Zone Of Polarizing Activity Research Articles

Neural expression of mouse Noelin-1/2 and comparison with other vertebrates

Noelins are secreted glycoproteins with important developmental functions in frogs and birds. Here, we present the expression pattern of the mouse homolog of Noelin-1/2 at E8–10 of development and compare this pattern to other vertebrates. Expression was observed in the neural plate and neural crest, as well as in the cranial ganglia. Later, expression is prominent in brain tissue and in the zone of polarizing activity in the limb.

Cholesterol modification of Hedgehog family proteins.

The Hedgehog (Hh) gene family encodes a group of secreted signaling molecules that are essential for growth and patterning of many different body parts of vertebrate and invertebrate embryos (1). Depending on the context, Hh signals can promote cell proliferation, prevent apoptosis, or induce specific cell fates. Hh family members can exert their effects not only on cells neighboring the source of the signal, but also over considerable distances (up to 30-cell diameters), acting in at least some cases as classic morphogens. Such morphogens are signaling molecules that diffuse from a source to form a concentration gradient over an extended area of the target field and elicit different responses from cells according to their position within the gradient, which in turn reflects the dosage of the ligand they are exposed to. In the fruit fly Drosophila, Hh patterns the embryonic ectoderm via short-range interactions with other signaling molecules (2). On the other hand, it employs two different strategies in larval wing imaginal discs: it induces a secondary signal (Decapentaplegic [Dpp]) locally, which then acts at a long range; and Hh itself diffuses over several cell diameters and acts as a morphogen (3–5). Unlike Drosophila, which has one member of the Hh family, mice have three — Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh) — with Shh being the best studied (Table ​(Table1).1). Shh is expressed at the ventral end of the neural tube (floor plate) and underlying notochord, and patterns the neural tube along its dorsoventral axis (6). Several pieces of evidence support the notion that this patterning is mediated by a direct morphogen activity of Shh. In vitro assays using undifferentiated neural tube explants demonstrate that different dosages of Shh can induce different cell types, where the relative dosages required to induce certain cell fates are consistent with their positions within the concentration gradient in vivo (7, 8). More recently, it has become possible to visualize the endogenous Shh gradient covering the ventral half of the neural tube by immunofluorescence and immunohistochemistry (9, 10). In addition, the cell-autonomous activation or inactivation of the pathway using mutant forms of receptor components results in autonomous changes of cell fates, confirming the direct role of Hh signaling (11, 12). The limb is another place where Shh may function as a morphogen. Shh is expressed in the posterior distal mesenchyme of the limb bud, called the zone of polarizing activity (ZPA), and makes a long-range gradient along the anteroposterior axis (13, 14). This concentration gradient is believed to be important in specifying digit identities across the limb bud, with high dosages of Shh close to the ZPA inducing posterior digits and low dosages inducing anterior digits (13, 15, 16). In addition to its importance during development, inappropriate activation of the Hh pathway has been implicated in human tumors such as basal cell carcinoma, medulloblastoma, fibrosarcoma, and rhabdomyosarcoma (17). Table 1 Mammalian Hh genes: some of their normal roles and pathological associations Not surprisingly, significant effort has been devoted to uncovering the molecular mechanism of this pathway (1). Genetic and biochemical studies have established the current model in which Hh receptor Patched (Ptc) is a negative regulator of the pathway repressing the downstream activator Smoothened (Smo), and binding of Hh to Ptc abrogates this inhibition, leading to cellular responses via specific transcription factors known in the fly as Ci and in the mouse as Gli. During the past decade, much excitement has been generated by the discovery of the unusual posttranslational modifications that the Hh protein undergoes (18): the addition of cholesterol (19), which is unprecedented, and palmitoylation (20), which is normally found in cytoplasmic proteins. Here, we will review our current understanding of the mechanism and biological relevance of the cholesterol modification of Hh, with additional discussion of the role of palmitoylation, which has come to light more recently.

Embryonic retinoic acid synthesis is required for forelimb growth and anteroposterior patterning in the mouse.

Numerous studies, often performed on avian embryos, have implicated retinoic acid (RA) in the control of limb bud growth and patterning. Here we have investigated whether the lack of endogenous RA synthesis affects limb morphogenesis in mutant mouse embryos deficient for the retinaldehyde dehydrogenase 2 (Raldh2/Aldh1a2). These mutants, which have no detectable embryonic RA except in the developing retina, die at E9.5-E10 without any evidence of limb bud formation, but maternal RA supplementation through oral gavage from E7.5 can extend their survival. Such survivors exhibit highly reduced forelimb rudiments, but apparently normal hindlimbs. By providing RA within maternal food, we found both a stage- and dose-dependency for rescue of forelimb growth and patterning. Following RA supplementation from E7.5 to 8.5, mutant forelimbs are markedly hypoplastic and lack anteroposterior (AP) patterning, with a single medial cartilage and 1-2 digit rudiments. RA provided until E9.5 significantly rescues forelimb growth, but cannot restore normal AP patterning. Increasing the RA dose rescues the hypodactyly, but leads to lack of asymmetry of the digit pattern, with abnormally long first digit or symmetrical polydactyly. Mutant forelimb buds are characterized by lack of expression or abnormal distal distribution of Sonic hedgehog (Shh) transcripts, sometimes with highest expression anteriorly. Downregulation or ectopic anterior expression of Fgf4 is also seen. As a result, genes such as Bmp2 or Hoxd genes are expressed symmetrically along the AP axis of the forelimb buds, and/or later, of the autopod. We suggest that RA signaling cooperates with a posteriorly restricted factor such as dHand, to generate a functional zone of polarizing activity (ZPA).

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.

Limb development in a "nonmodel" vertebrate, the direct-developing frog Eleutherodactylus coqui.

Mechanisms that mediate limb development are regarded as highly conserved among vertebrates, especially tetrapods. Yet, this assumption is based on the study of relatively few species, and virtually none of those that display any of a large number of specialized life-history or reproductive modes, which might be expected to affect developmental pattern or process. Direct development is an alternative life history found in many anuran amphibians. Many adult features that form after hatching in metamorphic frogs, such as limbs, appear during embryogenesis in direct-developing species. Limb development in the direct-developing frog Eleutherodactylus coqui presents a mosaic of apparently conserved and novel features. The former include the basic sequence and pattern of limb chondrogenesis, which are typical of anurans generally and appear largely unaffected by the gross shift in developmental timing; expression of Distal-less protein (Dlx) in the distal ectoderm; expression of the gene Sonic hedgehog (Shh) in the zone of polarizing activity (ZPA); and the ability of the ZPA to induce supernumerary digits when transplanted to the anterior region of an early host limb bud. Novel features include the absence of a morphologically distinct apical ectodermal ridge, the ability of the limb to continue distal outgrowth and differentiation following removal of the distal ectoderm, and earlier cessation of the inductive ability of the ZPA. Attempts to represent tetrapod limb development as a developmental "module" must allow for this kind of evolutionary variation among species.

Patterning Mechanisms Controlling Vertebrate Limb Development

Vertebrate limb buds are embryonic structures for which much molecular and cellular data are known regarding the mechanisms that control pattern formation during development. Specialized regions of the developing limb bud, such as the zone of polarizing activity (ZPA), the apical ectodermal ridge (AER), and the non-ridge ectoderm, direct and coordinate the development of the limb bud along the anterior-posterior (AP), dorsal-ventral (DV), and proximal-distal (PD) axes, giving rise to a stereotyped pattern of elements well conserved among tetrapods. In recent years, specific gene functions have been shown to mediate the organizing and patterning activities of the ZPA, the AER, and the non-ridge ectoderm. The analysis of these gene functions has revealed the existence of complex interactions between signaling pathways operated by secreted factors of the HH, TGF-beta/BMP, WNT, and FGF superfamilies, which interact with many other genetic networks to control limb positioning, outgrowth, and patterning. The study of limb development has helped to establish paradigms for the analysis of pattern formation in many other embryonic structures and organs.

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.

Differential expression of chicken CYP26 in anterior versus posterior limb bud in response to retinoic acid.

Multiple studies indicate that quantitative control of the levels of all-trans-retinoic acid (RA) in the vertebrate embryo is necessary for correct development. The function of RA in cells is regulated by a number of coordinated mechanisms. One of those mechanisms involves controls on the rate of RA catabolism. Recently, enzymes capable of catabolizing RA were found to constitute a new family, called CYP26, within the cytochrome P450 superfamily. CYP26 homologues have been isolated from human, mouse, zebra fish, and recently from the chick. In this study, we examined the regulation of chicken CYP26 (cCYP26) expression by RA during the early phase of chick limb outgrowth. In the anterior limb mesenchyme and apical ectodermal ridge (AER), cCYP26 expression was induced in a concentration dependent manner by implanting beads soaked in 0.1, 1, and 5 mg/ml RA. The RA-induced expression of cCYP26 in anterior limb mesenchyme and the AER was detected as early as 1 hr after treatment and was not affected by the presence of cycloheximide. In contrast to the anterior limb, the induction of cCYP26 was dramatically reduced (or absent) when RA beads were implanted in the posterior limb mesenchyme. Furthermore, induction of cCYP26 expression in the anterior mesenchyme was inhibited by transplantations of the zone of polarizing activity (ZPA) and by Shh-soaked beads. Our data suggest that different mechanisms regulate retinoid homeostasis in the AER and mesenchyme during limb bud outgrowth. J. Exp. Zool. 290:136-147, 2001.

Cholesterol Modification of Sonic Hedgehog Is Required for Long-Range Signaling Activity and Effective Modulation of Signaling by Ptc1

Sonic hedgehog (Shh) signaling from the posterior zone of polarizing activity (ZPA) is the primary determinant of anterior-posterior polarity in the vertebrate limb field. An active signal is produced by an autoprocessing reaction that covalently links cholesterol to the N-terminal signaling moiety (N-Shh p), tethering N-Shh p to the cell membrane. We have addressed the role played by this lipophilic modification in Shh-mediated patterning of mouse digits. Both the distribution and activity of N-Shh p indicate that N-Shh p acts directly over a few hundred microns. In contrast, N-Shh, a form that lacks cholesterol, retains similar biological activity to N-Shh p, but signaling is posteriorly restricted. Thus, cholesterol modification is essential for the normal range of signaling. It also appears to be necessary for appropriate modulation of signaling by the Shh receptor, Ptc1.

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 Sonic Hedgehog–Patched–Gli Pathway in Human Development and Disease

Embryogenesis is regulated by a number of complex signaling cascades, which are critical for normal development. One such pathway begins with a secreted protein called “SONIC HEDGEHOG” (SHH [MIM 600725]), which sets off a chain of events in target cells, leading to the activation and repression of target genes by transcription factors in the Gli family. Dysregulation of the Sonic hedgehog–Patched–Gli (Shh-Ptch-Gli) pathway leads to several human diseases, including birth defects and cancers. Elements of the Shh-Ptch-Gli pathway are highly conserved, indicating its essential role in development. However, Shh signaling is also apparently susceptible to evolutionary pressures. Thus, Shh signaling has been adapted to serve tremendously diverse functions in a wide array of animal models, in both embryonic and adult life. Shh and the downstream molecules involved in signal transduction vary, to some extent, between animals, as well. Although a great deal can be learned from the study of animal models, it is essential to not assume that what is true in one model system is necessarily true in another. Thus, this review focuses specifically on our current understanding of the Shh-Ptch-Gli pathway and its clinical significance in humans.

The expression of teneurin-4 in the avian embryo

The teneurins are a family of four transmembrane proteins expressed in the developing vertebrate nervous system, though the Drosophila teneurin ten-m is also a pair-rule gene. Whole-mount in situ hybridization was used to localize teneurin-4 transcripts in the chicken embryo. The earliest signal is detected at stage 19 in the somites and limb buds. By stage 20 teneurin-4 transcripts are detected in temporal periocular mesenchyme, branchial arches, diencephalon and somites. Teneurin-4 expression in the limbs changes dramatically during development. Between stages 19 and 21 teneurin-4 expression is concentrated proximally in the zone of polarizing activity. Between stages 24 and 26 teneurin-4 is expressed in the mesenchyme of the anterodistal part of the limb. As in Drosophila, vertebrate teneurins are expressed not only in the nervous system, but also in non-neuronal tissues during pattern formation.

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 – talpid2, 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.

Synergistic effects of FGF and non-ridge ectoderm on gene expression involved in the formation of the anteroposterior axis of the chick limb bud in cell culture.

Skeletal patterning of the vertebrate limb is controlled by the zone of polarizing activity (ZPA), apical ectodermal ridge (AER) and dorsal ectoderm. In the present study, to understand the involvement of fibroblast growth factor (FGF) and non-ridge ectoderm in anteroposterior (AP) axis formation, gene expression in chick limb bud mesenchymal cells in culture was investigated by reverse transcription-polymerase chain reaction and in situ hybridization. It was found that Shh expression was locally maintained in the mesenchymal cells underneath and near non-ridge ectoderm in coculture with the posterior mesenchymal cells and non-ridge ectoderm in the presence of FGF-4 by in situ hybridization. In Shh-expressing anterior limb bud mesenchymal cells cultured with non-ridge ectoderm, it was also discovered that Bmp-2 was activated in the presence of FGF-2, -4 and -8, while Hoxd-13 was activated in the presence of FGF-4 and that FGF-2 had a similar effect but FGF-8 did not. This result indicates that Hoxd-13 activation by SHH depends on non-ridge ectoderm and FGF-2 or FGF-4, and that there may be a difference in the effect on AP axis formation of the limb bud between FGF-2, -4 and -8. Possible roles of these genes and signal molecules in AP pattern formation are discussed.

The bHLH transcription factor dHAND controls Sonic hedgehog expression and establishment of the zone of polarizing activity during limb development

Limb outgrowth and patterning of skeletal elements are dependent on complex tissue interactions involving the zone of polarizing activity (ZPA) in the posterior region of the limb bud and the apical ectodermal ridge. The peptide morphogen Sonic hedgehog (SHH) is expressed specifically in the ZPA and, when expressed ectopically, is sufficient to mimic its functions, inducing tissue growth and formation of posterior skeletal elements. We show that the basic helix-loop-helix transcription factor dHAND is expressed posteriorly in the developing limb prior to Shh and subsequently occupies a broad domain that encompasses the Shh expression domain. In mouse embryos homozygous for a dHAND null allele, limb buds are severely underdeveloped and Shh is not expressed. Conversely, misexpression of dHAND in the anterior region of the limb bud of transgenic mice results in formation of an additional ZPA, revealed by ectopic expression of Shh and its target genes, and resulting limb abnormalities that include preaxial polydactyly with duplication of posterior skeletal elements. Analysis of mouse mutants in which Hedgehog expression is altered also revealed a feedback mechanism in which Hedgehog signaling is required to maintain the full dHAND expression domain in the developing limb. Together, these findings identify dHAND as an upstream activator of Shh expression and important transcriptional regulator of limb development.

Conditional inactivation of Fgf4 reveals complexity of signalling during limb bud development.

Development of the vertebrate limb bud depends on reciprocal interactions between the zone of polarizing activity (ZPA) and the apical ectodermal ridge (AER). Sonic hedgehog (SHH) and fibroblast growth factors (FGFs) are key signalling molecules produced in the ZPA and AER, respectively. Experiments in chicks suggested that SHH expression in the ZPA is maintained by FGF4 expression in the AER, and vice versa, providing a molecular mechanism for coordinating the activities of these two signalling centres. This SHH/FGF4 feedback loop model is supported by genetic evidence showing that Fgf4 expression is not maintained in Shh-/- mouse limbs. We report here that Shh expression is maintained and limb formation is normal when Fgf4 is inactivated in mouse limbs, thus contradicting the model. We also found that maintenance of Fgf9 and Fgf17 expression is dependent on Shh, whereas Fgf8 expression is not. We discuss a model in which no individual Fgf expressed in the AER (AER-Fgf) is solely necessary to maintain Shh expression, but, instead, the combined activities of two or more AER-Fgfs function in a positive feedback loop with Shh to control limb development.

Pattern formation and regulation of gene expressions in chick recombinant limbs

Recombinant limbs were performed by ensembling dissociated-reaggregated wing bud mesoderm inside an ectodermal hull. The zone of polarizing activity was excluded from the mesoderm used to perform the recombinant limbs (non-polarized recombinants), and grafted when desired (polarized recombinants). Reorganization of patterning progressively occurred in the newly formed progress zone under the influence of the apical ectodermal ridge (AER), explaining the proximo–distal gradient of morphogenesis observed in developed recombinant limbs. The AER, without the influence of the polarizing region (ZPA), was sufficient to direct outgrowth and appropriate proximo–distal patterning, as observed in the expression of the Hoxa-11 and Hoxa-13 genes. The development of the recombinant limbs coursed with symmetric AER and downregulation of Bmp expression in the mesoderm supporting a negative effect of Bmp signaling upon the apical ridge. The recombinant ectoderm maintained previously established compartments of gene expressions and organized a correct dorso-ventral patterning in the recombinant progress zone. Finally, the ZPA effect was only detected on Bmp expression and pattern formation along the antero-posterior axis.

SF/HGF is a mediator between limb patterning and muscle development.

Scatter factor/hepatocyte growth factor (SF/HGF) is known to be involved in the detachment of myogenic precursor cells from the lateral dermomyotomes and their subsequent migration into the newly formed limb buds. As yet, however, nothing has been known about the role of the persistent expression of SF/HGF in the limb bud mesenchyme during later stages of limb bud development. To test for a potential role of SF/HGF in early limb muscle patterning, we examined the regulation of SF/HGF expression in the limb bud as well as the influence of SF/HGF on direction control of myogenic precursor cells in limb bud mesenchyme. We demonstrate that SF/HGF expression is controlled by signals involved in limb bud patterning. In the absence of an apical ectodermal ridge (AER), no expression of SF/HGF in the limb bud is observed. However, FGF-2 application can rescue SF/HGF expression. Excision of the zone of polarizing activity (ZPA) results in ectopic and enhanced SF/HGF expression in the posterior limb bud mesenchyme. We could identify BMP-2 as a potential inhibitor of SF/HGF expression in the posterior limb bud mesenchyme. We further demonstrate that ZPA excision results in a shift of Pax-3-positive cells towards the posterior limb bud mesenchyme, indicating a role of the ZPA in positioning of the premuscle masses. Moreover, we present evidence that, in the limb bud mesenchyme, SF/HGF increases the motility of myogenic precursor cells and has a role in maintaining their undifferentiated state during migration. We present a model for a crucial role of SF/HGF during migration and early patterning of muscle precursor cells in the vertebrate limb.

Expression of the Sonic hedgehog (SHH ) gene during early human development and phenotypic expression of new mutations causing holoprosencephaly.

Holoprosencephaly (HPE), the most common developmental defect of the forebrain and the face, is genetically heterogeneous. One of the genes involved, Sonic hedgehog ( SHH ), on 7q36, has been identified as the first HPE-causing gene both in mouse and humans. In order to delineate the phenotype of specific SHH mutations, we described the expression of the SHH gene during early human embryogenesis and investigated the phenotype of novel SHH mutations. In situ hybridization studies were performed on paraffin-embedded human embryo sections at three different development stages. These studies show that SHH is expressed in the notochord, the floorplate, the brain, the zone of polarizing activity and the gut. We also report on the phenotype of four novel mutations identified in 40 HPE families (two in isolated HPE and two in familial HPE). Expressivity ranged from alobar HPE to microcephaly and hypoplasia of the pituitary gland in one family, and from HPE to an asymptomatic form in another family. No SHH mutation was found in six polymalformed cases combining HPE with other defects, such as skeletal, limb, cardiac, anal and/or renal anomalies. This study confirms the genetic heterogeneity of HPE, and further demonstrates that SHH mutations are associated with a broad spectrum of cerebral midline defects.

Constitutive Activation of Sonic Hedgehog Signaling in the Chicken Mutant talpid2: Shh-Independent Outgrowth and Polarizing Activity

We have examined the developmental properties of the polydactylous chicken mutant, talpid2. 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 talpid2 mutants. We show that preaxial talpid2 limb bud mesoderm has polarizing activity in the absence of detectable Shh mRNA. When the postaxial talpid2 limb bud (including all Shh-expressing cells) is removed, the preaxial cells reform a normal-shaped talpid2 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 talpid2 limb buds develop complete talpid2 limbs. After similar treatment, normal limb buds downregulate Fgf4, the preaxial cells do not regulate, and a truncated anteroposterior deficient limb forms. In talpid2 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 talpid2 in which leaky activation of the Shh signaling pathway occurs in the absence of Shh ligand.