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.     

Sonic hedgehog (SHH) specifies muscle pattern at tissue and cellular chick level, in the chick limb bud.

Development of the musculature in chick limbs involves tissue and cellular patterning. Patterning at the tissue level leads to the precise arrangement of specific muscles; at the cellular level patterning gives rise to the fibre type diversity in muscles. Although the data suggests that the information controlling muscle patterning is localised within the limb mesenchyme and not in the somitic myogenic precursor cells themselves, the mechanisms underlying muscle organisation have still to be elucidated. The anterior-posterior axis of the limb is specified by a group of cells in the posterior region of the limb mesenchyme, called the zone of polarizing activity (ZPA). When polarizing-region cells are grafted to the anterior margin of the bud, they cause mirror-image digit duplications to be produced. The effect of ZPA grafts can be reproduced by application of retinoic acid (RA) beads and by grafting sonic hedgehog (SHH)-expressing cells to the anterior margin of the limb. Although most previous studies have looked at changes of the skeletal patterning, ZPA and RA also affect muscle patterning. In this report, we investigated the role of SHH in tissue and cellular patterning of forearm wing muscles. Ectopic application of a localised source of SHH to the anterior margin of the wing, leading to complete digit duplication, is able to transform anterior forearm muscles into muscles with a posterior identity. Moreover, the ectopic source of SHH induces a mirror image duplication of the normal posterior muscles fibre types in the new posterior muscles. The reorganisation of the slow fibres can be detected before muscle mass cleavage has started; suggesting that the appropriate fibre type arrangement is in place before the splitting process can be observed.     

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.     

Immunolocalization of sonic hedgehog (Shh) in developing mouse lung.

Expression of sonic hedgehog (Shh) is required for normal development of the lung during embryogenesis. Loss of Shh expression in mice results in tracheoesophageal fistula, lung hypoplasia, and abnormal lung lobulation. To determine whether Shh may play a role later in lung morphogenesis, immunostaining for Shh was performed in mouse lung from embryonic day (E) 10.5 to postnatal day (PD) 24. Shh was detected in the distal epithelium of the developing mouse lung from E10.5 to E16.5. From E16.5 until PD15, Shh was present in epithelial cells in both the peripheral and conducting airways. Although all cells of the developing epithelium uniformly expressed Shh at E10.5, Shh expression was restricted to subsets of epithelial cells by E16.5. Between E16.5 and PD15, non-uniform Shh staining of epithelial cells was observed in the conducting airways in a pattern consistent with the distribution of non-ciliated bronchiolar cells (i.e., Clara cells) and the Clara cell marker CCSP. Shh did not co-localize with hepatocyte nuclear factor/forkhead homologue-4 (HFH-4), beta-tubulin, or with the presence of cilia. These results support the concept that Shh plays a distinct regulatory role in the lung later in morphogenesis, when it may influence formation or cytodifferentiation of the conducting airways.     

Local sonic hedgehog signaling regulates oligodendrocyte precursor appearance in multiple ventricular zone domains in the chick metencephalon

In the chick metencephalon, oligodendrocyte precursors arise in distinct domains of the ventricular zone. During development, the earliest oligodendrocyte precursors appear in the metencephalic ventral ventricular zone adjacent to the midline, consistent with their location in the spinal cord. In contrast to spinal cord, however, distinct domains in the lateral and dorsal metencephalic ventricular zone subsequently generate oligodendrocyte precursors. All oligodendrogenic domains of the metencephalon appear in close apposition to regions that transiently express sonic hedgehog (Shh). Inhibition studies demonstrate a functional requirement for Shh signaling in the early appearance of metencephalic oligodendrocyte precursors, while in vitro studies suggest a dose-dependent increase in the number of oligodendrocyte precursors in response to Shh. In purified cultures of oligodendrocyte precursors, Shh promotes cell survival and proliferation, suggesting that Shh can act directly on these cells. These data suggest that Shh may be responsible for the localized appearance of oligodendrocyte precursors throughout the CNS, irrespective of the dorso-ventral neural axis.     

Identification of sonic hedgehog as a candidate gene responsible for the polydactylous mouse mutant Sasquatch

The mouse mutants of the hemimelia-luxate group (lx, lu, lst, Dh, Xt, and the more recently identified Hx, Xpl and Rim4; [1] [2] [3] [4] [5]) have in common preaxial polydactyly and longbone abnormalities. Associated with the duplication of digits are changes in the regulation of development of the anterior limb bud resulting in ectopic expression of signalling components such as Sonic hedgehog (Shh) and fibroblast growth factor-4 (Fgf4), but little is known about the molecular causes of this misregulation. We generated, by a transgene insertion event, a new member of this group of mutants, Sasquatch (Ssq), which disrupted aspects of both anteroposterior (AP) and dorsoventral (DV) patterning. The mutant displayed preaxial polydactyly in the hindlimbs of heterozygous embryos, and in both hindlimbs and forelimbs of homozygotes. The Shh, Fgf4, Fgf8, Hoxd12 and Hoxd13 genes were all ectopically expressed in the anterior region of affected limb buds. The insertion site was found to lie close to the Shh locus. Furthermore, expression from the transgene reporter has come under the control of a regulatory element that directs a pattern mirroring the endogenous expression pattern of Shh in limbs. In abnormal limbs, both Shh and the reporter were ectopically induced in the anterior region, whereas in normal limbs the reporter and Shh were restricted to the zone of polarising activity (ZPA). These data strongly suggest that Ssq is caused by direct interference with the cis regulation of the Shh gene.     

The adventures of sonic hedgehog in development and repair. IV. Sonic hedgehog processing, secretion, and function in the stomach

Sonic hedgehog (Shh) is recognized as one of the main morphogens that regulates cell differentiation during early development of the stomach. In the adult stomach, Shh is expressed and secreted from the acid-producing parietal cells, where it is believed to play an essential role in gastric tissue homeostasis and normal differentiation of the epithelium. The present Themes article focuses on reviewing the literature and controversies surrounding the processing and secretion and the role of Shh in the adult stomach.     

Reduced thymocyte development in sonic hedgehog knockout embryos

The Hedgehog family of secreted intercellular signaling molecules are regulators of patterning and organogenesis during animal development. In this study we provide genetic evidence that Sonic Hedgehog (Shh) has a role in the control of murine T cell development. Analysis of Shh(-/-) mouse embryos revealed that Shh regulates fetal thymus cellularity and thymocyte differentiation. Shh is necessary for expansion of CD4(-)CD8(-) double-negative (DN) thymocytes and for efficient transition from the earliest CD44(+)CD25(-) DN population to the subsequent CD44(+)CD25(+) DN population and from DN to CD4(+)CD8(+) double-positive cells.     

Cooperation of sonic hedgehog enhancers in midline expression

In zebrafish, as in other vertebrates, the secreted signalling molecule Sonic hedgehog (Shh) is expressed in organiser regions such as the embryonic midline and the zona limitans intrathalamica (zli). To investigate the regulatory mechanisms underlying the pattern of shh expression, we carried out a systematic analysis of the intronic regulatory sequences of zebrafish shh using stable transgenesis. Deletion analysis identified the modules responsible for expression in the embryonic shield, the hypothalamus and the zli and confirmed the activities of previously identified notochord and floor plate enhancers. We detected a strong synergism between regulatory regions. The degree of synergy varied over time in the hypothalamus suggesting different mechanisms for initiation and maintenance of expression. Our data show that the pattern of shh expression in the embryonic central nervous system involves an intricate crosstalk of at least 4 different regulatory regions. When compared to the enhancer activities of the mouse Shh gene, we observed a remarkable divergence of function of structurally conserved enhancer sequences. The activating region ar-C (61% identical to SFPE2 in mouse Shh), for example, mediates floor plate expression in the mouse embryo while it directs expression in the forebrain and the notochord and only weakly in the floor plate in the zebrafish embryo. This raises doubts on the predictive power of phylogenetic footprinting and indicates a stunning divergence of function of structurally conserved regulatory modules during vertebrate evolution.     

The level of sonic hedgehog signaling regulates the complexity of cerebellar foliation

Foliation of the mouse cerebellum occurs primarily during the first 2 weeks after birth and is accompanied by tremendous proliferation of granule cell precursors (GCPs). We have previously shown that sonic hedgehog (Shh) signaling correlates spatially and temporally with fissure formation, and that Gli2 is the main activator driving Shh induced proliferation of embryonic GCPs. Here, we have tested whether the level of Shh signaling regulates the extent of cerebellar foliation. By progressively lowering signaling by removing Gli1 and Gli2 or the Shh receptor smoothened, we found the extent of foliation is gradually reduced, and that this correlates with a decrease in the duration of GCP proliferation. Importantly, the pattern of the remaining fissures in the mutants corresponds to the first fissures that form during normal development. In a complementary manner, an increase in the level and length of Shh signaling results in formation of an extra fissure in a position conserved in rat. The complexity of cerebellar foliation varies greatly between vertebrate species. Our studies have uncovered a mechanism by which the level and length of Shh signaling could be integral to determining the distinct number of fissures in each species.     

Cell generation times in normal and talpid3 mutant chick limb mesenchyme

A comparative study of the cell cycle and its parameters, and of limb pattern development, was carried out in normal and talpid3 wing buds. The relative duration of the G2 + M phase is the least variable. The change in the length of the G1 phase and the S phase is associated with the appearance of mucopolysaccharides in chondrogenic zones and of myosin in myogenic zones. The effect of the ta3 gene is failure of chondrogenesis-induced lengthening of the G1 phase to appear in the chondrogenic condensation in the limb mesenchyme. The data also raise the question of the possible role of the apical ectodermal ridge in control of the cell division rate in the sub-ridge region.     

Discovery of sonic hedgehog expression in postnatal growth plate chondrocytes: differential regulation of sonic and Indian hedgehog by retinoic acid

Sonic hedgehog (Shh) is a key signal protein in early embryological patterning of limb bud development. Its analog, Indian hedgehog (Ihh), primarily expressed during early cartilage development in prehypertrophic chondrocytes, regulates proliferation and suppresses terminal differentiation of postnatal growth plate (GP) chondrocytes. We report here for the first time that both Shh and Ihh mRNA are expressed in the GP of rapidly growing 6-week-old broiler-strain chickens. They are also expressed in other tissues such as articular chondrocytes, kidney, and bone. In situ hybridization and RT-PCR analyses reveal Shh in all zones of the GP, with peak expression in late hypertrophy. Using primary cultures of GP chondrocytes in serum-containing medium, we followed the patterns of Shh and Ihh mRNA expression as the cultures matured and mineralized. We find a cyclical expression of both hedgehog genes during the early period of culture development between day 10 and 14; when one is elevated, the other tended to be suppressed, suggesting that the two hedgehogs may play complementary roles during GP development. Retinoic acid (RA), a powerful modulator of gene expression in cell differentiation, stimulates GP chondrocytes toward terminal differentiation, enhancing mineral formation. We find that RA strongly suppresses Ihh, but enhances expression of Shh in this system. While Ihh suppresses maturation of GP chondrocytes to hypertrophy, we hypothesize that Shh acts to push these cells toward hypertrophy.     

Pattern regulation in the embryonic chick limb: Supernumerary limb formation with anterior (non-ZPA) limb bud tissue

The formation of supernumerary limbs and limb structures was studied by juxtaposing normally nonadjacent embryonic chick limb bud tissue. A “wedge” (ectoderm and mesoderm) of anterior or mid donor right wing bud (stage 21) was inserted in a slit made in a host right limb bud (stage 21) at the same position as its position of origin or to a more posterior position. The AER of the donor tissue and host wing bud were aligned with each other. Donor tissue was grafted with its dorsalventral polarity the same as the host's limb bud or reversed to that of the host's. Depending on the position of origin of the donor limb bud tissue and the position to which it was transplanted in a host, supernumerary wings or wing structures formed. Furthermore, depending on the orientation of the graft in the host, supernumerary limbs with either left or right asymmetry developed. The results of experiments performed here are considered in light of two current models which have been used to describe supernumerary limb formation: one based on local, short-range, cell-cell interactions and the other based on long-range positional signaling via a diffusible morphogen.     

The role of sonic hedgehog in normal and abnormal craniofacial morphogenesis.

There is growing evidence that implicates a role for Sonic hedgehog (SHH) in morphogenesis of the craniofacial complex. Mutations in human and murine SHH cause midline patterning defects that are manifested in the head as holoprosencephaly and cyclopia. In addition, teratogens such as jervine, which inhibit the response of tissues to SHH, also produce cyclopia. Thus, the loss of SHH signaling during early stages of neural plate patterning has a profound influence of craniofacial morphogenesis. However, the severity of these defects precludes analyses of SHH function during later stages of craniofacial development. We have used an embryonic chick system to study the role of SHH during these later stages of craniofacial development. Using a combination of surgical and molecular experiments, we show here that SHH is essential for morphogenesis of the frontonasal and maxillary processes (FNP and MXPs), which give rise to the mid- and upper face. Transient loss of SHH signaling in the embryonic face inhibits growth of the primordia and results in defects analogous to hypotelorism and cleft lip/palate, characteristics of the mild forms of holoprosencephaly. In contrast, excess SHH leads to a mediolateral widening of the FNP and a widening between the eyes, a condition known as hypertelorism. In severe cases, this widening is accompanied by facial duplications. Collectively, these experiments demonstrate that SHH has multiple and profound effects on the entire spectrum of craniofacial development, and perturbations in SHH signaling are likely to underlie a number of human craniofacial anomalies.     

Gene regulatory landscape of the sonic hedgehog locus in embryonic development

The organs of vertebrate species display a wide variety of morphology. A remaining challenge in evolutionary developmental biology is to elucidate how vertebrate lineages acquire distinct morphological features. Developmental programs are driven by spatiotemporal regulation of gene expression controlled by hundreds of thousands of cis-regulatory elements. Changes in the regulatory elements caused by the introduction of genetic variants can confer regulatory innovation that may underlie morphological novelties. Recent advances in sequencing technology have revealed a number of potential regulatory variants that can alter gene expression patterns. However, a limited number of studies demonstrate causal dependence between genetic and morphological changes. Regulation of Shh expression is a good model to understand how multiple regulatory elements organize tissue-specific gene expression patterns. This model also provides insights into how evolution of molecular traits, such as gene regulatory networks, lead to phenotypic novelty.     

cGMP enhances the sonic hedgehog response in neural plate cells.

The elaboration of distinct cell types during development is dependent on a small number of inductive molecules. Among these inducers is Sonic hedgehog (Shh), which, in combination with other factors, patterns the dorsoventral (DV) axis of the nervous system. The response of a cell is dependent in part on its complement of cyclic nucleotides. cAMP antagonizes Shh signaling, and we examined the influence of cGMP on the Shh response. Cells in chick neural plate explants respond to Shh by differentiating into ventral neural-cell types. Exposure of intermediate-zone explants to cGMP analogs enhanced their response to Shh in a dose-dependent manner. The Shh response was also enhanced in dorsal-zone explants exposed to chick natriuretic peptide (chNP), which stimulates cGMP production by membrane-bound guanylate cyclase (mGC). Addition of chNP to intermediate-zone explants did not enhance the Shh response, consistent with a reported lack of mGC in this region of the neural tube. Finally, the presence of a nitric oxide (NO)-sensitive guanylate cyclase (GC) was established by demonstrating cGMP immunoreactivity in neural tissue following NO stimulation of whole chick embryos. Intracellular levels of cGMP and cAMP may thus provide a mechanism through which other factors modulate the Shh response during neural development.     

Adenohypophysis formation in the zebrafish and its dependence on sonic hedgehog

Formation of the adenohypophysis in mammalian embryos occurs via an invagination of the oral ectoderm to form Rathke's pouch, which becomes exposed to opposing dorsoventral gradients of signaling proteins governing specification of the different hormone-producing pituitary cell types. One signal promoting pituitary cell proliferation and differentiation to ventral cell types is Sonic hedgehog (Shh) from the oral ectoderm. To study pituitary formation and patterning in zebrafish, we cloned four cDNAs encoding different pituitary hormones, prolactin (prl), proopiomelancortin (pomc), thyroid stimulating hormone (tsh), and growth hormone (gh), and analyzed their expression patterns relative to that of the pituitary marker lim3. prl and pomc start to be expressed at the lateral edges of the lim3 expression domain, before pituitary cells move into the head. This indicates that patterning of the pituitary anlage and terminal differentiation of pituitary cells starts while cells are still organized in a placodal fashion at the anterior edge of the developing brain. Following the expression pattern of prl and pomc during development, we show that no pituitary-specific invagination equivalent to Rathke's pouch formation takes place. Rather, pituitary cells move inwards together with stomodeal cells during oral cavity formation, with medial cells of the placode ending up posterior and lateral cells ending up anterior, resulting in an anterior-posterior, rather than a dorsoventral, patterning of the adenohypophysis. Carrying out loss- and gain-of-function experiments, we show that Shh from the ventral diencephalon plays a crucial role during induction, patterning, and growth of the zebrafish adenohypophysis. The phenotypes are very similar to those obtained upon pituitary-specific inactivation or overexpression of Shh in mouse embryo, suggesting that the role of Shh during pituitary development has been largely conserved between fish and mice, despite the different modes of pituitary formation in the two vertebrate classes.