Multiple roles for Hedgehog signaling in zebrafish pituitary development

The endocrine-secreting lobe of the pituitary gland, or adenohypophysis, forms from cells at the anterior margin of the neural plate through inductive interactions involving secreted morphogens of the Hedgehog (Hh), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) families. To better understand when and where Hh signaling influences pituitary development, we have analyzed the effects of blocking Hh signaling both pharmacologically (cyclopamine treatments) and genetically (zebrafish Hh pathway mutants). While current models state that Shh signaling from the oral ectoderm patterns the pituitary after placode induction, our data suggest that Shh plays a direct early role in both pituitary induction and patterning, and that early Hh signals comes from adjacent neural ectoderm. We report that Hh signaling is necessary between 10 and 15 h of development for induction of the zebrafish adenohypophysis, a time when shh is expressed only in neural tissue. We show that the Hh responsive genes ptc1 and nk2.2 are expressed in preplacodal cells at the anterior margin of the neural tube at this time, indicating that these cells are directly receiving Hh signals. Later (15-20 h) cyclopamine treatments disrupt anterior expression of nk2.2 and Prolactin, showing that early functional patterning requires Hh signals. Consistent with a direct role for Hh signaling in pituitary induction and patterning, overexpression of Shh results in expanded adenohypophyseal expression of lim3, expansion of nk2.2 into the posterior adenohypophysis, and an increase in Prolactin- and Somatolactin-secreting cells. We also use the zebrafish Hh pathway mutants to document the range of pituitary defects that occur when different elements of the Hh signaling pathway are mutated. These defects, ranging from a complete loss of the adenohypophysis (smu/smo and yot/gli2 mutants) to more subtle patterning defects (dtr/gli1 mutants), may correlate to human Hh signaling mutant phenotypes seen in Holoprosencephaly and other congenital disorders. Our results reveal multiple and distinct roles for Hh signaling in the formation of the vertebrate pituitary gland, and suggest that Hh signaling from neural ectoderm is necessary for induction and functional patterning of the vertebrate pituitary gland.     

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.     

Conserved expression control and shared activity between cognate T-box genes Tbx2 and Tbx3 in connection with Sonic hedgehog signaling during Xenopus eye development

Tbx2 and Tbx3 are considered to be cognate genes within a Tbx2/3/4/5 subfamily of T-box genes and are expressed in closely overlapping areas in a variety of tissues, including the eye. Herein, we show that misexpression of Tbx2 and Tbx3 in Xenopus embryos gave rise to defective eye morphogenesis, which was reminiscent of the defect caused by attenuated Sonic hedgehog (Shh) signaling. Indeed, Tbx2/3 misexpression suppressed Gli1, Gli2, Ptc2 and Pax2, mediators or targets of Hedgehog (Hh) signals. From these data, Tbx2/3 may have a shared function in inhibiting Gli-dependent Shh signaling during eye development. Conversely, the expression of Tbx2/3 was severely affected by both Shh and a putative dominant negative form of Hh, as well as by both transactivator and transrepressor forms of Gli-fusion proteins, suggesting that the expression of Tbx2/3 may be regulated by a Gli-dependent Hh signal transduction pathway. Because the Shh signal has been considered to play crucial roles in the formation of the proximal-distal and dorsal-ventral axes in the eyes, these findings about the mutual regulatory mechanism between Tbx2/3 and Gli-dependent Hh signaling provide valuable insight into the cause of the localized expression of Tbx2/3 and their role during the formation of these axes. In addition, our findings also imply the conserved regulation and shared activity between the cognate genes of Tbx2 and Tbx3.     

A SHH-independent regulation of Gli3 is a significant determinant of anteroposterior patterning of the limb bud

The family of GLI proteins (GLI1-3) comprises the intracellular mediators of the hedgehog pathway, which regulates a myriad of developmental processes, one of which is limb development. Whereas GLI1 and GLI2 seem to be dispensable during limb development, GLI3 is especially crucial since all GLI3-associated human congenital diseases comprise limb malformations. Furthermore, Gli3^−/− mouse embryos exhibit pronounced polydactyly in conjunction with a loss of digit identities.Here we examined how the quantity of GLI3 contributes to its function by using different Gli3 mutants in order to vary overall GLI3 levels. In addition, we made use of the Gli3^Δ699 allele, which encodes a C-terminally truncated version of GLI3, thus mimicking the processed GLI3 isoform (GLI3R). The Gli3^Δ699 mutant made it feasible to analyze isoform-specific contributions of GLI3 within the context of anteroposterior patterning of the limb bud. We revealed a so far unappreciated variation in the quantitative demand for GLI3 within different phases and aspects of distal limb formation. In addition, our analyses provide evidence that unprocessed full-length GLI3 is dispensable for anteroposterior patterning of the limb bud. Instead, digit identities are most likely defined by GLI3 repressor activity alone. Furthermore, we present evidence that the anteroposterior grading of GLI3 activity by the action of SHH is supported by a prototype patterning, which regulates Gli3 independently from SHH.     

On the role of the connective tissue in the patterning of the chick limb musculature

Summary By modifying the temporal relationship between connective tissue and myogenic cell invasion during early limb bud development new evidence of the organizing role of the connective tissue was obtained.Muscle cell-deprived wing buds were allowed to grow up to stages 22 to 27 of Hamburger and Hamilton, when they received a transplant of quail myogenic cells (somitic mesoderm or wing premuscular mass) into the dorsal face of their presumptive upper arm. Muscular arrangement in forearm and hand was analyzed 4 days later. In 8 out of 14 of those cases which had received a graft of premuscular mass before stage 25 of Hamburger and Hamilton, muscle development took place distally to the graft-site in accordance with the wing segment.     

Role of RhoC in digit morphogenesis during limb development

Here we report a new role for the small GTPase RhoC in the control of limb chondrogenesis. Expression of rhoC is a precocious marker of the zeugopodial and digit blastemas and is induced by treatments with TGFbetas preceding the formation of ectopic digits. As development progresses, expression of rhoC outlines the growing distal tip of the digits, and marks the regions of interphalangeal joint formation. Functional experiments show that RhoC is a negative regulator of chondrogenesis, which controls digit outgrowth and joint segmentation. These functions appear to be mediated by reorganization of the actin cytoskeleton and modification of the adhesive properties of the mesenchymal cells.     

Palmitoylation of Hedgehog proteins by Hedgehog acyltransferase: roles in signalling and disease

Hedgehog acyltransferase (Hhat), a member of the membrane-bound O-acyltransferase (MBOAT) family, catalyses the covalent attachment of palmitate to the N-terminus of Hedgehog proteins. Palmitoylation is a post-translational modification essential for Hedgehog signalling. This review explores the mechanisms involved in Hhat acyltransferase enzymatic activity, similarities and differences between Hhat and other MBOAT enzymes, and the role of palmitoylation in Hedgehog signalling. In vitro and cell-based assays for Hhat activity have been developed, and residues within Hhat and Hedgehog essential for palmitoylation have been identified. In cells, Hhat promotes the transfer of palmitoyl-CoA from the cytoplasmic to the luminal side of the endoplasmic reticulum membrane, where Shh palmitoylation occurs. Palmitoylation is required for efficient delivery of secreted Hedgehog to its receptor Patched1, as well as for the deactivation of Patched1, which initiates the downstream Hedgehog signalling pathway. While Hhat loss is lethal during embryogenesis, mutations in Hhat have been linked to disease states or abnormalities in mice and humans. In adults, aberrant re-expression of Hedgehog ligands promotes tumorigenesis in an Hhat-dependent manner in a variety of different cancers, including pancreatic, breast and lung. Targeting hedgehog palmitoylation by inhibition of Hhat is thus a promising, potential intervention in human disease.     

Sonic hedgehog signaling during digit pattern duplication after application of recombinant protein and expressing cells

HoxD expression and cartilage pattern formation were compared after application of a recombinant amino-terminal peptide of Sonic hedgehog protein (Shh-N) and implantation of cells expressing the Sonic hedgehog (Shh) gene. During digit duplication after implantation of a Shh-N-soaked bead, BMP-2 and Patched expression was transiently induced in the anterior limb mesenchyme 20 h after grafting, but was reduced to the basal level 48 h after grafting. On the contrary, when Shh-expressing cells were grafted to the anterior limb bud, expression domains of the BMP-2 and Patched genes were initially induced in the restricted region in close proximity to the grafted cells. Induced expression of BMP-2 and Patched was maintained in the anterior-peripheral region of the limb bud for 42 h after grafting. In either case, HoxD12 and HoxD13 were consistently induced in the anterior-distal limb mesenchyme, accompanying mirror-image duplication of the digit pattern. Induction and maintenance of HoxD expression were consistent with the resultant digit pattern. A steep gradient of Shh activity provided by Shh-expressing cells is most adequate to induce complete digit pattern, as compared to the shallow gradient provided by Shh-N protein released from a bead. These results suggest that positional identity is respecified by Shh-N activity within the first 24 h during digit duplication, and that Shh-N on its own is not acting as a long-range signaling molecule to determine positional identity at a distance in the limb bud.     

Gli2 and Gli3 play distinct roles in the dorsoventral patterning of the mouse hindbrain

Sonic Hedgehog (Shh) signaling plays a critical role during dorsoventral (DV) patterning of the developing neural tube by modulating the expression of neural patterning genes. Overlapping activator functions of Gli2 and Gli3 have been shown to be required for motoneuron development and correct neural patterning in the ventral spinal cord. However, the role of Gli2 and Gli3 in ventral hindbrain development is unclear. In this paper, we have examined DV patterning of the hindbrain of Shh(-/-), Gli2(-/-) and Gli3(-/-) embryos, and found that the respective role of Gli2 and Gli3 is not only different between the hindbrain and spinal cord, but also at distinct rostrocaudal levels of the hindbrain. Remarkably, the anterior hindbrain of Gli2(-/-) embryos displays ventral patterning defects as severe as those observed in Shh(-/-) embryos suggesting that, unlike in the spinal cord and posterior hindbrain, Gli3 cannot compensate for the loss of Gli2 activator function in Shh-dependent ventral patterning of the anterior hindbrain. Loss of Gli3 also results in a distinct patterning defect in the anterior hindbrain, including dorsal expansion of Nkx6.1 expression. Furthermore, we demonstrate that ventral patterning of rhombomere 4 is less affected by loss of Gli2 function revealing a different requirement for Gli proteins in this rhombomere. Taken together, these observations indicate that Gli2 and Gli3 perform rhombomere-specific function during DV patterning of the hindbrain.     

Gestational ethanol exposure disrupts the expression of FGF8 and Sonic hedgehog during limb patterning

BACKGROUND: Ethanol is known to induce a wide variety of gestational anomalies, including skeletal malformations. Gestational ethanol exposure in mice has been shown to induce postaxial digit loss (ectrodactyly). How ethanol induces limb malformations is not understood. To better understand how ethanol effects limb development, we have utilized a transgenic line of mice that expresses beta-galactosidase in the apical ectodermal ridge (AER) of the limbs throughout gestation. METHODS: Pregnant female mice were injected with 2.9, 3.4, or 3.9 gm/kg ethanol at E9.3 and E9.5; embryos were isolated at E11.25, stained for beta-galactosidase activity, and evaluated for AER defects. Based upon the pattern of defects seen, expression of FGF8 in the AER and Sonic hedgehog in the postaxial mesoderm was evaluated by in situ hybridization. RESULTS: Two distinct phenotypes were seen in response to ethanol that were dose dependent. At 2.9 gm/kg ethanol, the most prevalent phenotype was a mislocalization of the AER to regions both dorsal and ventral to the midline. A higher dosage of 3.4 gm/kg ethanol did not increase the mislocalization phenotype, but resulted in a higher frequency of postaxial loss of the AER and associated mesenchymal tissue. The highest dosage utilized (3.9 gm/kg) resulted in a high frequency of both preaxial and postaxial loss of the AER. Through in situ hybridization, we found that ethanol exposure resulted in a concomitant reduction in FGF8 expression in the AER and Sonic hedgehog expression from the zone of polarizing activity (ZPA). CONCLUSIONS: We propose a model where ethanol disrupts the AER/ZPA positive feedback loop to induce postaxial malformations. Preaxial malformations seen at higher ethanol dosage suggest FGF8 as a critical target of ethanol in producing limb defects.     

Thalamic development induced by Shh in the chick embryo

Patterning of the early neural tube is achieved in part by the inductive signals, which arise from neuroepithelial signaling centers. The zona limitans intrathalamica (ZLI) is a neuroepithelial domain in the alar plate of the diencephalon which separates the prethalamus from the thalamus. The ZLI has recently been considered to be a possible secondary organizer, effecting its inductions via sonic hedgehog (Shh), a signaling molecule which drives morphogenetic information for the thalamus. Using experimental embryological techniques involving the generation of chimeric embryos, we show that the formation of the ZLI in the diencephalic alar plate is due to an interaction between the prechordal and epichordal plate neuroepithelia. We also provide evidence that Shh expression in the ZLI underlies the morphogenetic activity of this putative diencephalic organizer. Ectopic Shh led to the auto-induction of its own gene expression in host cells, as well as to the expression of other genes involved in diencephalic regionalization and histogenesis. Analysis of long-term surviving embryos after Shh ectopic expression demonstrated that Shh was able to induce thalamic structures and local overgrowth. Overall, these results indicate that Shh expressed in the ZLI plays an important role in diencephalic growth and in the development of the thalamus.