Constitutive activation of sonic hedgehog signaling in the chicken mutant talpid(2): Shh-independent outgrowth and polarizing activity.

We have examined the developmental properties of the polydactylous chicken mutant, talpid(2). Ptc, Gli1, Bmp2, Hoxd13, and Fgf4 are expressed throughout the anteroposterior axis of the mutant limb bud, despite normal Shh expression. The expression of Gli3, Ihh, and Dhh appears to be normal, suggesting that the Shh signaling pathway is constitutively active in talpid(2) mutants. We show that preaxial talpid(2) limb bud mesoderm has polarizing activity in the absence of detectable Shh mRNA. When the postaxial talpid(2) limb bud (including all Shh-expressing cells) is removed, the preaxial cells reform a normal-shaped talpid(2) limb bud (regulate). However, a Shh-expressing region (zone of polarizing activity) does not reform; nevertheless Fgf4 expression in the apical ectodermal ridge is maintained. Such reformed talpid(2) limb buds develop complete talpid(2) limbs. After similar treatment, normal limb buds downregulate Fgf4, the preaxial cells do not regulate, and a truncated anteroposterior deficient limb forms. In talpid(2) limbs, distal outgrowth is independent of Shh and correlates with Fgf4, but not Fgf8, expression by the apical ectodermal ridge. We propose a model for talpid(2) in which leaky activation of the Shh signaling pathway occurs in the absence of Shh ligand.     

The expression pattern of the chicken homeobox-containing gene GHox-7 in developing polydactylous limb buds suggests its involvement in apical ectodermal ridge-directed outgrowth of limb mesoderm and in programmed cell death

Abstract. The limb buds of the polydactylous mutant embryos, talpid ² and diplopodia -5, possess expanded distal apexes surmounted by prolongated thickened apical ectodermal ridges that promote the outgrowth and formation of digits from both the anterior and posterior mesoderm of the mutant limb buds. The chicken homeobox-containing gene GHox-7 exhibits an expanded domain of expression throughout the expanded subridge mesoderm of the mutant limb buds, providing support for the hypothesis that GHox-7 expression by subridge mesenchymal cells is involved in the outgrowth-promoting effect of the apical ectodermal ridge. During normal limb development GHox-7 is also expressed by the mesoderm in the proximal anterior nonchondrogenic periphery of the limb bud, which includes, but is not limited to the anterior necrotic zone. GHox-7 is also expressed in the posterior necrotic zone at the mid-proximal posterior edge of the limb bud. In contrast, GHox-7 is not expressed in either the proximal anterior or posterior peripheral mesoderm of talpid ² and diplopodia -5 limb buds which lack proximal anterior and posterior necrotic zones. Furthermore, retinoic acid-coated bead implants, which diminish cell death in the anterior necrotic zone, elicit a local inhibition of GHox-7 expression in the proximal anterior peripheral mesoderm. These results support the suggestion that GHox-7 may be involved in defining regions of programmed cell death during limb development. Furthermore, these studies indicate that the distal subridge and proximal anterior nonchondrogenic mesodermal domains of GHox-7 expression are independently regulated.     

Cell movement and adhesion in the developing chick wing bud: studies on cultured mesenchyme cells from normal and talpid mutant embryos.

Mesenchyme fragments from early wing buds of normal and talpid3 mutant chick embryos were explanted for culture in plastic Petri dishes and the behaviour of individual cells as they moved out on to the plastic surface was studied by time-lapse ciné photography, followed by statistical analysis. Two parameters of cell movement were recorded: (1) the distances moved over measured 100-s intervals and (2) the length of time each cell spent at rest before moving on. The average speed of movement over the whole path tracked for each cell, inclusive of time at rest, was significantly greater in normal than talpid3 cells. There was no significant difference between normal and mutant cells in the average distance mover per 100-s step, equivalent to the speed over the whole path exclusive of time at rest, but the percentage of time spent at rest was significantly less in normal than in talpid3 cells. This difference appears to be related to a difference in cell morphology, since it was observed that the mutant cells were more flattened than normals, with very extensive ruffled membranes and short spiky microvilli all round the cell periphery. The relation of these differences in cell morphology and behaviour in vitro to the production of the characteristically fan-shaped limb bud outgrowth and altered pattern of cartilage elements in the developing mutant limb bud is discussed.     

Expression of ptc and gli genes in talpid3 suggests bifurcation in Shh pathway

talpid3 is an embryonic-lethal chicken mutation in a molecularly un-characterised autosomal gene. The recessive, pleiotropic phenotype includes polydactylous limbs with morphologically similar digits. Previous analysis established that hox-D and bmp genes, that are normally expressed posteriorly in the limb bud in response to a localised, posterior source of Sonic Hedgehog (Shh) are expressed symmetrically across the entire anteroposterior axis in talpid3 limb buds. In contrast, Shh expression itself is unaffected. Here we examine expression of patched (ptc), which encodes a component of the Shh receptor, and is probably itself a direct target of Shh signalling, to establish whether talpid3 acts in the Shh pathway. We find that ptc expression is significantly reduced in talpid3 embryos. We also demonstrate that talpid3 function is not required for Shh signal production but is required for normal response to Shh signals, implicating talpid3 in transduction of Shh signals in responding cells. Our analysis of expression of putative components of the Shh pathway, gli1, gli3 and coupTFII shows that genes regulated by Shh are either ectopically expressed or no longer responsive to Shh signals in talpid3 limbs, suggesting possible bifurcation in the Shh pathway. We also describe genetic mapping of gli1, ptc, shh and smoothened in chickens and confirm by co-segregation analysis that none of these genes correspond to talpid3.     

Generation of mice with functional inactivation of talpid3, a gene first identified in chicken

Specification of digit number and identity is central to digit pattern in vertebrate limbs. The classical talpid(3) chicken mutant has many unpatterned digits together with defects in other regions, depending on hedgehog (Hh) signalling, and exhibits embryonic lethality. The talpid(3) chicken has a mutation in KIAA0586, which encodes a centrosomal protein required for the formation of primary cilia, which are sites of vertebrate Hh signalling. The highly conserved exons 11 and 12 of KIAA0586 are essential to rescue cilia in talpid(3) chicken mutants. We constitutively deleted these two exons to make a talpid3(-/-) mouse. Mutant mouse embryos lack primary cilia and, like talpid(3) chicken embryos, have face and neural tube defects but also defects in left/right asymmetry. Conditional deletion in mouse limb mesenchyme results in polydactyly and in brachydactyly and a failure of subperisoteal bone formation, defects that are attributable to abnormal sonic hedgehog and Indian hedgehog signalling, respectively. Like talpid(3) chicken limbs, the mutant mouse limbs are syndactylous with uneven digit spacing as reflected in altered Raldh2 expression, which is normally associated with interdigital mesenchyme. Both mouse and chicken mutant limb buds are broad and short. talpid3(-/-) mouse cells migrate more slowly than wild-type mouse cells, a change in cell behaviour that possibly contributes to altered limb bud morphogenesis. This genetic mouse model will facilitate further conditional approaches, epistatic experiments and open up investigation into the function of the novel talpid3 gene using the many resources available for mice.     

Immunohistochemical localization of cyclic AMP during normal and abnormal chick and mouse limb development

This paper describes the immunohistochemical localization of cAMP during limb chondrogenesis in talpid3 chick, brachypod mouse, and normal embryos. Comparisons were made between chick wing buds at Stages 22, 25, and 30, and mouse hind limb buds at Days 11, 12.5 and 14. At Stage 22, the normal mesenchyme in the chick displayed areas of bright fluorescence compared to a lesser intense and more evenly distributed fluorescence in talpid3. Sections of the central region from normal Stage 25 limb buds exhibited an intense fluorescence that was uniformly distributed, whereas, in talpid3 staining was more mosaic with some areas fluorescing brightly and others showing little fluorescence. At Stage 30 the staining pattern was similar between normal and talpid3, with the fluorescence being brighter in the cartilage tissue than in the surrounding soft tissue. Difference in the staining patterns of normal and brachypod limb tissue were not detectable. At Days 11 and 12.5, tissue from both genotypes displayed a very bright, uniform fluorescence. In the 14-day hind limb buds, the staining patterns were comparable to those observed in Stage 30 chick wing buds. However, under in vitro conditions conducive for the expression of the chondrogenic phenotype, differences in the intensity and extensiveness of fluorescent staining were detectable in cultures derived from 12-day normal and brachypod hind limb mesenchyme. Compared to the control, the uneven distribution of immunofluorescence in the talpid3 limb buds and the differences in intensity and extensiveness of fluorescence in the brachypod cultures support the hypothesis that cAMP is involved in limb cartilage differentiation.     

Role of the chicken homeobox-containing genes GHox-4.6 and GHox-8 in the specification of positional identities during the development of normal and polydactylous chick limb buds.

During early stages of normal chick limb development, the homeobox-containing (HOX) gene GHox-4.6 is expressed throughout the posterior mesoderm of the wing bud from which most of the skeletal elements including the digits will develop, whereas GHox-8 is expressed in the anterior limb bud mesoderm which will not give rise to skeletal elements. In the present study, we have examined the expression of GHox-4.6 and GHox-8 in the wing buds of two polydactylous mutant chick embryos, diplopodia-5 and talpid2, from which supernumerary digits develop from anterior limb mesoderm, and have also examined the expression of these genes in response to polarizing zone grafts and retinoic acid-coated bead implants which induce the formation of supernumerary digits from anterior limb mesoderm. We have found that the formation of supernumerary digits from the anterior mesoderm in mutant and experimentally induced polydactylous limb buds is preceded by the ectopic expression of GHox-4.6 in the anterior mesoderm and the coincident suppression of GHox-8 expression in the anterior mesoderm. These observations suggest that the anterior mesoderm of the polydactylous limb buds is "posteriorized" and support the suggestion that GHox-8 and GHox-4.6, respectively, are involved in specifying the anterior non-skeletal and posterior digit-forming regions of the limb bud. Although the anterior mesodermal domain of GHox-8 expression is severely impaired in the mutant and experimentally induced polydactylous limb buds, this gene is expressed by the prolonged, thickened apical ectodermal ridges of the polydactylous limb buds that extend along the distal anterior as well as the distal posterior mesoderm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential susceptibility of midbrain and spinal cord patterning to floor plate defects in the talpid2 mutant

The chick talpid2 mutant displays polydactylous digits attributed to defects of the Hedgehog (HH) signaling pathway. We examined the talpid2 neural tube and show that patterning defects in the spinal cord and the midbrain are distinct from each other and from the limb. Unlike the Sonic Hedgehog (SHH) source in the limb, the SHH-rich floor plate (FP) is reduced in the talpid2 midbrain. This is accompanied by a severe depletion of medial cell populations that encounter high concentrations of SHH, an expansion of lateral cell populations that experience low concentrations of SHH and a broad deregulation of HH's principal effectors (PTC1, GLI1, GLI2, GLI3). Together with the failure of SHH misexpression to rescue the talpid2 phenotype, these results suggest that talpid2 is likely to have a tissue-autonomous, bidirectional (positive and negative) role in HH signaling that cannot be attributed to the altered expression of several newly cloned HH pathway genes (SUFU, DZIP1, DISP1, BTRC). Strikingly, FP defects in the spinal cord are accompanied by relatively normal patterning in the talpid2 mutant. We propose that this differential FP dependence may be due to the prolonged apposition of the notochord to the spinal cord, but not the midbrain during development.     

The anatomy, ultrastructure and fluid dynamics of the developing vasculature of the embryonic chick wing bud

The spatiotemporal sequence of vascular pattern development in the embryonic chick wing bud and surrounding shoulder, flank and belly regions is detailed for Hamburger-Hamilton (1951) stages 20-25. Vasculature was microinjected with an unreactive aqueous tracer (aniline blue), and major traffic patterns were visualized. Formation of extensive avascular regions and the emergence of chondrogenic phenotypes are correlated with the retreat of the vasculature from the wing core. Ultrastructural studies of vascular cells show that vessels remain monolayered and undifferentiated until stage 25, after the adult vascular pattern has been laid down. Vascular cytodifferentiation occurs only in the cells of the brachial artery until stage 35, with the veins and capillaries retaining an 'early' morphology. This vascular pattern may be an important component reflecting or directing limb pattern development.     

Craniofacial development in the talpid3 chicken mutant

The talpid(3) chicken mutant has a pleiotropic phenotype including polydactyly and craniofacial abnormalities. Limb polydactyly in talpid(3) suggests a gain of Hedgehog (Hh) signaling, whereas, paradoxically, absence of midline facial structures suggests a loss of Hh function. Here we analyze the status of Shh signaling in the talpid(3) mutant head. We show that Shh expression domains are lost from the talpid(3) head--in hindbrain, midbrain, zona limitans intrathalamica, and stomodeal ectoderm--and that direct targets of Hedgehog signaling, Ptc1, Ptc2, and Gli1, are also absent even in areas associated with primary Shh expression. These data suggest that the talpid(3) mutation leads to defective activation of the Shh pathway and, furthermore, that tissue-to-tissue transduction of Shh expression in the developing head depends on Hh pathway activation. Failure to activate the Shh pathway can also explain absence of floor plate and Hnf-3beta and Netrin-1 expression in midbrain and hindbrain and absence of Fgf-8 expression in commissural plate. Other aspects of gene expression in the talpid(3) head, however, suggest misspecification, such as maintenance of floor plate-like gene expression in telencephalon. In branchial arches and lower jaw, where Shh is expressed, changes in expression of genes involved in patterning and mesodermal specification suggest both gain and loss of Hedgehog function. Thus, analysis of gene expression in talpid(3) head shows that, as in talpid(3) limb, expression of some genes is lost, while others are ectopically expressed. Unlike the limb, many head regions depend on Hh induction of a secondary domain of Shh expression, and failure of this induction in talpid(3), together with the inability to activate the Shh pathway, explain the loss-of-function head phenotype. This gene expression analysis in the talpid(3) head also confirms and extends knowledge of the importance of Shh signaling and the balance between activation and repression of Shh targets in many aspects of craniofacial morphogenesis.     

Expression and regulation of chicken fibroblast growth factor homologous factor (FHF)-4 at the base of the developing limbs

Fibroblast growth factor homologous factors (FHFs) have been implicated in limb and nervous system development. In this paper we describe the expression of the cFHF-4 gene during early chicken development. cFHF-4 is expressed in the paraxial mesoderm, lateral ridge, and, most prominently, in the posterior-dorsal side of the base of each limb bud. The expression pattern of cFHF-4 at the base of the limbs is not altered by tissue grafts containing the zone of polarizing activity (ZPA), by implants of Shh-expressing cells, or by implants of beads containing retinoic acid, nor does it depend on the distal growth of the limb as it is not altered in limb buds that are surgically truncated. In three chicken mutants affecting limb patterning - talpid(2), limbless, and wingless - altered patterns of cFHF-4 expression are correlated with abnormal nerve plexus formation and altered patterns of limb bud innervation. Similarly, ectopic expression of cFHF-4 is correlated with a local induction of limb-like innervation patterns when beads containing FGF-2 are implanted in the flank. In these experiments, both ectopic innervation and ectopic expression of cFHF-4 in the flank were observed regardless of the size of the FGF-2-induced outgrowths. By contrast, ectopic expression of Shh and HoxD13 are seen only in the larger FGF-2-induced outgrowths. Taken together, these data suggest that cFHF-4 regulates or is coregulated with early events related to innervation at the base of the limbs.     

Transient expression of a cell surface heparan sulfate proteoglycan (syndecan) during limb development

Syndecan is an integral membrane proteoglycan that contains both heparan sulfate and chondroitin sulfate chains and that links the cytoskeleton to interstitial extracellular matrix components, including collagen and fibronectin. Immunohistochemistry with a monoclonal antibody directed to the core protein of the syndecan ectodomain has been used to analyze the distribution of this proteoglycan in the developing mouse limb bud and in high-density cultures of limb mesenchyme cells. By Day 9 of gestation when the limb buds are just apparent, syndecan is detected on cells throughout the limb region, including both ectodermal and mesenchymal components. This distribution does not change as the limb bud elongates along its proximodistal axis, except for its reduction in the apical ectodermal ridge. By Day 11, the intensity of immunofluorescence in the central core decreases relative to other regions. By Day 13 immunostaining is lost in the regions destined for chondrogenesis and myogenesis but persists in the limb ectoderm and peripheral and distal mesenchyme. In the limb mesenchyme cell cultures, syndecan is initially undetected, but is found throughout the culture by 24 hr. With further culture the antigen becomes reduced in chondrogenic foci and in association with myogenic cells. When chick limb ectoderm is placed on the high-density cultures, immunoreactivity in the mouse mesenchyme is enhanced suggesting that epithelial-mesenchymal interactions modulate syndecan expression in the limb bud. Based on analysis of 35S-labeled syndecan from the cultures, syndecan from limb mesenchyme cells contains more glycosaminoglycan chains and is larger in size than the previously described polymorphic forms of syndecan from various epithelia. The high affinity of syndecan for components of the extracellular matrix and its distribution in the early limb bud are consistent with a role in maintaining the morphologic integrity of the limb bud during the period of initiation and rapid outgrowth, and in preventing the onset of chondrogenesis.     

Distribution of Retinoids Applied Exogenously to Chick Limb Buds: an Autoradiographic Analysis

An autoradiographic analysis was undertaken to examine the localization of retinoids applied exogenously to chick limb buds. Ion-exchange beads (AGI-X2) containing a tritium-labeled synthetic retinoid, Am80, were implanted to various regions of chick wing buds. This synthetic retinoid is known to induce a duplicated limb pattern as retinoic acid (RA) does. One to 24 hours after the application, wing buds were fixed, sectioned, and prepared for autoradiography. Heavy labeling was observed in the peripheral region of the wing mesoderm, but no gradient along the antero-posterior axis was found.
These results suggest that the peripheral region of the limb bud may be important for the morphogenetic function of RA. Tissue-bound retinoids may not form an antero-posterior concentration gradient when retinoids are added to the anterior margin of the chick limb bud.     

Distribution of retinoids in the chick limb bud: analysis with monoclonal antibody

Retinoic acid induces anteroposterior duplicate formation in developing chick limb bud, and it may be a natural morphogen involved in limb pattern formation. Retinoic acid is produced from retinol locally in the limb bud via retinal, and thus, to elucidate the distribution of these retinoids in the limb bud seems to be important for the understanding of the morphogen formation. We produced a monoclonal antibody against the retinoids with BSA-RA (bovine serum albumin-retinoic acid) conjugate for antigen, and investigated the distribution of retinoids in the chick limb bud. The antibody predominantly bound to retinoic acid, but weakly to retinol and retinal. Retinoids appeared in the limb bud at stage 18 and were distributed through stages 20-24, when the pattern formation in distal mesoderm was in progress. Initially they were found evenly in the whole mesoderm, but disappeared gradually from core mesoderm and remained only in the region of peripheral mesoderm at stage 24. At stage 26, retinoids were detected only in ectoderm. These results support the idea that the retinoids actually play roles in limb pattern formation and suggest that the retinoids in the peripheral mesoderm are important for pattern formation. Further, the role of retinoids in epidermis development at later limb bud stages is also suggested.     

Initial limb budding is independent of apical ectodermal ridge activity; evidence from a limbless mutant

Outgrowth of normal chick limb bud mesoderm is dependent on the presence of a specialized epithelium called the apical ectodermal ridge. This ectodermal ridge is induced by the mesoderm at about the time of limb bud formation. The limbless mutation in the chick affects apical ectodermal ridge formation in the limb buds of homozygotes. The initial formation of the limb bud appears to be unaffected by the mutation but no ridge develops and further outgrowth, which is normally dependent on the ridge, does not take place. As a result, limbless chicks develop without limbs. In the present study, which utilized a pre-limb-bud recombinant technique, limbless mesoderm induced an apical ectodermal ridge in grafted normal flank ectoderm. However, at stages when normal flank ectoderm is capable of responding to ridge induction, limbless flank ectoderm did not form a ridge or promote outgrowth of a limb in response to normal presumptive wing bud mesoderm. We conclude from this that the limbless mutation affects the ability of the ectoderm to form a ridge. In addition, because the limbless ectoderm has no morphological ridge and no apparent ridge activity (i.e. it does not stabilize limb elements in stage-18 limb bud mesoderm), the limbless mutant demonstrates that the initial formation of the limb bud is independent of apical ectodermal ridge activity.     

Experimental manipulation leading to induction of dorsal ectodermal ridges on normal limb buds results in a phenocopy of the eudiplopodia chick mutant

Elongation of chick limb buds depends on the presence of the apical ectodermal ridge which is induced by subjacent limb bud mesoderm. Recombination experiments have shown that the limb bud mesoderm loses the capacity to induce ridges by late stage 17. Moreover, in normal limb development only one ridge forms. However, in the eudiplopodia chick mutant accessory ectodermal ridges form on the dorsal surface of limb buds as late as stage 22. Tissue recombinant experiments show that the mutation affects the ectoderm, extending the time it responds to ridge induction (Fraser and Abbott, 1971a, Fraser and Abbott, 1971b while the mesoderm is normal. The result is polydactyly, with extra digits dorsal to the normal digits. Because eudiplopodia limb bud dorsal mesoderm can induce ridges at stage 22 but is unaffected by the gene, genetically normal dorsal limb bud mesoderm may also be able to induce ridges after stage 17. To test this possibility we grafted stages 14–18 flank ectoderm to normal limb bud dorsal mesoderm and found that mesoderm from stages 17 through 20 was able to induce a ridge and subsequently dorsal digits developed. Limbs with duplicate digits were similar to eudiplopodia limbs. In other experiments, stage 18, 19, and 20 leg bud dorsal ectoderm did not form ridges when grafted to leg bud dorsal mesoderm of the same stage, indicating a lack of response to the mesoderm. Finally, the inductive capacity of limb bud mesoderm appeared to be reduced compared to mesoderm at pre-limb bud stages. These experiments demonstrate a spatially generalized potential in limb bud dorsal mesoderm to induce ridges during the stages when the apical ridge is induced. The determination of where the ridge will form and the acquired inability of limb bud dorsal ectoderm to respond to induction by underlying mesoderm are necessary early pattern forming events which assure that a single proximodistal limb axis will form.     

The blood vessels of the regenerating limb of the adult newt,Triturus

The vessels of the forelimb stump and regenerate were perfused with Prussian blue and studied as whole mounts and in histological sections to reveal the condition and disposition of the blood vessels in various stages of forelimb regeneration in the adult newt, Triturus viridescens. The development of the vessels in the regenerate seemed to be comparable in all its essential features to that which has been described for the normal developing limb in urodele, chick and pig embryos. The first signs of regeneration of the vessels are seen during wound healing when fine sprouts appear from the old vessels near the amputation wound. These grow and anastomose, but are limited to the transition region between old and new tissues and avoid the growing blastema during the early stages of regeneration. As the regenerate enlarges into a conical structure vessels invade the proximal part of the growth and avoid the distal regions. It is only during the stages of histogenesis and morphogenesis that vessels grow into more distal regions. The regions of most active enlargement of the early or later regenerate are those most poorly vascularized. These results are discussed against the background of the activity of certain enzymes during regeneration. In the advanced regenerate, preferential channels are consolidated until in the palette and digital stages the pattern of the blood vessels resembles that of the normal limb.