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To understand the developmental mechanism that determines limb size and the consequent limb-to-trunk proportions in the tetrapod body, we investigated the role of the paraxial mesoderm in the specification of the limb and flank fields in the chick embryo. We found that the paraxial mesoderm subjacent to the limb field can affect the size of the limb bud along the anterior–posterior and proximal–distal axes. We also found that the paraxial mesoderm subjacent to the flank plays roles in suppressing the emergence and growth of the limb bud and in promoting flank-specific apoptosis in the lateral plate mesoderm. Our results suggest that signals from the paraxial mesoderm specify the limb and flank fields in the competent lateral plate mesoderm. Developmental Dynamics 240:1639–1649, 2011. © 2011 Wiley-Liss, Inc.

Limb development has long provided an excellent model for understanding the genetic principles driving embryogenesis. Studies utilizing chick and mouse have led to new insights into limb patterning and morphogenesis. Recent research has centered on the regulatory networks underlying limb development. Here, we discuss the hierarchical, overlapping, and iterative roles of Pbx family members in appendicular development that have emerged from genetic analyses in the mouse. Pbx genes are essential in determining limb bud positioning, early bud formation, limb axes establishment and coordination, and patterning and morphogenesis of most elements of the limb and girdle. Pbx proteins directly regulate critical effectors of limb and girdle development, including morphogen-encoding genes like Shh in limb posterior mesoderm, and transcription factor-encoding genes like Alx1 in pre-scapular domains. Interestingly, at least in limb buds, Pbx appear to act not only as Hox cofactors, but also in the upstream control of 5' HoxA/D gene expression.

While the paired forelimb and hindlimb buds of vertebrates are initially morphologically homogeneous, as the limb progenitors differentiate, each individual tissue element attains a characteristic limb-type morphology that ultimately defines the constitution of the forelimb or hindlimb. This review focuses on contemporary understanding of the regulation of limb bud initiation and formation of limb-type specific morphologies and how these regulatory mechanisms evolved in vertebrates. We also attempt to clarify the definition of the terms limb-type identity and limb-type morphology that have frequently been used interchangeably. Over the last decade, three genes, Tbx4, Tbx5, and Pitx1, have been extensively studied for their roles in limb initiation and determining limb-type morphologies. The role of Tbx4 and Tbx5 in limb initiation is clearly established. However, their putative role in the generation of limb-type morphologies remains controversial. In contrast, all evidence supports a function for Pitx1 in determination of hindlimb morphologies.

In Xenopus laevis, limb buds start to develop at a later point of the larval stage, prior to metamorphosis. This onset of limb development in Xenopus is totally different from that in amniotes such as birds and mammals, in which limb buds emerge at an early stage of embryogenesis, in parallel with other organogenesis. We investigated limb myogenesis in Xenopus, focusing on myogenic gene expression, myogenic ability of limb bud cells in the early stage, and the origin of myogenic precursor cells in the limb bud. The Xenopus early limb bud contains myoD/cardiac actin-positive and pax3/pax7-negative cells. Interestingly, results of transplantation experiments have revealed that this early limb bud contains myogenic precursor cells. In order to know the contribution of myogenic cells in somites to myogenic precursor cells in the early limb bud, we used a Cre-LoxP system for tracing over a long period. The results of fate tracing for myogenic cells in somites of the Xenopus embryo suggested that early-specified myogenic cells in somites do not contribute to limb muscle in Xenopus. Taken together, the results suggest that limb muscle development in Xenopus has characteristics of initiation and early events distinct from those of other vertebrate clades.

Ptprv is a member of the transmembrane tyrosine phosphatase gene family reported to be expressed in osteoblasts and gonads. To better define the developmental and tissue specificity of Ptprv expression, we generated knock-in mice expressing a nuclear localised β-galactosidase reporter under the control of resident Ptprv regulatory elements. Histochemical staining of Ptprv-nLacZ mice revealed that Ptprv expression is readily detectable in the foetal gonadal ridge of both sexes and in adult gonads where it is localised to Sertoli cells of the testis and celomic epithelial cells of the ovaries. During early limb development, Ptprv expression is prominent in the apical ectodermal ridge of the limb bud. At latter stages of development, Ptprv is predominantly expressed in the perichondrial and periosteal region of long bones, known as the bone collar. In contrast to previous indications from in vitro studies, there is little if any expression in mature osteoblasts in vivo. Analysis of Ptprv mRNA localisation by in situ hybridization in parallel with molecular markers of chondrocytes and osteoblasts confirmed the specific expression of Ptprv in immature bone collar cells. The specificity of Ptprv expression in these cells may be a useful tool to elucidate their role in the transition of skeletal elements from cartilage template to bone. Developmental Dynamics 229:826–834, 2004. © 2004 Wiley-Liss, Inc.

Tg737 mutant mice exhibit pathologic conditions in numerous tissues along with skeletal patterning defects. Herein, we characterize the skeletal pathologic conditions and confirm a role for Tg737 in skeletal patterning through transgenic rescue. Analyses were conducted in both the hypomorphic Tg737orpk allele that results in duplication of digit one and in the null Tg737Δ2-3βGal allele that is an embryonic lethal mutation exhibiting eight digits per limb. In early limb buds, Tg737 expression is detected throughout the mesenchyme becoming concentrated in precartilage condensations at later stages. In situ analyses indicate that the Tg737orpk mutant limb defects are not associated with changes in expression of Shh, Ihh, HoxD11–13, Patched, BMPs, or Glis. Likewise, in Tg737Δ2-3βGal mutant embryos, there was no change in Shh expression. However, in both alleles, Fgf4 was ectopically expressed on the anterior apical ectodermal ridge. Collectively, the data argue for a dosage effect of Tg737 on the limb phenotypes and that the polydactyly is independent of Shh misexpression. Developmental Dynamics 227:78–90, 2003. © 2003 Wiley-Liss, Inc.

Dach1 is a mouse homologue of the Drosophila dachshund gene, which is a key regulator of cell fate determination during eye, leg, and brain development in the fly. We have investigated the expression and growth factor regulation of Dach1 during pre- and postnatal skeletal development in the mouse limb to understand better the function of Dach1. Dach1 was expressed in the distal mesenchyme of the early embryonic mouse limb bud and subsequently became restricted to the tips of digital cartilages. Dach1 protein was localized to postmitotic, prehypertrophic, and early hypertrophic chondrocytes during the initiation of ossification centers, but Dach1 was not expressed in growth plates that exhibited extensive ossification. Dach1 colocalized with Runx2/Cbfa1 in chondrocytes but not in the forming bone collar or primary spongiosa. Dach1 also colocalized with cyclin-dependent kinase inhibitors p27 (Kip1) and p57 (Kip2) in chondrocytes of the growth plate and in the epiphysis before the formation of the secondary ossification center. Because fibroblast growth factors (FGF), bone morphogenetic proteins (BMP), and hedgehog molecules (Hh) regulate skeletal patterning of the limb bud and chondrocyte maturation in developing endochondral bones, we investigated the regulation of Dach1 by these growth and differentiation factors. Expression of Dach1 in 11 days postcoitus mouse limb buds in organ culture was up-regulated by implanting beads soaked in FGF1, 2, 8, or 9 but not FGF10. BMP4-soaked beads down-regulated Dach1 expression, whereas Shh and bovine serum albumin had no effect. Furthermore, FGF4 or 8 could substitute for the apical ectodermal ridge in maintaining Dach1 expression in the limb buds. Immunolocalization of FGFR2 and FGFR3 revealed overlap with Dach1 expression during skeletal patterning and chondrocyte maturation. We conclude that Dach1 is a target gene of FGF signaling during limb skeletal development, and Dach1 may function as an intermediary in the FGF signaling pathway regulating cell proliferation or differentiation. Published 2002 Wiley-Liss, Inc.

The interactions of Sonic hedgehog (Shh) and fibroblast growth factor (FGF) play important roles in vertebrate limb pattern formation. In the posterior region of the chick limb bud, Shh and FGF-4 each maintain expression in a positive feedback loop. In the anterior region, Shh can also induce Fgf-4 expression in the anterior apical ectodermal ridge. However, the possibility of Shh induction by FGF protein is unclear. Because many experiments to analyze gene expression have been carried out by using the forelimb bud of the chick embryo, we investigated gene expression of the cells in the anterior region of the chick hindlimb bud after FGF-4 application and compared the results with those for the forelimb bud. When an FGF-4-containing bead was implanted into the anterior region of a stage 20 hindlimb bud, ectopic expression of Shh was induced in the mesenchyme beneath the anterior end of the apical ectodermal ridge at 36 hr after implantation. Subsequent to Shh activation, Hoxd13 was also observed in the anterior-distal region of the limb bud. Furthermore, FGF-4 implantation to the hindlimb bud caused additional digit formation accompanying respecification of positional value in the anterior tissue. Ectopic Shh was induced in cells located distal to the FGF-4 bead, and the cells of the flank region did not contribute to ectopic Shh induction. On the other hand, no ectopic Shh and Hoxd13 expression was detected by grafting an FGF-4 bead into the forelimb bud. Although FGF-4 implantation to the forelimb bud occasionally induced extra digit 2 formation, no embryos had an extra digit 3 or digit 4, and many specimens exhibited normal skeletal pattern. These results demonstrate the difference between the fore- and hindlimb buds in the cell competence of Shh induction in response to FGF-4, suggesting the possibility that the responsiveness of mesenchymal cells in signaling molecules is not the same in the fore- and hindlimb buds.

Programmed cell death (PCD) is considered one of the most important cellular processes in the morphogenesis of organs and tissues during animal development. Although the embryonic limb has been established as a classic model for the study of PCD, detailed studies on this process' contribution to morphogenesis are still lacking. In the present work, using modern computer-aided techniques, we estimated the contribution of PCD to mouse limb morphogenesis. For the detection of apoptotic cell death, we stained whole embryonic limbs with acridine orange or, in some instances, used the TUNEL technique, and visualized the tissues by confocal laser scanning microscopy. We found that cell death patterns are dynamic during limb development, and occur in gradients oriented with the main limb axes, anteroposterior, dorsoventral and distoproximal. Interdigital apoptosis in the autopod was initially detected at the most distal region, and then more proximally as development proceeded. Interestingly, we found that digit separation is more pronounced on the dorsal side, contrary to what is expected from the apoptotic cell distribution, which shows more abundant cell death in the ventral region. Using 2-D and 3-D models, we found that most digit individualization occurs rather by digit growth than by interdigital cell death. Therefore, digits do not mainly individualize by degeneration of preformed interdigital tissue, but probably by a dynamic balance between proliferation and cell death, reducing interdigital growth, which results in protrusion of digits. We determined the expression pattern of fgf-8 during the period of digit individualization, as the product of this gene could participate in defining the limb growth pattern. Initially, fgf-8 expression was coincident with the apical ectodermal ridge, but when cell death was first detected in the interdigits, fgf-8 expression became restricted to the tip of the growing digits. Therefore, FGF-8 could be one of the factors responsible for differential digit-interdigit growth, and might also act as a survival factor on interdigital tissue. We also found that the expression patterns of rar-beta, bmp-2, bmp-4, bmp-7, msx-1, and msx-2 genes, proposed to be involved in the activation of interdigital cell death, did not overlap with, or were not highly expressed in the major zones of cell death in the developing limb.

Mesenchymal cells from different stages of chick limb buds sort out in monolayer culture, suggesting the presence of different cell affinities dependent on their positions along the proximodistal axis. However, it is still not clear which molecules are responsible for the sorting-out. Here, we propose that N-cadherin, a cell-adhesion molecule, is involved in the sorting-out and is likely to be a component of the mechanism of proximodistal patterning in the developing limb. N-cadherin proteins accumulate in the distal region of the chick limb bud as limb development proceeds. In monolayer culture of distal mesenchymal cells, the stage-dependent levels of N-cadherin proteins are maintained during cell sorting. The results of this study have also demonstrated that an anti-N-cadherin monoclonal antibody, NCD-2, clearly inhibits the cell sorting. Moreover, removal of the apical ectodermal ridge or retinoic-acid treatment of distal cells, which results in a change in the pattern of sorting-out, inhibits the accumulation of N-cadherin proteins, suggesting that the distribution of these proteins is related to the positional identity that gives rise to the different shape and number of cartilage elements along the proximodistal axis.

Dac is a novel nuclear factor in mouse and humans that shares homology with Drosophila dachshund (dac). Alignment with available sequences defines a conserved box of 117 amino acids that shares weak homology with the proto-oncogene Ski and Sno. Dac expression is found in various neuroectodermal and mesenchymal tissues. At early developmental stages Dac is expressed in lateral mesoderm and in neural crest cells. In the neural plate/tube Dac expression is initially seen in the prosencephalon and gets gradually restricted to the presumptive neocortex and the distal portion of the outgrowing optic vesicle. Furthermore, Dac transcripts are detected in the mesenchyme underlying the Apical Ectodermal Ridge (AER) of the extending limb bud, the dorsal root ganglia and chain ganglia, and the mesenchyme of the growing genitalia. Dac expression in the Gli 3 mutant extra toes (Xt/Xt) shows little difference compared to the expression in wild-type limb buds. In contrast, a significant expansion of Dac expression are observed in the anterior mesenchyme of the limb buds of hemimelic extra toes (Hx/+) mice. FISH analysis reveals that human DAC maps to chromosome 13q22.3-23 and further fine-mapping defined a position of the DAC gene at 54cM or 13q21.1, a locus that associates with mental retardation and skeletal abnormalities.

Pattern formation of the devel- oping vertebrate limb is mainly controlled by the zone of polarizing activity (ZPA) and the apical ectodermal ridge (AER) which may act as sources of diffusing morphogens. These sources are tightly interconnected and maintained by positive feedback and, together with the established role of Wnt7a on the dorsal side of the bud, they con- stitute a Cartesian reference frame for the pro- cesses of patterning and growth of the limb bud. As an input to our model we have used the local extent and temporal activity of the AER source as it is reflected by Fgf4 expression in the ridge. We have assumed that this source produces a mor- phogen which diffuses in the three-dimensional limb field and degradates by first-order kinetics. When in a cell the morphogen concentration ex- ceeds a particular threshold value, a gene is switched on. To every threshold corresponds a specific gene. In the following we introduce an or- der of increasing concentration thresholds corre- sponding to the sequence of Hoxa-10,11, and 13 genes (threshold collinearity). With this simple rule of correspondence we can reproduce both spatial and temporal collinearities of Hoxa gene expression. This outcome may be the first direct observable effect of a putative morphogen in the developing limb. The expression patterns are es- sentially transient, and they are followed by se- quential refinements which lead to the final limb structures. Furthermore, the continuous flow of the morphogen through the progress zone guar- antees the coherent course of patterning and limb growth. Several experiments are proposed for ad- ditional tests of the validity of the model and the eventual reversibility of Hoxu gene expression. 0 1996 Wiley-Lisa, Inc.

IGF-I and members of the FGF family have been implicated in the reciprocal interactions between the apical ectodermal ridge (AER) and underlying subridge mesoderm that are required for outgrowth of the developing limb bud. Several FGFs are expressed by the AER and appear to mediate its outgrowth and patterning effects on the subridge mesoderm. IGF-I is expressed by the subridge mesoderm that is growing out in response to the AER, and exogenous IGF-I can promote the outgrowth of the subridge mesoderm in the absence of the AER. Maintenance of IGF-I expression by the subridge mesoderm is dependent on the AER. Here we report that exogenous FGF-2 and FGF-4 can substitute for the AER in maintaining IGF-I expression by the posterior subridge mesoderm of the chick limb bud. Furthermore, we have found that neutralizing antibodies against IGF-I inhibit the ability of FGFs to promote the outgrowth and proliferation of the posterior subridge mesoderm cultured in the absence of the AER. These results indicate that FGFs secreted by the AER maintain IGF-I expression by the subridge mesoderm, and that the ability of FGFs to promote the outgrowth and proliferation of limb mesoderm is dependent on IGF-I activity. Interestingly, however, the ability of FGFs to maintain the expression of the homeobox containing gene Msx-1 by the subridge mesoderm is not dependent on IGF-I activity.

The chicken mutant talpid3 (ta3) has polydactylous limbs with up to 7-8 morphologically similar digits. This lack of antero-posterior polarity in digit pattern is correlated with symmetrical expression of genes of the HoxD complex. We determined the distribution of polarizing activity in limb buds of the chick mutant ta3 by assessing the ability of mesenchyme from various positions along the antero-posterior axis to induce digit duplications when grafted anteriorly into a normal limb. Cells with highest polarizing activity were found at the posterior margin of the wing as in the polarizing region of normal limb buds. However, in contrast to normal limb buds, ta3 anterior mesenchyme also had low polarizing activity. Application of retinoic acid or a polarizing region graft to the anterior of ta3 limb buds changed digit morphology but did not induce digit duplications or digits with any characteristic a-p pattern. To determine which genes are associated with polarizing activity and which are associated with patterning of the digits, we examined expression of the genes Sonic hedgehog (shh), Bmp-2, and Bmp-7, whose expression is normally confined to the posterior margin of the early wing bud and is associated with the polarizing region. In addition, we determined the distribution of Fgf-4 transcripts which in normal limb buds are restricted to the posterior part of the apical ectodermal ridge. In ta3 limb buds, shh expression is restricted to the posterior limb mesenchyme, which has high polarizing activity, but is not expressed in regions which have low polarizing activity. In contrast, Bmp-2 and Bmp-7 are expressed uniformly along the a-p axis. Fgf-4 transcripts are present throughout the apical ectodermal ridge in ta3 limb buds. In the ta3 mutant, there is both an abnormal distribution of signalling activity and response to polarizing signals. In addition, the dissociation between the expression of shh and Bmps suggests distinct roles for the encoded molecules in signalling and response in a-p patterning of limb buds.

We developed and characterized antibodies specific for FGF-2 and used them to locate FGF-2 during chick embryo development. A series of micrographs demonstrated the progression of FGF-2 staining during development of the different tissues and organs. FGF-2 was present in the ectoderm covering the entire embryo, muscle cells, nervous system, neural crest cells, and mesonephros. FGF-2 was also present in the limb from initiation of budding through differentiation. The limb ectoderm and subjacent mesoderm showed the strongest immunostaining, with lower levels in the center of the bud. However, the distribution of FGF-2 positive cells in the mesoderm was not homogeneous. This heterogeneity was not due to cell cycle specific distribution of FGF-2 protein, as flow cytometric analysis showed that FGF-2-positive cells were distributed throughout the cell cycle. However, the amount of anti-FGF-2 fluorescence varied most during G1, consistent with the possibility that FGF-2 is low after M phase and increases during G1. A bioassay was used to demonstrate FGF-2 levels in the wing ectoderm were approximately 2.7-fold greater than in the mesoderm. We propose that the location of FGF-2 in the embryo is consistent with a role in epithelial-mesenchymal interactions; in the limb bud it may prevent differentiation and permit limb outgrowth and subsequent expression of patterning events.

We have studied the expression patterns of the chick homeobox-containing genes, GHox-7 and GHox-8, in the talpid2 (ta2) chick mutant whose limbs have abnormal pattern. These studies provide new insight into how homeobox gene expression and limb patterning may be related. This is the first study demonstrating a natural change in GHox-7 and GHox-8 along the anteroposterior axis. While GHox-7 is expressed asymmetrically in normal limb buds, it is expressed at a uniform level across the anteroposterior axis of ta2 limb buds. GHox-8 is expressed in anterior mesoderm of normal limb buds, but is un-detectable in ta2 limb bud mesoderm. These data are consistent with the subtle anteroposterior polarity in ta2 limbs, and allow us to propose that ta2 limb buds lack anterior positional information, but have a narrow range of posterior positional values. We suggest that in normal limb buds GHox-8 may establish the anterior limb bud boundary. Furthermore, we point out that coexpression of GHox-7 and GHox-8 in normal anterior limb bud mesoderm can be correlated with the reduced apical ridge maintenance activity of this tissue, while the lack of coexpression in ta2 limb buds is correlated with the strong ridge maintenance activity in the mutant's anterior limb bud mesoderm. Last, ta2 limbs contain no dying cells in their anterior and posterior border mesoderm; nevertheless, they express GHox-7 in these regions. These data challenge the proposal that this gene determines cell death. © 1992 Wiley-Liss, Inc.

Homeobox-containing genes have been implicated in a variety of patterning events during vertebrate limb development. In an attempt to isolate cDNAs corresponding to 5′ members of the chicken HOX 4 cluster of homeobox-containing genes, a cDNA library constructed from mRNAs expressed during early stages of chick limb development was screened with probes generated by the polymerase chain reaction (PCR) using oligonucleotide primers corresponding to sequences in the homeoboxes of the human HOX4C and HOX4F genes, the human homologs of Hox-4.4 and Hox-4.6. This screening resulted in the isolation of full length cDNAs for the chicken homolog of HOX4F (cognate of mouse Hox-4.6), which we have termed GHox-4.6, and the chicken homolog of human HOX1I, which we have named GHox-1i, a paralog of Hox-4.6 in the HOX 1 cluster. The homeodomains encoded by GHox-4.6 and GHox- li differ by only three amino acids, and the two proteins show extensive similarity along their entire lengths. Despite their sequence similarity, in situ hybridization analysis has revealed that GHox-4.6 and GHox-li exhibit strikingly different spatial patterns of expression during embryonic chick limb development. At early stages of limb development (stages 20–22), GHox-4.6 transcripts are present in high amounts throughout the posterior half of the limb mesoderm and are absent from the anterior half of the mesoderm, an expression pattern consistent with the possible involvement of GHox-4.6 in the specification of posterior positional identity. In contrast, GHox-li exhibits no distinct anterior-posterior polarity of expression at stage 22, but rather is expressed in high amounts throughout the mesenchyme of the limb bud. At later stages of development (stage 25), GHox-li continues to be expressed in high amounts throughout the undifferentiated mesenchyme subjacent to the apical ectodermal ridge, and, in addition, is expressed in the mesodermal cells in the proximal peripheral regions of the limb bud subjacent to the ectoderm which are differentiating into nonchondrogenic lineages. Conversely, little or no expression of GHox-li is detectable in the proximal central core of the limb bud where chondrogenic differentiation is occur-ring. Thus, GHox-li is expressed by the undifferentiated subridge mesenchymal cells and proximal peripheral mesenchymal cells of the limb bud that are being inhibited from undergoing chondrogenesis by the apical ectodermal ridge and nonridge ectoderm. These observations suggest the possibility that GHox-li may be involved in mediating the antichondrogenic effect of limb ectoderm, and that the cessation of GHox-li expression may be required for chondrogenic differentiation to occur in the proximal central core of the limb bud.