Evidence that the limb bud ectoderm is required for survival of the underlying mesoderm

The limb forms from a bud of mesoderm encased in a hull of ectoderm that grows out from the flank of the embryo. Coordinated signaling between the limb mesoderm and ectoderm is critical for normal limb outgrowth and patterning. The apical ectodermal ridge (AER), found at the distal tip, is a rich source of signaling molecules and has been proposed to specify distal structures and maintain the survival of cells in the underlying distal mesoderm. The dorsal and ventral non-AER ectoderm is also a source of signaling molecules and is important for dorsal–ventral patterning of the limb bud. Here we determine if this ectoderm provides cell survival signals by surgically removing the dorsal or ventral ectoderm during early chicken limb bud development and assaying for programmed cell death. We find that, similar to the AER, removal of the dorsal or ventral non-AER ectoderm results in massive cell death in the underlying mesoderm. In addition, although a re-epithelialization occurs, we find perturbations in the timing of Shh expression and, for the case of the dorsal ectoderm removal, defects in soft tissue and skeletal development along the proximal–distal axis. Furthermore, ectoderm substitution experiments show that the survival signal produced by the dorsal limb ectoderm is specific. Thus, our results argue that the non-AER ectoderm, like the AER, provides a specific survival signal to the underlying mesoderm that is necessary for normal limb development and conclusions drawn from experiments in which the non-AER ectoderm is removed, need to take into consideration this observation.     

Manifestation of the limb prepattern: limb development in the absence of sonic hedgehog function

The secreted protein encoded by the Sonic hedgehog (Shh) gene is localized to the posterior margin of vertebrate limb buds and is thought to be a key signal in establishing anterior-posterior limb polarity. In the Shh(-/-) mutant mouse, the development of many embryonic structures, including the limb, is severely compromised. In this study, we report the analysis of Shh(-/-) mutant limbs in detail. Each mutant embryo has four limbs with recognizable humerus/femur bones that have anterior-posterior polarity. Distal to the elbow/knee joints, skeletal elements representing the zeugopod form but lack identifiable anterior-posterior polarity. Therefore, Shh specifically becomes necessary for normal limb development at or just distal to the stylopod/zeugopod junction (elbow/knee joints) during mouse limb development. The forelimb autopod is represented by a single distal cartilage element, while the hindlimb autopod is invariably composed of a single digit with well-formed interphalangeal joints and a dorsal nail bed at the terminal phalanx. Analysis of GDF5 and Hoxd11-13 expression in the hindlimb autopod suggests that the forming digit has a digit-one identity. This finding is corroborated by the formation of only two phalangeal elements which are unique to digit one on the foot. The apical ectodermal ridge (AER) is induced in the Shh(-/-) mutant buds with relatively normal morphology. We report that the architecture of the Shh(-/-) AER is gradually disrupted over developmental time in parallel with a reduction of Fgf8 expression in the ridge. Concomitantly, abnormal cell death in the Shh(-/-) limb bud occurs in the anterior mesenchyme of both fore- and hindlimb. It is notable that the AER changes and mesodermal cell death occur earlier in the Shh(-/-) forelimb than the hindlimb bud. This provides an explanation for the hindlimb-specific competence to form autopodial structures in the mutant. Finally, unlike the wild-type mouse limb bud, the Shh(-/-) mutant posterior limb bud mesoderm does not cause digit duplications when grafted to the anterior border of chick limb buds, and therefore lacks polarizing activity. We propose that a prepattern exists in the limb field for the three axes of the emerging limb bud as well as specific limb skeletal elements. According to this model, the limb bud signaling centers, including the zone of polarizing activity (ZPA) acting through Shh, are required to elaborate upon the axial information provided by the native limb field prepattern.     

Retinoic acid synthesis controlled by Raldh2 is required early for limb bud initiation and then later as a proximodistal signal during apical ectodermal ridge formation

We present evidence for the existence of two phases of retinoic acid (RA) signaling required for vertebrate limb development. Limb RA synthesis is under the control of retinaldehyde dehydrogenase-2 (Raldh2) expressed in the lateral plate mesoderm, which generates a proximodistal RA signal during limb outgrowth. We report that Raldh2(-/-) embryos lack trunk mesodermal RA activity and fail to initiate forelimb development. This is associated with deficient expression of important limb determinants Tbx5, Meis2, and dHand needed to establish forelimb bud initiation, proximal identity, and the zone of polarizing activity (ZPA), respectively. Limb expression of these genes can be rescued by maternal RA treatment limited to embryonic day 8 (E8) during limb field establishment, but the mutant forelimbs obtained at E10 display a significant growth defect associated with a smaller apical ectodermal ridge (AER), referred to here as an apical ectodermal mound (AEM). In these RA-deficient forelimbs, a ZPA expressing Shh forms, but it is located distally adjacent to the Fgf8 expression domain in the AEM rather than posteriorly as is normal. AER formation in Raldh2(-/-) forelimbs is rescued by continuous RA treatment through E10, which restores RA to distal ectoderm fated to become the AER. Our findings indicate the existence of an early phase of RA signaling acting upstream of Tbx5, Meis2, and dHand, followed by a late phase of RA signaling needed to expand AER structure fully along the distal ectoderm. During ZPA formation, RA acts early to activate expression of dHand, but it is not required later for Shh activation.     

The mouse polydactylous mutation,luxate (lx), causes anterior shift of the anteroposterior border in the developing hindlimb bud

Pattern formation along the anterior-posterior axis of the vertebrate limb is established upon activation of Sonic Hedgehog (SHH) in the zone of polarizing activity (ZPA). Since many mouse mutants with preaxial polydactyly show ectopic expression of Shh at the anterior margin of the limb buds, it has been thought to be a primary defect caused by these mutations. We show here that the mouse mutation luxate (lx) exhibits dose-dependent reduction in the size of the Fgf8 expression domain in the ectoderm from the initial stage of limb development. This aberration was independent of Fgf10 expression in the limb mesenchyme. Shh was induced in the mesenchyme underlying the posterior end of the Fgf8 expression domain, indicating an anterior shift of Shh expression in lx hindlimb buds. Prior to the ectopic induction of Shh, the expression domains of genes downstream from Shh, namely dHAND, Gli1, Ptc and Gre, which are normally expressed in posterior mesenchyme of limb buds, expanded anteriorly on the lx hindlimb buds. Conversely, the expression domains of anterior mesenchymal markers such as Gli3and Alx4 decreased in size. Thus, ectopic Shh is not a primary defect of the lx mutation. Rather, our results indicate that the lx mutation affects the positioning of the anteroposterior border in developing hindlimb buds.     

The chick oligozeugodactyly (ozd) mutant lacks sonic hedgehog function in the limb

We have analyzed a new limb mutant in the chicken that we name oligozeugodactyly (ozd). The limbs of this mutant have a longitudinal postaxial defect, lacking the posterior element in the zeugopod (ulna/fibula) and all digits except digit 1 in the leg. Classical recombination experiments show that the limb mesoderm is the defective tissue layer in ozd limb buds. Molecular analysis revealed that the ozd limbs develop in the absence of Shh expression, while all other organs express Shh and develop normally. Neither Ptc1 nor Gli1 are detectable in mutant limb buds. However, Bmp2 and dHAND are expressed in the posterior wing and leg bud mesoderm, although at lower levels than in normal embryos. Activation of Hoxd11-13 occurs normally in ozd limbs but progressively declines with time. Phase III of expression is more affected than phase II, and expression is more severely affected in the more 5' genes. Interestingly, re-expression of Hoxd13 occurs at late stages in the distal mesoderm of ozd leg buds, correlating with formation of digit 1. Fgf8 and Fgf4 expression are initiated normally in the mutant AER but their expression is progressively downregulated in the anterior AER. Recombinant Shh protein or ZPA grafts restore normal pattern to ozd limbs; however, retinoic acid fails to induce Shh in ozd limb mesoderm. We conclude that Shh function is required for limb development distal to the elbow/knee joints, similar to the Shh(-/-) mouse. Accordingly we classify the limb skeletal elements as Shh dependent or independent, with the ulna/fibula and digits other than digit 1 in the leg being Shh dependent. Finally we propose that the ozd mutation is most likely a defect in a regulatory element that controls limb-specific expression of Shh.     

Differential cell affinity and sorting of anterior and posterior cells during outgrowth of recombinant avian limb buds

To examine the role of position-specific differences in cell-cell affinity, recombinant limb buds composed of dissociated and reaggregated cells derived from anterior (A) and posterior (P) limb bud fragments were analyzed. Dissociated anterior and/or posterior cells were differentially labeled, and their behavior was analyzed during recombinant limb bud outgrowth. We find that anterior and posterior cells sort out from one another to form alternating anterior and posterior stripes of cells that extend distally along the proximal-distal axis. These alternating stripes are prominent across the A/P axis in whole-mount preparations of recombinant limb buds after 48 h of outgrowth when the presumptive autopod is dorsal-ventrally flattened and digit rudiments are not evident. After 96 h, when digital and interdigital regions are clearly defined, we find evidence that A/P stripes do not follow obvious anatomical boundaries. The formation of A/P stripes is not inhibited by grafts of ZPA tissue, suggesting that polarizing activity does not influence cell-cell affinity early in limb outgrowth. In vitro studies provide evidence that cell sorting is not dependent on the limb bud ectoderm or the AER; however, cells sort out without organizing into stripes. Gene expression studies using anterior-specific (Alx-4) and posterior-specific (Shh, Bmp-2, and Hoxd-13) marker genes failed to reveal expression domains that corresponded to stripe formation. Control recombinant limb buds composed of anterior, central, or posterior mesenchyme formed digits in a position-specific manner. A/P recombinant limb buds that develop to later stages form digits that are characteristic of central recombinant limbs. These data provide the first definitive evidence of A/P cell sorting during limb outgrowth in vivo and suggest that differential cell affinities play a role in modulating cell behavior during distal outgrowth.     

FGF7 and FGF10 directly induce the apical ectodermal ridge in chick embryos.

During vertebrate limb development, the apical ectodermal ridge (AER) plays a vital role in both limb initiation and distal outgrowth of the limb bud. In the early chick embryo the prelimb bud mesoderm induces the AER in the overlying ectoderm. However, the direct inducer of the AER remains unknown. Here we report that FGF7 and FGF10, members of the fibroblast growth factor family, are the best candidates for the direct inducer of the AER. FGF7 induces an ectopic AER in the flank ectoderm of the chick embryo in a different manner from FGF1, -2, and -4 and activates the expression of Fgf8, an AER marker gene, in a cultured flank ectoderm without the mesoderm. Remarkably, FGF7 and FGF10 applied in the back induced an ectopic AER in the dorsal median ectoderm. Our results suggest that FGF7 and FGF10 directly induce the AER in the ectoderm both of the flank and of the dorsal midline and that these two regions have the competence for AER induction. Formation of the AER of the dorsal median ectoderm in the chick embryo is likely to appear as a vestige of the dorsal fin of the ancestors.     

Tbx18 and boundary formation in chick somite and wing development

The chicken Tbx gene, Tbx18, is expressed in lateral plate mesoderm, limb, and developing somites. Here we show that Tbx18 is expressed transiently in axial mesenchyme during somite segmentation. We present evidence from overexpression and transplantation experiments that Tbx18 controls fissure formation in the late stages of somite maturation. In presumptive wing lateral plate mesoderm, ectopic Tbx18 expression leads to anterior extension of the wing bud. These results suggest that Tbx18 is involved in producing mesodermal boundaries, generating in paraxial mesoderm morphological boundaries between somites and in lateral plate mesoderm a wing- or non-wing-forming boundary.     

Level-specific role of paraxial mesoderm in regulation of Tbx5/Tbx4 expression and limb initiation

Tetrapod limbs, forelimbs and hindlimbs, emerge as limb buds during development from appropriate positions along the rostro-caudal axis of the main body. In this study, tissue interactions by which rostro-caudal level-specific limb initiation is established were analyzed. The limb bud originates from the lateral plate located laterally to the paraxial mesoderm, and we obtained evidence that level-specific tissue interactions between the paraxial mesoderm and the lateral plate mesoderm are important for the determination of the limb-type-specific gene expression and limb outgrowth. When the wing-level paraxial mesoderm was transplanted into the presumptive leg region, the wing-level paraxial mesoderm upregulated the expression of Tbx5, a wing marker gene, and down regulated the expression of Tbx4 and Pitx1, leg marker genes, in the leg-level lateral plate. The wing-level paraxial mesoderm relocated into the leg level also inhibited outgrowth of the hindlimb bud and down regulated Fgf10 and Fgf8 expression, demonstrating that the wing-level paraxial mesoderm cannot substitute for the function of the leg-level paraxial mesoderm in initiation and outgrowth of the hindlimb. The paraxial mesoderm taken from the neck- and flank-level regions also had effects on Tbx5/Tbx4 expression with different efficiencies. These findings suggest that the paraxial mesoderm has level-specific abilities along the rostro-caudal axis in the limb-type-specific mechanism for limb initiation.     

Targeted misexpression of constitutively active BMP receptor-IB causes bifurcation, duplication, and posterior transformation of digit in mouse limb

Members of bone morphogenetic proteins (BMPs) play important roles in many aspects of vertebrate embryogenesis. In developing limbs, BMPs have been implicated in control of anterior-posterior patterning, outgrowth, chondrogenesis, and apoptosis. These diverse roles of BMPs in limb development are apparently mediated by different BMP receptors (BMPR). To identify the developmental processes in mouse limb possibly contributed by BMP receptor-IB (BMPR-IB), we generated transgenic mice misexpressing a constitutively active Bmpr-IB (caBmpr-IB). The transgene driven by the mouse Hoxb-6 promoter was ectopically expressed in the posterior mesenchyme of the forelimb bud, the lateral plate mesoderm, and the whole mesenchyme of the hindlimb bud. While the forelimbs appeared normal, the transgenic hindlimbs exhibited several phenotypes, including bifurcation, preaxial polydactyly, and posterior transformation of the anterior digit. However, the size of bones in the transgenic limbs seemed unaltered. Defects in sternum and ribs were also found. The bifurcation in the transgenic hindlimb occurred early in the limb development (E10.5) and was associated with extensive cell death in the mesenchyme and occasionally in the apical ectodermal ridge (AER). Sonic hedgehog (Shh) and Patched (Ptc) expression appeared unaffected in the transgenic limb buds, suggesting that the BMPR-IB mediated signaling pathway is downstream from Shh. However, ectopic Fgf4 expression was found in the anterior AER, which may account for the duplication of the anterior digit. An ectopic expression of Gremlin found in the transgenic limb bud would be responsible for the ectopic Fgf4 expression. The observations that Hoxd-12 and Hoxd-13 expression patterns were extended anteriorly provide a molecular basis for the posterior transformation of the anterior digit. Together these results suggest that BMPR-IB is the endogenous receptor to mediate the role of BMPs in anterior-posterior patterning and apoptosis in mouse developing limb. In addition, BMPR-IB may represent a critical component in the Shh/FGF4 feedback loop by regulating Gremlin expression.     

Some distal limb structures develop in mice lacking Sonic hedgehog signaling

Patterning of the limb is coordinated by the complex interplay of three signaling regions: the apical ectodermal ridge (AER), the zone of polarizing activity (ZPA), and the non-ridge limb ectoderm. Complex feedback loops exist between Shh in the ZPA, Bmps and their antagonists in the adjacent mesenchyme, Wnt7a in the dorsal ectoderm and Fgfs in the AER. In contrast to the previously reported complete absence of digits in Shh(-/-) mice, we show that one morphologically distinct digit, with a well-delineated nail and phalanges, forms in Shh(-/-) hindlimbs, while intermediate structures are severely truncated and fused. The presence of distal autopod elements is consistent with weak expression of Hoxd13 in Shh(-/-) hindlimbs. Shh(-/-) forelimbs in contrast have one distal cartilage element, a less-well differentiated nail and fused intermediate bones. Interestingly, Ihh is expressed at the tip of Shh mutant limbs and could account for formation of distal structures. In contrast to previous studies we also demonstrate that Shh signaling is required for maintenance of normal Fgf8 expression, since expression of Fgf8, unlike some other AER marker genes, is rapidly lost from anterior to posterior after E10.5, with only a small domain of Fgf8 expression remaining posteriorly. Furthermore, loss of expanded Fgf8 expression is paralleled by a collapse of the handplate. Our data show that development of most intermediate elements of the hindlimb skeleton are Shh-dependent, and that Shh signaling is required for anterior-posterior expansion of the AER in both limbs and for the subsequent branching of zeugopod and autopod elements. Finally, we show that Shh is also required for outgrowth of the limb ectoderm and thus for the formation of a distinct limb compartment.     

The dominant hemimelia mutation uncouples epithelial-mesenchymal interactions and disrupts anterior mesenchyme formation in mouse hindlimbs.

Epithelial-mesenchymal interactions are essential for both limb outgrowth and pattern formation in the limb. Molecules capable of communication between these two tissues are known and include the signaling molecules SHH and FGF4, FGF8 and FGF10. Evidence suggests that the pattern and maintenance of expression of these genes are dependent on a number of factors including regulatory loops between genes expressed in the AER and those in the underlying mesenchyme. We show here that the mouse mutation dominant hemimelia (Dh) alters the pattern of gene expression in the AER such that Fgf4, which is normally expressed in a posterior domain, and Fgf8, which is expressed throughout are expressed in anterior patterns. We show that maintenance of Shh expression in the posterior mesenchyme is not dependent on either expression of Fgf4 or normal levels of Fgf8 in the overlying AER. Conversely, AER expression of Fgf4 is not directly dependent on Shh expression. Also the reciprocal regulatory loop proposed for Fgf8 in the AER and Fgf10 in the underlying mesenchyme is also uncoupled by this mutation. Early during the process of limb initiation, Dh is involved in regulating the width of the limb bud, the mutation resulting in selective loss of anterior mesenchyme. The Dh gene functions in the initial stages of limb development and we suggest that these initial roles are linked to mechanisms that pattern gene expression in the AER.     

Bmp4 in limb bud mesoderm regulates digit pattern by controlling AER development

In the developing limb, Bmp4 is expressed in the apical ectodermal ridge (AER) and underlying mesoderm. Insight into the function of Bmp4 in limb development has been hampered by the early embryonic lethality of Bmp4 null embryos. We directly investigated Bmp4 using a conditional null allele of Bmp4 and the Prx1(cre) transgene to inactivate Bmp4 in limb bud mesoderm. The limb bud mesoderm of Prx1(cre);Bmp4 mutants was defective in production of Bmp4 but still competent to respond to Bmp signaling. Prx1(cre);Bmp4 mutant embryos had defective digit patterning including hindlimb preaxial polydactyly with posterior digit transformations. The Prx1(cre);Bmp4 mutants also had postaxial polydactyly with digit five duplications. Bmp4 mutant limbs had delayed induction and maturation of the AER that resulted in expanded Shh signaling. Moreover, the AER persisted longer in the Bmp4 mutant limb buds exposing the forming digits to prolonged Fgf8 signaling. Our data show that Bmp4 in limb mesoderm regulates AER induction and maturation and implicate signaling from the AER in regulation of digit number and identity.     

Cooperative Activation of HoxD Homeobox Genes by Factors from the Polarizing Region and the Apical Ridge in Chick Limb Morphogenesis

When a mouse zone of polarizing activity (ZPA) at the posterior margin of the limb bud was grafted into the anterior margin of the chick limb bud, the expressions of the chick homeobox genes HoxD12 and D13 were induced prior to the formation of chick extra digits. This induction was observed in a restricted domain close to both the grafted mouse ZPA and the chick apical ectodermal ridge (AER). When the posterior half of the AER was removed, the normal expression was diminished in the distaloposterior region. Thus, it is likely that at least two distinct factors, one from the ZPA and the other from the AER, act cooperatively to provide positional information to induce the sequential expression of the HoxD genes.