Since their isolation in the early 1990s, members of the Hedgehog family of intercellular signaling proteins have come to be recognized as key mediators of many fundamental processes in embryonic development. Their activities are central to the growth, patterning, and morphogenesis of many different regions within the body plans of vertebrates and insects, and most likely other invertebrates. In some contexts, Hedgehog signals act as morphogens in the dose-dependent induction of distinct cell fates within a target field, in others as mitogens regulating cell proliferation or as inducing factors controlling the form of a developing organ. These diverse functions of Hedgehog proteins raise many intriguing questions about their mode of operation. How do these proteins move between or across fields of cells? How are their activities modulated and transduced? What are their intracellular targets? In this article we review some well-established paradigms of Hedgehog function inDrosophila and vertebrate development and survey the current understanding of the synthesis, modification, and transduction of Hedgehog proteins. Embryological studies over much of the last century that relied primarily on the physical manipulation of cells within the developing embryo or fragments of the embryo in culture, provided many compelling examples for the primacy of cell–cell interactions in regulating invertebrate and vertebrate development. The subsequent identification of many of the signaling factors that mediate cellular communication has led to two general conclusions. First, although there are many important signals, most of these fall into a few large families of secreted peptide factors: theWnt (Wodarz and Nusse 1998), fibroblast growth factor (Szebenyi and Fallon 1999), TGFsuperfamily (Massague and Chen 2000), plateletderived growth factor (Betsholtz et al. 2001), ephrin (Bruckner and Klein 1998), and Hedgehog families. Second, parallel studies in invertebrate and vertebrate systems have shown that although the final outcome might look quite different (e.g., a fly vs. a mouse), there is a striking conservation in the deployment of members of the same signaling families to regulate development of these seemingly quite different organisms. This review focuses on one of the most intriguing examples of this phenomenon, that of the Hedgehog family. As with many of the advances in our understanding of the genetic regulation of animal development, hedgehog (hh) genes owe their discovery to the pioneering work of Nusslein-Volhard and Wieschaus (1980). In their screen for mutations that disrupt the Drosophila larval body plan, these authors identified several that cause the duplication of denticles (spiky cuticular processes that decorate the anterior half of each body segment) and an accompanying loss of naked cuticle, characteristic of the posterior half of each segment (see Fig. 1). The ensuing appearance of a continuous lawn of denticles projecting from the larval cuticle evidently suggested the spines of a hedgehog to the discoverers, hence the origin of the name of one of these genes. Other loci identified by mutants with this phenotype included armadillo, gooseberry, and wingless (wg). In contrast, animals mutant for the aptly named naked gene showed the converse phenotype, with denticle belts replaced by naked cuticle in every segment. On the basis of these mutant phenotypes, Nusslein-Volhard and Wieschaus (1980) proposed that these so-called segment-polarity genes regulate pattern within each of the segments of the larval body, individual genes acting within distinct subregions of the emerging segmental pattern. The first important breakthrough in unraveling how segment-polarity genes act came in the mid-1980s with the cloning of two members of the class, wingless and engrailed (en). Wg was shown to be the ortholog of the vertebrate proto-oncogene int1 (subsequently renamed Wnt1 and the founder member of the Wnt family of secreted peptide factors; Rijsewijk et al. 1987), whereas the sequence of en revealed that it encodes a homeodomaincontaining transcription factor (Fjose et al. 1985; Poole et al. 1985). Intriguingly, the two genes were found to be expressed in adjacent narrow stripes of cells in each segment (Martinez Arias et al. 1988). A close spatial relationship between Wnt1 and En expression domains was also reported in the primordial midbrain and hindbrain of the vertebrate embryo (McMahon et al. 1992). AnalyWe dedicate this review to the memory of our dear friend and colleague Rosa Beddington, whose encouragement led to our initial collaboration. 3Corresponding authors. E-MAIL p.w.ingham@sheffield.ac.uk; FAX 0114-222-288. E-MAIL amcmahon@biosun.harvard.edu; FAX (617) 496-3763. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ gad.938601.

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Hedgehog signaling in animal development: paradigms and principles.

Neural circuits are assembled with remarkable precision during embryonic development, and the selectivity inherent in their formation helps to define the behavioural repertoire of the mature organism. In the vertebrate central nervous system, this developmental program begins with the differentiation of distinct classes of neurons from progenitor cells located at defined positions within the neural tube. The mechanisms that specify the identity of neural cells have been examined in many regions of the nervous system and reveal a high degree of conservation in the specification of cell fate by key signalling molecules.

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Neuronal specification in the spinal cord: inductive signals and transcriptional codes

https://arep.med.harvard.edu/pdf/Wichterle02.pdf

Directed Differentiation of Embryonic Stem Cells into Motor Neurons

The PATCHED (PTC) gene encodes a Sonic hedgehog (Shh) receptor and a tumor suppressor protein that is defective in basal cell nevus syndrome (BCNS). Functions of PTC were investigated by inactivating the mouse gene. Mice homozygous for the ptc mutation died during embryogenesis and were found to have open and overgrown neural tubes. Two Shh target genes, ptc itself and Gli, were derepressed in the ectoderm and mesoderm but not in the endoderm. Shh targets that are, under normal conditions, transcribed ventrally were aberrantly expressed in dorsal and lateral neural tube cells. Thus Ptc appears to be essential for repression of genes that are locally activated by Shh. Mice heterozygous for the ptc mutation were larger than normal, and a subset of them developed hindlimb defects or cerebellar medulloblastomas, abnormalities also seen in BCNS patients.

http://science.sciencemag.org/content/sci/277/5329/1109.full.pdf

Altered Neural Cell Fates and Medulloblastoma in Mouse patched Mutants

Why are tumours heterogeneous, in terms of cell phenotype and proliferative potential, even in cases in which all cells are derived from a single clone? Ongoing mutagenesis can partially explain this heterogeneity, but it also seems that some tumours arise from small populations of 'cancer stem cells' that give rise to phenotypically diverse cancer cells, with less proliferative potential. These cancer stem cells are likely to arise from mutations that dysregulate normal stem-cell self-renewal. Using this information, it might be possible to devise more effective therapies.

Applying the principles of stem-cell biology to cancer

Targeted gene disruption in the mouse shows that the Sonic hedgehog (Shh) gene plays a critical role in patterning of vertebrate embryonic tissues, including the brain and spinal cord, the axial skeleton and the limbs. Early defects are observed in the establishment or maintenance of midline structures, such as the notochord and the floorplate, and later defects include absence of distal limb structures, cyclopia, absence of ventral cell types within the neural tube, and absence of the spinal column and most of the ribs. Defects in all tissues extend beyond the normal sites of Shh transcription, confirming the proposed role of Shh proteins as an extracellular signal required for the tissue-organizing properties of several vertebrate patterning centres.

Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function.

Essential role for Sonic hedgehog during hair follicle morphogenesis.

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II consists of tandem repeats of the consensus heptapeptide YSPTSPS. Deletion studies in tissue culture cells have indicated that the CTD plays an essential role in transcription, although the nature of this essential function remains unclear. About half of the CTD can be deleted without affecting the viability of cells in tissue culture. Paradoxically, the dispensable CTD repeats are precisely conserved among all mammals whose CTD sequences are known. To determine whether the mammalian CTD is important in transcription during mouse development, we developed a gene targeting approach to introduce deletions into the CTD coding region of mouse embryonic stem (ES) cells. To maintain a functional Rpo2-1 gene, the neo marker in the targeting vector was positioned outside of the Rpo2-1 transcribed region, 1.2 kb from the site of the CTD deletion. G418-resistant clones were screened for co-integration of the CTD deletion, and the resulting ES lines were used to create germline chimeric mice. Stable heterozygous lines were established and mated to produce animals homozygous for the CTD deletion. We show here that mice homozygous for a deletion of thirteen of the 52 heptapeptide repeats are smaller than wild-type littermates and have a high rate of neonatal lethality. Surviving adults, although small, appear morphologically normal and are fertile. This result suggests that the CTD plays a role in regulating growth during mammalian development. The gene targeting approach described here should be useful for making further deletions in the CTD and may be of general applicability where it is desirable to engineer specific mutations in the germline of mice.

Ying Litingtung

Papers

Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function.

Essential role for Sonic hedgehog during hair follicle morphogenesis.

Growth retardation and neonatal lethality in mice with a homozygous deletion in the C-terminal domain of RNA polymerase II.

Targeted disruption of Sonic hedgehog reveals an essential role in hair follicle morphogenesis