Smoothened, thickveins and the genetic control of cell cycle and cell fate in the developing Drosophila eye

The Hedgehog and Decapentaplegic pathways have several well-characterized functions in the developing Drosophila compound eye, including initiation and progression of the morphogenetic furrow. Other functions involve control of cell cycle and cell survival as well as cell type specification. Here we have used the mosaic clone analysis of null mutations of the smoothened and thickveins genes (which encode the receptors for these two signals) both alone and in combination, to study cell cycle and cell fate in the developing eye. We conclude that both pathways have several, but differing roles in furrow induction and cell fate and survival, but that neither directly affects cell type specification.     

Zebrafish slow muscle cell migration induces a wave of fast muscle morphogenesis

The specification and morphogenesis of slow and fast twitch muscle fibers are crucial for muscle development. In zebrafish, Hedgehog is required for slow muscle fiber specification. However, less is known about signals that promote development of fast muscle fibers, which constitute the majority of somitic cells. We show that when Hedgehog signaling is blocked, fast muscle cell elongation is disrupted. Using genetic mosaics, we show that Hedgehog signal perception is required by slow muscle cells but not by fast muscle cells for fast muscle cell elongation. Furthermore, we show that slow muscle cells are sufficient to pattern the medial to lateral wave of fast muscle fiber morphogenesis even when fast muscle cells cannot perceive the Hedgehog signal. Thus, the medial to lateral migration of slow muscle fibers through the somite creates a morphogenetic signal that patterns fast muscle fiber elongation in its wake.     

Back and forth between cell fate specification and movement during vertebrate gastrulation

Animal body plan arises during gastrulation and organogenesis by the coordination of inductive events and cell movements. Several signaling pathways, such as BMP, FGF, Hedgehog, Nodal, and Wnt have well-recognized instructive roles in cell fate specification during vertebrate embryogenesis. Growing evidence indicates that BMP, Nodal, and FGF signaling also regulate cell movements, and that they do so through mechanisms distinct from those that specify cell fates. Moreover, pathways controlling cell movements can also indirectly influence cell fate specification by regulating dimensions and relative positions of interacting tissues. The current challenge is to delineate the molecular mechanisms via which the major signaling pathways regulate cell fate specification and movements, and how these two processes are coordinated to ensure normal development.     

Robustness of positional specification by the Hedgehog morphogen gradient

Spatial gradients of Hedgehog signalling play a central role in many patterning events during animal development, regulating cell fate determination and tissue growth in a variety of tissues and developmental stages. Experimental evidence suggests that many of the proteins responsible for regulating Hedgehog signalling and transport are themselves targets of Hedgehog signalling, leading to multiple levels of feedback within the system. We use mathematical modelling to analyse how these overlapping feedbacks combine to regulate patterning and potentially enhance robustness in the Drosophila wing imaginal disc. Our results predict that the regulation of Hedgehog transport and stability by glypicans, as well as multiple overlapping feedbacks in the Hedgehog response network, can combine to enhance the robustness of positional specification against variability in Hedgehog levels. We also discuss potential trade-offs between robustness and additional features of the Hedgehog gradient, such as signalling range and size regulation.     

The you gene encodes an EGF-CUB protein essential for Hedgehog signaling in zebrafish

Hedgehog signaling is required for many aspects of development in vertebrates and invertebrates. Misregulation of the Hedgehog pathway causes developmental abnormalities and has been implicated in certain types of cancer. Large-scale genetic screens in zebrafish have identified a group of mutations, termed you-class mutations, that share common defects in somite shape and in most cases disrupt Hedgehog signaling. These mutant embryos exhibit U-shaped somites characteristic of defects in slow muscle development. In addition, Hedgehog pathway mutations disrupt spinal cord patterning. We report the positional cloning of you, one of the original you-class mutations, and show that it is required for Hedgehog signaling in the development of slow muscle and in the specification of ventral fates in the spinal cord. The you gene encodes a novel protein with conserved EGF and CUB domains and a secretory pathway signal sequence. Epistasis experiments support an extracellular role for You upstream of the Hedgehog response mechanism. Analysis of chimeras indicates that you mutant cells can appropriately respond to Hedgehog signaling in a wild-type environment. Additional chimera analysis indicates that wild-type you gene function is not required in axial Hedgehog-producing cells, suggesting that You is essential for transport or stability of Hedgehog signals in the extracellular environment. Our positional cloning and functional studies demonstrate that You is a novel extracellular component of the Hedgehog pathway in vertebrates.     

Lineage-tracing cells born in different domains along the PD axis of the developing Drosophila leg.

Patterning of the developing limbs by the secreted signaling proteins Wingless, Hedgehog and Dpp takes place while the imaginal discs are growing rapidly. Cells born in regions of high ligand concentration may be displaced through growth to regions of lower ligand concentration. We have used a novel lineage-tagging method to address the reversibility of cell fate specification by morphogen gradients. We find that responses to Hedgehog and Dpp in the wing disc are readily reversible. In the leg, we find that cells readily adopt more distal fates, but do not normally shift from distal to proximal fate. However, they can do so if given a growth advantage. These results indicate that cell fate specification by morphogen gradients remains largely reversible while the imaginal discs grow. In other systems, where growth and patterning are uncoupled, nonreversible specification events or 'ratchet' effects may be of functional significance.     

pitx3 defines an equivalence domain for lens and anterior pituitary placode

Hedgehog signaling is required for formation and patterning of the anterior pituitary gland. However, the role of Hedgehog in pituitary precursor cell specification and subsequent placode formation is not well understood. We analyzed pituitary precursor cell lineages and find that pitx3 and distal-less3b (dlx3b) expression domains define lens and pituitary precursor positions. We show that pitx3 is required for pituitary pre-placode formation and cell specification, whereas dlx3b and dlx4b are required to restrict pituitary placode size. In smoothened mutant embryos that cannot transduce Hedgehog signals, median pituitary precursors are mis-specified and form an ectopic lens. Moreover, overexpression of sonic hedgehog (shh) blocks lens formation, and derivatives of lens precursors express genes characteristic of pituitary cells. However, overexpression of shh does not increase median pituitary placode size nor does it upregulate patched (ptc) expression in pituitary precursors during early somitogenesis. Our study suggests that by the end of gastrulation, pitx3-expressing cells constitute an equivalence domain of cells that can form either pituitary or lens, and that a non-Hedgehog signal initially specifies this placodal field. During mid-somitogenesis, Hedgehog then acts on the established median placode as a necessary and sufficient signal to specify pituitary cell types.     

Intra-endodermal interactions are required for pancreatic beta cell induction

The cellular origin of signals that regulate pancreatic beta cell induction is not clearly defined. Here, we investigate the seeming paradox that Hedgehog/Smoothened signaling functions during gastrulation to promote pancreatic beta cell development in zebrafish, yet has an inhibitory role during later stages of pancreas development in amniotes. Our cell transplantation experiments reveal that in zebrafish, Smoothened function is not required in beta cell precursors. At early somitogenesis stages, when the zebrafish endoderm first forms a sheet, pancreatic beta cell precursors lie closest to the midline; however, the requirement for Smoothened lies in their lateral neighbors, which ultimately give rise to the exocrine pancreas and intestine. Thus, pancreatic beta cell induction requires Smoothened function cell-nonautonomously during gastrulation, to allow subsequent intra-endodermal interactions. These results clarify the function of Hedgehog signaling in pancreas development, identify an unexpected cellular source of factors that regulate beta cell specification, and uncover complex patterning and signaling interactions within the endoderm.     

Hedgehog induces opposite changes in turnover and subcellular localization of patched and smoothened

Secreted signaling proteins of the Hedgehog family organize spatial pattern during animal development. Two integral membrane proteins have been identified with distinct roles in Hedgehog signaling. Patched functions in Hedgehog binding, and Smoothened functions in transducing the signal. Current models view Patched and Smoothened as a preformed receptor complex that is activated by Hedgehog binding. Here we present evidence that Patched destabilizes Smoothened in the absence of Hedgehog. Hedgehog binding causes removal of Patched from the cell surface. In contrast, Hedgehog causes phosphorylation, stabilization, and accumulation of Smoothened at the cell surface. Comparable effects can be produced by removing Patched from cells by RNA-mediated interference. These findings raise the possibility that Patched acts indirectly to regulate Smoothened activity.     

Life, death and Sonic hedgehog

The secreted glycoprotein Sonic hedgehog (SHH), a vertebrate homologue of the Drosophila segment polarity gene Hedgehog, is essential for the development of diverse tissues during embryogenesis. Studies of SHH function during neural tube and somite development have focused on its role in specifying the dorsoventral polarity of these structures, but a recent report by Ahlgren and Bronner-Fraser(1) supports the possibility that SHH has additional functions in cell survival and cell proliferation. Perturbation of SHH signaling after the early dorsoventral specification of the cranial neural tube leads to increased cell death in both the neural tube and the neural crest. This implies that SHH is continually required as a trophic and/or mitogenic factor during brain development, and expands the variety of cellular responses to SHH signaling. BioEssays 22:499-502, 2000.     

New "hogs" in Hedgehog transport and signal reception

A recent paper in Cell (Yao et al., 2006) and two papers in Developmental Cell (Tenzen et al., 2006; Zhang et al., 2006) identify a new receptor component for Hedgehog, a key morphogen in embryonic development. Many other proteins that bind to Hedgehog in the extracellular matrix or on the cell surface have been identified. In light of these recent discoveries, we discuss how these factors control the stability, transport, reception, and availability of Hedgehog in modulating Hedgehog-mediated responses.     

Hedgehog signalling: how to get from Smo to Ci and Gli

The secreted morphogens of the Hedgehog family have important roles in normal development as well as in associated pathologies, including cancer. The Hedgehog signalling pathway has been studied in Drosophila and is thought to be conserved in vertebrates. Hedgehog elicits a signalling response that activates Smoothened (Smo). There is evidence of differences between Drosophila and vertebrates concerning signalling downstream of Smo, as well as in Smo itself. Here, we discuss this evidence and its importance for investigations of the pathway and related biology, as well as for the development of drugs targeting components of the pathway for treatment of associated pathologies.     

A restrictive role for Hedgehog signalling during otic specification in Xenopus

Vertebrate inner ear development is initiated by the specification of the otic placode, an ectodermal structure induced by signals from neighboring tissue. Although several signaling molecules have been identified as candidate otic inducers, many details of the process of inner ear induction remain elusive. Here, we report that otic induction is responsive to the level of Hedgehog (Hh) signaling activity in Xenopus, making use of both gain- and loss-of-function approaches. Ectopic activation of Hedgehog signaling resulted in the development of ectopic vesicular structures expressing the otic marker genes XPax-2, Xdll-3, and Xwnt-3A, thus revealing otic identity. Induction of ectopic otic vesicles was also achieved by misexpression of two different inhibitors of Hh signaling: the putative Hh antagonist mHIP and XPtc1deltaLoop2, a dominant-negative form of the Hh receptor Patched. In addition, misexpression of XPtc1deltaLoop2 as well as treatment of Xenopus embryos with the specific Hh signaling antagonist cyclopamine resulted in the formation of enlarged otic vesicles. In summary, our observations suggest that a defined level of Hh signaling provides a restrictive environment for otic fate in Xenopus embryos.     

Proteins of the Hedgehog signaling pathway as therapeutic targets against cancer

The Hedgehog pathway plays a crucial role in growth and patterning during embryonic development and is involved in stem cell maintenance and proliferation in adult tissues. Mutations that increase the overall activity of the pathway are often associated with a higher incidence of cancers. This article focuses on the mutations, misfoldings and deregulations of the Hedgehog pathway proteins that have been reported to be involved in different tumors, and on small molecules targeting these proteins shown to slow down the growth of certain tumors in various animal models. We propose that proteomics could be a powerful tool to identify new targets of the Hedgehog pathway, enabling the discovery of effective and novel treatments for cancers.