Bmp2, Bmp4 and Bmp7 are co-required in the mouse AER for normal digit patterning but not limb outgrowth

Outgrowth and patterning of the vertebrate limb requires a functional apical ectodermal ridge (AER). The AER is a thickening of ectodermal tissue located at the distal end of the limb bud. Loss of this structure, either through genetic or physical manipulations results in truncation of the limb. A number of genes, including Bmps, are expressed in the AER. Previously, it was shown that removal of the BMP receptor Bmpr1a specifically from the AER resulted in complete loss of hindlimbs suggesting that Bmp signaling in the AER is required for limb outgrowth. In this report, we genetically removed the three known AER-expressed Bmp ligands, Bmp2, Bmp4 and Bmp7 from the AER of the limb bud using floxed conditional alleles and the Msx2-cre allele. Surprisingly, only defects in digit patterning and not limb outgrowth were observed. In triple mutants, the anterior and posterior AER was present but loss of the central region of the AER was observed. These data suggest that Bmp ligands expressed in the AER are not required for limb outgrowth but instead play an essential role in maintaining the AER and patterning vertebrate digits.     

Doubleridge,a mouse mutant with defective compaction of the apical ectodermal ridge and normal dorsal-ventral patterning of the limb

doubleridge is a transgene-induced mutation characterized by polydactyly and syndactyly of the forelimbs. The transgene insertion maps to the proximal region of chromosome 19. During embryonic development of the mutant forelimb, delayed elevation and compaction of the apical ectodermal ridge (AER) produces a ridge that is abnormally broad and flat. Fgf8 expression persists in the ventral forelimb ectoderm of the mutant until E10.5. Strong expression of Fgf8 and other markers at the borders of the AER at E11.5 gives the appearance of a double ridge. At E11.5, apoptotic cells are distributed across the broadened ridge, but at E13.5, there is reduced apoptosis in the interdigital regions. The Shh expression domain is widely spaced at the posterior margin of the AER. The doubleridge AER is morphologically similar to that of En1 null mice, but the expression of En1 and Wnt7a is properly restricted in doubleridge, and the dorsal and ventral structures are correctly determined. doubleridge thus exhibits an unusual limb phenotype combining abnormal compaction of the AER with normal dorsal/ventral patterning.     

Essential mesenchymal role of small GTPase Rac1 in interdigital programmed cell death during limb development

Developing vertebrate limbs are often utilized as a model for studying pattern formation and morphogenetic cell death. Herein, we report that conditional deletion of Rac1, a member of the Rho family of proteins, in mouse limb bud mesenchyme led to skeletal deformities in the autopod and soft tissue syndactyly, with the latter caused by a complete absence of interdigital programmed cell death. Furthermore, the lack of interdigital programmed cell death and associated syndactyly was related to down-regulated gene expression of Bmp2, Bmp7, Msx1, and Msx2, which are known to promote apoptosis in the interdigital mesenchyme. Our findings from Rac1 conditional mutants indicate crucial roles for Rac1 in limb bud morphogenesis, especially interdigital programmed cell death.     

The apical ectodermal ridge (AER) can be re-induced by wounding, wnt-2b, and fgf-10 in the chicken limb bud

Little effort has been made to apply the insights gained from studies of amphibian limb regeneration to higher vertebrates. During amphibian limb regeneration, a functional epithelium called the apical ectodermal cap (AEC) triggers a regenerative response. As long as the AEC is induced, limb regeneration will take place. Interestingly, similar responses have been observed in chicken embryos. The AEC is an equivalent structure to the apical ectodermal ridge (AER) in higher vertebrates. When a limb bud is amputated it does not regenerate; however, if the AER is grafted onto the amputation surface, damage to the amputated limb bud can be repaired. Thus, the AER/AEC is able to induce regenerative responses in both amphibians and higher vertebrates. It is difficult, however, to induce limb regeneration in higher vertebrates. One reason for this is that re-induction of the AER after amputation in higher vertebrates is challenging. Here, we evaluated whether AER re-induction was possible in higher vertebrates. First, we assessed the sequence of events following limb amputation in chick embryos and compared the features of limb development and regeneration in amphibians and chicks. Based on our findings, we attempted to re-induce the AER. When wnt-2b/fgf-10-expressing cells were inserted concurrently with wounding, successful re-induction of the AER occurred. These results open up new possibilities for limb regeneration in higher vertebrates since AER re-induction, which is considered a key factor in limb regeneration, is now possible.     

Extended exposure to Sonic hedgehog is required for patterning the posterior digits of the vertebrate limb

Sonic hedgehog (Shh) is a key signal in establishing different digit fates along the anterior-posterior axis of the vertebrate limb bud. Although the anterior digits appear to be specified by differential concentrations of Shh in a traditional, morphogen-like response, recent studies have suggested that posterior digits are specified by an extended time of exposure to Shh rather than, or in addition to, a threshold concentration of Shh. This model for digit patterning depends upon continued Shh signaling in the posterior limb through mid-to-late bud stages. We find that cyclopamine, a potent antagonist of Shh signaling, can down-regulate hedgehog target genes in the posterior limb throughout the time Shh is expressed, indicating that continued active Shh signaling indeed takes place. To further explore the relative roles of time and concentration of Shh during limb development, we carried out two additional series of experiments. To test the effect of limiting the time, but not the amount of Shh produced, we treated chick embryos with the hedgehog antagonist cyclopamine at various stages of limb development. We find that short exposures to Shh result in specification of only the most anterior digits and that more posterior digits are specified sequentially with increasing times of uninterrupted Shh activity. To test the effect of limiting the level of Shh produced, but not the time of exposure, we genetically modified Shh production in mice. As previously shown, reducing both the concentration of Shh produced and the duration of Shh exposure results in a loss of posterior digits. We find that maintaining a low level of Shh production throughout the normal time frame of ZPA signaling results in a near complete restoration of the posterior-most digits. These data are consistent with, and lend additional support to, the model that concentration of Shh seen and duration of exposure both contribute to the dose-dependent specification of digit identities, but for the posterior-most digits the temporal component is the more critical parameter.     

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.     

The recombinant limb as a model for the study of limb patterning, and its application to muscle development

The recombinant limb is a model system that has proved fruitful for analyzing epithelial-mesenchymal interactions and understanding the functional properties of the components of the limb bud. Here we present an overview of some of the insights obtained through the use of this technique. Among these are the understanding that fore or hind limb identity is inherent to the limb bud mesoderm, that the apical ectodermal ridge (AER) is a permissive signaling center and that the limb bud ectoderm plays a central role in the control of dorsoventral polarity. Recombinant limb studies have also allowed the identification of the affected tissue component in several limb mutants. More recently this model has been applied to the study of regulation of gene expressions related to patterning. In this report we use recombinant limbs to analyze pattering of the Pax3 expressing limb muscle cell lineage in the early stages of limb development. In recombinant limbs made without the zone of polarizing activity (ZPA), myoblasts appear intermingled with other mesodermal cells at the beginning of the recombinant limb development. Rapidly thereafter, the muscle precursors segregate and organize around the central forming chondrogenic core of the recombinant. Although this segregation is reminiscent of that occurring during normal development, the myoblasts in the recombinant fail to proliferate appropriately and also fail to migrate distally. Consequently, the muscle pattern in the recombinant limb is defective indicating that normal patterning cues are absent. However, recombinant limbs polarized with a ZPA exhibited a larger mass of muscle cells and a more normal morphogenesis, supporting a role for this signaling center in limb muscle development. Finally, we have ruled out host somite contributions to recombinant limbs by grafting chick recombinant limbs to quail hosts. This initial report demonstrates the value of the recombinant limb model system for dissecting the environmental cues required for normal muscle limb patterning. Received: 31 August 1998 / Accepted: 29 September 1998     

Smad2/3 activities are required for induction and patterning of the neuroectoderm in zebrafish

Smad2 and Smad3, two essential nuclear effectors of transforming growth factor (Tgf)-β signals, have been found to be implicated in mesoderm and endoderm development in vertebrate embryos. However, their roles in the induction and patterning of the neuroectoderm are not well established. In this study, we show that interference with Smad2/3 activities in zebrafish embryos, by injecting dnsmad3b mRNA encoding a dominant negative Smad3b mutant, inhibits the expression of the early neural markers sox2 and sox3 at the onset of gastrulation and results in reduction of the anterior neuroectodermal marker otx2 as well as the posterior neuroectodermal marker hoxb1b during late gastrulation, suggesting a role of Smad2/3 activities in neural induction. Conversely, excess Smad2/3 activities, caused by injecting smad3b mRNA, lead to an enhancement of sox2 and sox3 expression in the ventral domains but an inhibition of their expression in the dorsalmost region at early stages. Overexpression of smad3b also causes ventral expansion of the otx2 and hoxb1b expression domains accompanied with rostral shift of the hoxb1b domain at late gastrulation stages. Collectively, these data indicate that Smad2/3 activities are required for neural induction and neuroectodermal posteriorization in zebrafish. Knockdown of chordin partially inhibits effect of smad3b overexpression on neural induction, implying that Smad2/3 exert their effect on neural induction in part by regulating the expression of Bmp antagonists. Furthermore, down-regulation or up-regulation of Smad2/3 activities in MZoep mutant embryos, which lack the organizer and mesendodermal tissues due to deficiency of Nodal signaling, still affects induction and patterning of the neuroectoderm, suggesting that Smad2/3 activities are implicated in neural development in the absence of the organizer and mesendodermal tissues. We additionally demonstrate that Smad2/3 activities cooperate with Wnt and Fgf signals in neural development. Thus, Smad2/3 activities play important roles not only in mesendodermal development but also in neural development during early vertebrate embryogenesis.     

Expression and regulation of antioxidant enzymes in the developing limb support a function of ROS in interdigital cell death

Vertebrate limb development is a well-studied model of apoptosis; however, little is known about the intracellular molecules involved in activating the cell death machinery. We have shown that high levels of reactive oxygen species (ROS) are present in the interdigital 'necrotic' tissue of mouse autopod, and that antioxidants can reduce cell death. Here, we determined the expression pattern of several antioxidant enzymes in order to establish their role in defining the areas with high ROS levels. We found that the genes encoding the superoxide dismutases and catalase are expressed in autopod, but they are downregulated in the interdigital regions at the time ROS levels increased and cell death was first detected. The possible role of superoxide and/or peroxide in activating cell death is supported by the protective effect of a superoxide dismutase/catalase mimetic. Interestingly, we found that peroxidase activity and glutathione peroxidase-4 gene (Gpx4) expression were restricted to the non-apoptotic tissue (e.g., digits) of the developing autopod. Induction of cell death with retinoic acid caused an increase in ROS and decrease in peroxidase activity. Even more inhibition of glutathione peroxidase activity leads to cell death in the digits, suggesting that a decrease in antioxidant activity, likely due to Gpx4, caused an increase in ROS levels, thus triggering apoptosis.     

Mouse R-spondin2 is required for apical ectodermal ridge maintenance in the hindlimb

The R-spondin (Rspo) family of proteins consists of secreted cysteine-rich proteins that can activate β-catenin signaling via the Frizzled/LRP5/6 receptor complex. Here, we report that targeted inactivation of the mouse Rspo2 gene causes developmental limb defects, especially in the hindlimb. Although the initiation of the expression of apical ectodermal ridge (AER)-specific genes, including fibroblast growth factor 8 (FGF8) and FGF4 occurred normally, the maintenance of these marker expressions was significantly defective in the hindlimb of Rspo2(−/−) mice. Consistent with the ligand role of R-spondins in the Wnt/β-catenin signaling pathway, expression of Axin2 and Sp8, targets for β-catenin signaling, within AER was greatly reduced in Rspo2(−/−) embryos. Furthermore, sonic hedgehog (Shh) signaling within the hindlimbs of Rspo2(−/−) mice was also significantly decreased. Rspo2 is expressed in the AER of all limb buds, however the stunted phenotype is significantly more severe in the hindlimbs than the forelimbs and strongly biased to the left side. Our findings strongly suggest that Rspo2 expression in the AER is required for AER maintenance likely by regulating Wnt/β-catenin signaling.     

Ganglion cells are required for normal progenitor- cell proliferation but not cell-fate determination or patterning in the developing mouse retina

The vertebrate retina develops from an amorphous sheet of dividing retinal progenitor cells (RPCs) through a sequential process that culminates in an exquisitely patterned neural tissue. A current model for retinal development posits that sequential cell-type differentiation is the result of changes in the intrinsic competence state of multipotent RPCs as they advance in time and that the intrinsic changes are influenced by continuous changes in the extracellular environment. Although several studies support the proposition that newly differentiated cells alter the extrinsic state of the developing retina, it is still far from clear what role they play in modifying the extracellular environment and in influencing the properties of RPCs. Here, we specifically ablate retinal ganglion cells (RGCs) as they differentiate, and we determine the impact of RGC absence on retinal development. We find that RGCs are not essential for changing the competence of RPCs, but they are necessary for maintaining sufficient numbers of RPCs by regulating cell proliferation via growth factors. Intrinsic rather than extrinsic factors are likely to play the critical roles in determining retinal cell fate.     

R-spondin2 expression in the apical ectodermal ridge is essential for outgrowth and patterning in mouse limb development

Mouse R-spondin2 (Rspo2) is a member of the R-spondin protein family, which is characterized by furin-like cysteine-rich domains and a thrombospondin type 1 repeat. R-spondin is a secreted molecule that activates Wnt/ β -catenin signaling. Rspo2 -deficient mice were generated to investigate the function of mouse Rspo2 during embryonic development. The homozygous mutant forelimb showed defects in distal phalanges and nail structures, and the digits were anomalous in shape. The homozygous mutant hindlimb showed more severe malformations, including lack of digits and zeugopod components. Rspo2 is expressed in the apical ectodermal ridge (AER) of the developing limb. Fgf8 expression in the AER was significantly lower in the homozygous mutant forelimb than in the wild-type forelimb and it was disturbed along the dorsoventral axis. In the homozygous mutant hindlimb, Fgf8 and Fgf4 expression in the posterior AER and Sonic hedgehog expression in the zone of polarizing activity (ZPA) were reduced. The homozygous mutant hindlimb also showed expansion of Wnt7a expression in the dorsal ectoderm toward the ventral side. This study shows that Rspo2 is critical for maintenance of the AER and for growth and patterning in limb development.     

En1 and Wnt7a interact with Dkk1 during limb development in the mouse

Wnt signaling plays an essential role in induction and development of the limb. Missing digits are one consequence of the reduced Wnt signaling in Wnt7a null mice, while extra digits result from excess Wnt signaling in mice null for the Wnt antagonist Dkk1. The extra digits and expanded apical ectodermal ridge (AER) of Dkk1-deficient mice closely resemble En1 null mice. To evaluate the in vivo interaction between En1 and the canonical Wnt signaling pathway, we generated double and triple mutants combining the hypomorphic doubleridge allele of Dkk1 with null alleles of En1 and Wnt7a. Reducing Dkk1 expression in Dkk1d/+Wnt7a-/- double mutants prevented digit loss, indicating that Wnt7a acts through the canonical pathway during limb development. Reducing Dkk1 levels in Dkk1d/dEn1-/- double mutants resulted in severe phenotypes not seen in either single mutant, including fused bones in the autopod, extensive defects of the zeugopod, and loss of the ischial bone. The subsequent elimination of Wnt7a in Dkk1d/dEn1-/-Wnt7a-/- triple mutants resulted in correction of most, but not all, of these defects. The failure of Wnt7a inactivation to completely correct the limb defects of Dkk1d/dEn1-/- double mutants indicates that Wnt7a is not the only gene regulated by En1 during development of the mouse limb.     

Changes in Nkx2.1, Sox2, Bmp4, and Bmp16 expression underlying the lung‐to‐gas bladder evolutionary transition in ray‐finned fishes

The key to understanding the evolutionary origin and modification of phenotypic traits is revealing the responsible underlying developmental genetic mechanisms. An important organismal trait of ray‐finned fishes is the gas bladder, an air‐filled organ that, in most fishes, functions for buoyancy control, and is homologous to the lungs of lobe‐finned fishes. The critical morphological difference between lungs and gas bladders, which otherwise share many characteristics, is the general direction of budding during development. Lungs bud ventrally and the gas bladder buds dorsally from the anterior foregut. We investigated the genetic underpinnings of this ventral‐to‐dorsal shift in budding direction by studying the expression patterns of known lung genes (Nkx2.1, Sox2, and Bmp4) during the development of lungs or gas bladder in three fishes: bichir, bowfin, and zebrafish. Nkx2.1 and Sox2 show reciprocal dorsoventral expression patterns during tetrapod lung development and are important regulators of lung budding; their expression during bichir lung development is conserved. Surprisingly, we find during gas bladder development, Nkx2.1 and Sox2 expression are inconsistent with the hypothesis that they regulate the direction of gas bladder budding. Bmp4 is expressed ventrally during lung development in bichir, akin to the pattern during mouse lung development. During gas bladder development, Bmp4 is not expressed. However, Bmp16, a paralogue of Bmp4, is expressed dorsally in the developing gas bladder of bowfin. Bmp16 is present in the known genomes of Actinopteri (ray‐finned fishes excluding bichir) but absent from mammalian genomes. We hypothesize that Bmp16 was recruited to regulate gas bladder development in the Actinopteri in place of Bmp4.     

ADF/cofilin proteins translocate to mitochondria during apoptosis but are not generally required for cell death signaling

Non-muscle cofilin (n-cofilin) is a member of the ADF/cofilin family of actin depolymerizing proteins. Recent studies reported a mitochondrial translocation of n-cofilin during apoptosis. As these studies also revealed impaired cytochrome c release and a block in apoptosis upon small interfering RNA-mediated n-cofilin knockdown, n-cofilin was postulated to be essential for apoptosis induction. To elucidate the general importance of ADF/cofilin activity for apoptosis, we exposed mouse embryonic fibroblasts deficient for n-cofilin, ADF (actin depolymerizing factor), or all ADF/cofilin isoforms to well-characterized apoptosis inducers. Cytochrome c release, caspase-3 activation, and apoptotic chromatin condensation were unchanged in all mutant fibroblasts. Thus, we conclude that ADF/cofilin activity is not generally required for induction or progression of apoptosis in mammalian cells. Interestingly, mitochondrial association of ADF and n-cofilin during apoptosis was preceded by, and dependent on, actin that translocated by a yet unknown mechanism to mitochondria during cell death.     

Fgfr2 and Fgfr3 are not required for patterning and maintenance of the midbrain and anterior hindbrain

The mid-/hindbrain organizer (MHO) is characterized by the expression of a network of genes, which controls the patterning and development of the prospective midbrain and anterior hindbrain. One key molecule acting at the MHO is the fibroblast growth factor (Fgf) 8. Ectopic expression of Fgf8 induces genes that are normally expressed at the mid-/hindbrain boundary followed by the induction of midbrain and anterior hindbrain structures. Inactivation of the Fgf receptor (Fgfr) 1 gene, which was thought to be the primary transducer of the Fgf8 signal at the MHO, in the mid-/hindbrain region, leads to a deletion of dorsal structures of the mid-/hindbrain region, whereas ventral tissues are less severely affected. This suggests that other Fgfrs might be responsible for ventral mid-/hindbrain region development. Here we report the analysis of Fgfr2 conditional knockout mice, lacking the Fgfr2 in the mid-/hindbrain region and of Fgfr3 knockout mice with respect to the mid-/hindbrain region. In both homozygous mouse mutants, patterning of the mid-/hindbrain region is not altered, neuronal populations develop normal and are maintained into adulthood. This analysis shows that the Fgfr2 and the Fgfr3 on their own are dispensable for the development of the mid-/hindbrain region. We suggest functional redundancy of Fgf receptors in the mid-/hindbrain region.     

Fibroblast growth factor receptor 2-IIIb acts upstream of Shh and Fgf4 and is required for limb bud maintenance but not for the induction of Fgf8, Fgf10, Msx1, or Bmp4

Mice deficient for FgfR2-IIIb were generated by placing translational stop codons and an IRES-LacZ cassette into exon IIIb of FgfR2. Expression of the alternatively spliced receptor isoform, FgfR2-IIIc, was not affected in mice deficient for the IIIb isoform. FgfR2-IIIb(-/-) (lac)(Z) mice survive to term but show dysgenesis of the kidneys, salivary glands, adrenal glands, thymus, pancreas, skin, otic vesicles, glandular stomach, and hair follicles, and agenesis of the lungs, anterior pituitary, thyroid, teeth, and limbs. Detailed analysis of limb development revealed an essential role for FgfR2-IIIb in maintaining the AER. Its absence did not prevent expression of Fgf8, Fgf10, Bmp4, and Msx1, but did prevent induction of Shh and Fgf4, indicating that they are downstream targets of FgfR2-IIIb activation. In the absence of FgfR2-IIIb, extensive apoptosis of the limb bud ectoderm and mesenchyme occurs between E10 and E10.5, providing evidence that Fgfs act primarily as survival factors. We propose that FgfR2-IIIb is not required for limb bud initiation, but is essential for its maintenance and growth.