by either the evaluation of other genes that were synexpressed or with an assessment of downstream genes. RESULTS: In the C57BL/6N limb bud following maternal RA administration, gene-specific perturbations were observed within hours of the RA injection in the posterior pre-AER (apical ectodermal ridge) (Fgf8, Dlx3, Bmp4, Sp8, but not Dlx2 or p63), whereas these genes were normally expressed in the SWV/Fnn limb bud. Furthermore, although RA caused comparable reductions of Shh expression between the strains in the 12 h after administration, some Shh downstream genes were differentially expressed (e.g., Gli1, Ptc, and Hoxd13), whereas others were not (e.g., Fgf4, Bmp4, and Gremlin). CONCLUSIONS: It is proposed that altered gene expression in both pre-AER and mesoderm is involved in the pathogenesis of postaxial digit loss, and that because the alterations in the pre-AER occur relatively early in the temporal sequence of events, those changes are candidates for an initiating factor in the malformation. Birth Defects Research (Part A) 2007. © 2007 Wiley-Liss, Inc.

Wnt/beta-catenin signaling is involved in the formation of the apical ectodermal ridge (AER) during vertebrate limb development. Although Wnt3a is a potent ligand for chick AER formation, whether chick Wnt3a can induce Fgf8 expression in chick embryos is unclear and the Wnt ligand involved in chick AER formation remains unknown. Here, we examined whether another Wnt3a isoform is expressed in the AER, and whether Wnt3 contributes to AER formation in chick as well as mouse embryos. We found that chick Wnt3 was not expressed in the presumptive limb ectoderm at the early stages of AER formation. Using 5'-rapid amplification of cDNA ends, we isolated another chick Wnt3a transcript. This novel variant, Wnt3a variant 2, induced Fgf8 in the limb ectoderm and activated the beta-catenin pathway in vivo and in vitro. These data showed that Wnt3a variant 2 is an active form of chick Wnt3a that regulates chick AER formation.

Fibroblast growth factors play critical roles in many aspects of embryo patterning that are conserved across broad phylogenetic distances. To help understand the evolution of fibroblast growth factor functions, we identified members of the Fgf8/17/18-subfamily in the three-spine stickleback Gasterosteus aculeatus, and investigated their evolutionary relationships and expression patterns. We found that fgf17b is the ortholog of tetrapod Fgf17, whereas the teleost genes called fgf8 and fgf17a are duplicates of the tetrapod gene Fgf8, and thus should be called fgf8a and fgf8b. Phylogenetic analysis supports the view that the Fgf8/17/18-subfamily expanded during the ray-fin fish genome duplication. In situ hybridization experiments showed that stickleback fgf8 duplicates exhibited common and unique expression patterns, indicating that tissue specialization followed the gene duplication event. Moreover, direct comparison of stickleback and zebrafish embryonic expression patterns of fgf8 co-orthologs suggested lineage-specific independent subfunction partitioning and the acquisition or the loss of ortholog functions. In tetrapods, Fgf8 plays an important role in the apical ectodermal ridge of the developing pectoral appendage. Surprisingly, differences in the expression of fgf8a in the apical ectodermal ridge of the pectoral fin bud in zebrafish and stickleback, coupled with the role of fgf16 and fgf24 in teleost pectoral appendage show that different Fgf genes may play similar roles in limb development in various vertebrates.

The development of the vertebrate limb is a highly regulated process. The outgrowth and patterning of the vertebrate limb depends on the Apical Ectodermal Ridge (AER), one of two important signaling centers in the developing limb bud. The AER expresses several families of genes involved in limb development including Fibroblast Growth Factor-8, the distal-less homeobox transcription factors (Dlx), and the muscle segment homeobox transcription factors (Msx). Previous studies have shown Dlx-5 expression in the AER during stages 18-31 (E 3-7.5) in the developing chick limb. Dlx-3 and Msx-2 have also been shown to play a possible role in the AER during vertebrate limb formation. The aim of this study was to determine the ontogeny of mRNA expression patterns of Dlx5, Dlx-3, and Msx-2 during limb development of the chicken (E 0-17) using reverse transcription polymerase chain reaction (RT-PCR). Both Dlx-3 and Dlx-5 expression were first detected on E 2 and increased in expression through E 17. This is consistent...

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.

The signaling pathways leading to growth and patterning of various organs are tightly controlled during the development of any organism. These control mechanisms usually involve the utilization of feedback- and pathway-specific antagonists where the pathway induces the expression of its own antagonist. Sef is a feedback antagonist of fibroblast growth factor (FGF) signaling, which has been identified recently in zebrafish and mammals. Here, we report the isolation of chicken Sef (cSef) and demonstrate the conserved nature of the regulatory relationship with FGF signaling. In chick embryos, Sef is expressed in a pattern that coincides with many known sites of FGF signaling. In the developing limb, cSef is expressed in the mesoderm underlying the apical ectodermal ridge (AER) in the region known as the progress zone. cSef message first appeared after limb budding and AER formation. Expression was intense at stages of rapid limb outgrowth, and gradually decreased to almost undetectable levels when differentiation was clearly apparent. Gain- and loss-of-function experiments showed that FGFs differentially regulate the expression of cSef in various tissues. Thus, removal of the AER down-regulated cSef expression, and FGF2 but not FGF4 or FGF8 beads substituted for the AER in maintaining cSef expression. At sites where cSef is not normally expressed, FGF4 and FGF2, but not FGF8 beads, induced cSef expression. Our results demonstrate the complexity of cSef regulation by FGFs and point to FGF2 as a prime candidate in regulating cSef expression during normal limb development. The spatiotemporal pattern of cSef expression during limb development suggests a role for cSef in regulating limb outgrowth but not limb initiation. Developmental Dynamics 233:301–312, 2005. © 2005 Wiley-Liss, Inc.

Hyperphalangy is a digit morphology in which increased numbers of phalanges are arranged linearly within a digit beyond the plesiomorphic condition. We analyse patterns and processes of hyperphalangy by considering previous definitions and occurrences of hyperphalangy among terrestrial and secondarily aquatic extant and fossil taxa (cetaceans, ichthyosaurs, plesiosaurs and mosasaurs), and recent studies that elucidate the factors involved in terrestrial autopod joint induction. Extreme hyperphalangy, defined as exceeding a threshold condition of 4/6/6/6/6, is shown only to be found among secondarily aquatic vertebrates with a flipper limb morphology. Based on this definition, hyperphalangy occurs exclusively in digits II and III among extant cetaceans. Previous reports of cetacean embryos having more phalanges than adults is clarified and shown to be based on cartilaginous elements not ossified phalanges. Developmental prerequisites for hyperphalangy include lack of cell death in interdigital mesoderm (producing a flipper limb) and maintenance of a secondary apical ectodermal ridge (AER), which initiates digit elongation and extra joint patterning. Factors of the limb-patterning pathways located in the interdigital mesoderm, including bone morphogenetic proteins (BMPs), BMP antagonists, fibroblast growth factors (FGFs), growth/differentiation factor-5 (GDF-5), Wnt-14 and ck-erg, are implicated in maintenance of the flipper limb, secondary AER formation, digit elongation and additional joint induction leading to hyperphalangy.

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.

Mice lacking transcription factor AP-2α exhibit defects in the formation of the head, body wall, heart, neural tube, eye, and limbs, reflecting important sites of AP-2α expression in the developing embryo. AP-2α is also expressed in the postnatal mammary gland and has been linked to tumor progression and defects in growth regulation in the breast. We have used a transgenic mouse approach to identify tissue-specific cis-acting sequences associated with expression of the human AP-2α gene. Our analysis indicates that multiple elements located throughout the gene contribute to expression in the trigeminal ganglia, spinal cord, mammary gland, and epidermis. A discrete cis-element located within the fifth intron is required for expression in the face and limbs, and we have derived a permanent line of AP-2α::lacZ transgenic mice to assess expression of this latter enhancer throughout morphogenesis. We also introduced this transgene into an AP-2α–null mouse background and detected subtle alterations of its expression within the progress zone and apical ectodermal ridge of the forelimbs. Similar changes in lacZ expression were observed within the zeugopod, and these correlated with defects in radius condensation in AP-2α–knockout mice. Taken together, these findings indicate that cell:cell communication within the forelimb is altered in the absence of AP-2α and reveal novel regulatory potential for AP-2α in limb development. Developmental Dynamics 228:194–207, 2003. © 2003 Wiley-Liss, Inc.

Split hand foot malformation (SHFM) is a congenital limb malformation presenting with a median cleft of the hand and/or foot, syndactyly and polydactyly. SHFM is genetically heterogeneous with four loci mapped to date. Murine Dactylaplasia (Dac) is phenotypically similar, and it has been mapped to a syntenic region of 10q24, where SHFM3 has been localized. Structural alterations of the gene-encoding dactylin, a constituent of the ubiquitinization pathway, leading to reduced levels of transcript have been identified in Dac. Here, we report a significant decrease of Dactylin transcript in several individuals affected by SHFM. This observation supports a central role for dactylin in the pathogenesis of SHFM.

Kruppel-like factor 4 (Klf4) belongs to the family of transcription factors that are thought to be involved in the regulation of epithelial and germ cell differentiation, based on their expression in postproliferative cells of the skin, gut, and testes. Gene ablation experiments suggest that Klf4 plays a role in keratinocyte differentiation, since mice lacking Klf4 fail to establish proper barrier function and, as a consequence, die postnatally due to dehydration. Recent studies have shown that Klf4 is also expressed in postnatal male mice, in postmeiotic sperm cells undergoing terminal differentiation into sperm cells. However, prior to the current study, the expression pattern of Klf4 during early and mid-embryogenesis had not been examined. Here we demonstrate that Klf4 transcripts can be detected from embryonic day 4.5 (E4.5) on in the developing conceptus, and that Klf4 expression before E10 is restricted to extraembryonic tissues. The embryo proper displays a highly dynamic and changing Klf4 signal from E10 of murine development on. In addition to being expressed in a stripe of mesenchymal cells extending from the forelimb bud rostrally over the branchial arches to the developing eye, Klf4 is also expressed in the mesenchyme surrounding the nasal pit at day E11.5. In addition, Klf4 has been detected in the apical ectodermal ridge and adjacent mesenchymal cells in the limb buds, and in mesenchymal cells of the developing body wall in trunk areas. These findings suggest that Klf4 plays an important role in regulating cellular proliferation, which underlies the morphogenetic changes that shape the developing embryo.

Signalling interactions between the polarizing region, which produces SHH, and the apical ectodermal ridge, which produces FGFs, are essential for outgrowth and patterning of vertebrate limbs. However, mechanisms that mediate translation of early positional information of cells into anatomy remain largely unknown. In particular, the molecular and cellular basis of digit morphogenesis are not fully understood, either in terms of the formation of the different digits along the antero-posterior axis or in the way digits stop growing once pattern formation has been completed. Here we will review recent data about digit development. Manipulation of morphogenetic signals during digit formation, including application of SHH interdigitally, has shown that digit primordia possess a certain plasticity, and that digit anatomy becomes irreversibly fixed during morphogenesis. The process of generation of joints and thus segmentation and formation of digit tips is also discussed.

Background Caspases play a pivotal role in the regulation and execution of apoptosis, an essential process during limb development. Caspase 8 activation is usually downstream of the Fas/FasL death receptors, whereas caspase 9 mediates the mitochondrial signaling pathway of apoptosis. Caspase 3 is an effector caspase. Previous studies have shown that the exposure of embryonic murine limbs in vitro to 4-hydroperoxycyclophosphamide (4-OOHCPA), an activated analog of the anticancer alkylating agent, cyclophosphamide, induced limb malformations and apoptosis. The goal of this study was to determine the role of caspases in mediating apoptosis in this model system. Methods Limb buds from gestational day 12 CD-1 mice were excised and cultured in roller bottles in a chemically defined medium for up to 6 days in the absence or presence of 4-OOHCPA. Apoptosis was indicated by internucleosomal DNA fragmentation, as detected by TUNEL staining. The profile of caspase activation was characterized by Western blot analysis and immunohistochemistry of control and treated limbs. To determine the consequences to limb morphology of inhibiting caspase activation, DEVD-CHO, a caspase-3 inhibitor, was added to the cultures. Results Limbs cultured in the presence of 4-OOHCPA were growth retarded and malformed; apoptosis was increased in the apical ectodermal ridge and interdigital areas. Western blot analysis showed that 4-OOHCPA exposure did not activate procaspases 8 or 9 in limbs. In contrast, procaspase-3 cleavage was increased in a concentration and time-dependent manner after exposure of limbs to 4-OOHCPA. Immunoreactive activated caspase-3 was localized in the interdigital areas and the apical ectodermal ridge region in control limbs; staining in these areas and in the interdigital areas was increased dramatically in limbs exposed to 4-OOHCPA. Inhibition of caspase 3 activation with DEVD-CHO partially protected limbs from insult with 4-OOHCPA. Conclusion Caspase-dependent and caspase-independent pathways of cell death are both important is mediating the abnormal limb development triggered by insult with 4-OOHCPA. Teratology 66:288–299, 2002. © 2002 Wiley-Liss, Inc.

In the developing chick wing, the use of antisense oligodeoxynucleotides to transiently knock down the expression of the gap junction protein, connexin43 (Cx43), results in limb patterning defects, including deletion of the anterior digits. To understand more about how such defects arise, the effects of transient Cx43 knockdown on the expression patterns of several genes known to play pivotal roles in limb formation were examined. Sonic hedgehog (Shh), which is normally expressed in the zone of polarizing activity (ZPA) and is required to maintain both the ZPA and the apical ectodermal ridge (AER), was found to be downregulated in treated limbs within 30 h. Bone morphogenetic protein-2 (Bmp-2), a gene downstream of Shh, was similarly downregulated. Fibroblast growth factor-8 expression, however, was unaltered 30 h after treatment but was greatly reduced at 48 h post-treatment, when the AER begins to regress. Expressions of Bmp-4 and Muscle segment homeobox-like gene (Msx-1) were not affected at any of the time points examined. Cx43 expression is therefore involved in some, but not all patterning cascades, and appears to play a role in the regulation of ZPA activity.

Since the origin of tetrapod limbs about 450 million years ago, every Class of land vertebrate - reptiles, birds, amphibians, and mammals - has undergone some degree of digit loss from the ancestral count of five. Even within mammals many species have independently reduced the number of digits as a locomotor adaptation. The repeated convergence on digit loss indicates it is perhaps mechanistically simple, yet little is known of the mechanisms or whether developmental constraints ensure the same mechanisms are re-deployed in divergent species. Here we show in the three-toed jerboa, horse, camel, and pig that the mechanisms of digit loss are varied and yet utilize recurring themes. Common characteristics of digit loss include downregulated expression of the growth factor fgf8 in the apical ectodermal ridge in the anterior and posterior limb of all species with reduced digits. We also see massively upregulated cell death in tissue that fails to become digits in the jerboa, horse, and camel. In the three-toed...

During limb development, fibroblast growth factors (FGFs) from the apical ectodermal ridge (AER) regulate the expression of Sonic hedgehog (SHH) in the underlying posterior distal mesoderm. The mol...

Congenital malformations of the extremities are conspicuous and have been described through the ages. Over the past decade, a wealth of knowledge has been generated regarding the genetic regulation of limb development and the underlying molecular mechanisms. Recent studies have identified several of the signaling molecules, growth factors, and transcriptional regulators involved in the initiation and maintenance of the apical ectodermal ridge (AER) as well as the molecular markers defining the three axes of the developing limb. Studies of abnormal murine phenotypes have uncovered the role played by genes such as p63 and Dactylin in the maintenance of AER activity. These phenotypes resemble human malformations and in this review we describe the underlying mechanisms and clinical associations of split hand/foot malformation and ectrodactyly-ectodermal dysplasia-cleft lip/palate syndrome, which have both been associated with mutations in the p63 gene.

Retinoic acid, when administered to pregnant mice on d 11.0 of gestation, causes limb skeletal abnormalities consisting of reduced digital number, shortening of the long bones and delayed ossification. We show here that these effects are correlated with a decrease in cell proliferation within 5 h of retinoic acid administration, specifically in the posterior half of the distal limb bud mesenchyme, from which the distal skeletal elements are generated. There is a specific downregulation of Fgf4, a gene known to be involved in limb bud outgrowth and expressed only in the posterior part of the apical ectodermal ridge; Fgf8, which is expressed throughout the apical ectodermal ridge, is unaffected. The reduction in Fgf4 expression is not accompanied by downregulation of Shh, nor of its receptor and downstream target gene Ptc, suggesting that the skeletal reduction defects induced by retinoic acid are mediated specifically by FGF4-induced skeletogenic mesenchymal cell proliferation.

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.

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.