Drosophila Segment Polarity Gene Research Articles

Combinatorial Activity of Pair-Rule Proteins on the Drosophila gooseberry Early Enhancer

The early expression of the Drosophila segment polarity gene gooseberry (gsb) is under the control of the pair-rule genes. We have identified a 514-bp enhancer which reproduces the early gsb expression pattern in transgenic flies. The transcription factor Paired (Prd) is the main activator of this enhancer in all parasegments of the embryo. It binds to paired- and homeodomain-binding sites, which are segregated on the enhancer. Using site-directed mutagenesis, we have identified sites critical for Prd activity. Negative regulation of this enhancer is mediated by the Even-skipped protein (Eve) in the odd-numbered parasegments and by the combination of Fushi-tarazu (Ftz) and Odd-skipped proteins in the even-numbered parasegments. The organisation of the Prd-binding sites, as well as the necessity for intact DNA binding sites for both paired- and homeodomains, suggests a molecular model whereby the two DNA-binding domains of the Prd protein cooperate in transcriptional activation of gsb. This positive activity appears to be in competition with Eve and Ftz on Prd homeodomain-binding sites.

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.

Dispatched protein releases Hedgehog from cells

A novel Drosophila segment polarity gene is identified and shown to be required for release of Hedgehog protein from secreting cells.

Naked cuticle encodes an inducible antagonist of Wnt signalling.

During animal development, cells have to respond appropriately to localized secreted signals. Proper responses to Hedgehog, transforming growth factor-beta, epidermal growth factor and fibroblast growth factor/Ras signals require cognate inducible antagonists such as Patched, Dad, Argos and Sprouty. Wnt signals are crucial in development and neoplasia. Here we show that naked cuticle (nkd), a Drosophila segment-polarity gene, encodes an inducible antagonist for the Wnt signal Wingless (Wg). In fly embryos and imaginal discs nkd transcription is induced by Wg. In embryos, decreased nkd function has an effect similar to excess Wg; at later stages such a decrease appears to have no effect. Conversely, overproduction of Nkd in Drosophila and misexpression of Nkd in the vertebrate Xenopus laevis result in phenotypes resembling those of loss of Wg/Wnt function. nkd encodes a protein with a single EF hand (a calcium-binding motif) that is most similar to the recoverin family of myristoyl switch proteins. Nkd may therefore link ion fluxes to the regulation of the potency, duration or distribution of Wnt signals. Signal-inducible feedback antagonists such as nkd may limit the effects of Wnt proteins in development and disease.

Circadian Rhythm of Patched1 Transcription in the Pineal Regulated by Adrenergic Stimulation and cAMP

The tumor suppressor patched1 (PTC1), a product of the mammalian homologue of the Drosophila segment polarity gene patched, is a receptor for hedgehog (HH) and is crucial for embryonic development. Although little is known about the signal transduction pathways leading to the activation of ptc1, increased ptc1 transcription has always been associated with elevated HH activity and decreased activity of cAMP-dependent protein kinase A. Here, we demonstrate that in the mammalian pineal gland, ptc1 expression exhibits a dramatic diurnal rhythm with peak expression at midnight. ptc1 mRNA expression in the pineal is regulated by a clock mechanism mediated by the superior cervical ganglion. Most importantly, ptc1 transcription can be induced by agents activating the cAMP signal transduction pathway both in vivo and in vitro and appears to be independent of HH signaling.

Segment polarity genes in neuroblast formation and identity specification during Drosophila neurogenesis.

The relatively simple central nervous system (CNS) of the Drosophila embryo provides a useful model system for investigating the mechanisms that generate and pattern complex nervous systems. Central to the generation of different types of neurons by precursor neuroblasts is the initial specification of neuroblast identity and the Drosophila segment polarity genes, genes that specify regions within a segment or repeating unit of the Drosophila embryo, have emerged recently as significant players in this process. During neurogenesis the segment polarity genes are expressed in the neuroectodermal cells from which neuroblasts delaminate and they continue to be expressed in neuroblasts and their progeny. Loss-of-function mutations in these genes lead to a failure in the formation of neuroblasts and/or specification of neuroblast identity. Results from several recent studies suggest that regulatory interactions between segment polarity genes during neurogenesis lead to an increase in the number of neuroblasts and specification of different identities to neuroblasts within a population of cells.

The patched signaling pathway in tumorigenesis and development: lessons from animal models.

The identification of mutations in the human homolog of the Drosophila segment polarity gene Patched in basal cell carcinoma has sparked intense interest in the role of this gene in human disorders. The transmembrane protein Patched is a receptor for the morphogene Sonic Hedgehog. Sonic Hedgehog/Patched signaling involves another transmembrane protein, Smoothened, and its intracellular effectors, including the proto-oncogene GLI1. During the past 2 years it has become evident that mutations in Patched or in one of the components of its signaling pathway contribute to the formation of several common human tumors. It is now well established that Patched is a tumor suppressor gene. The Sonic Hedgehog/Patched/Smoothened signaling pathway is thus rapidly emerging as one of the most important regulators of oncogenic transformation. This pathway also plays an important role during mammalian embryonic development. This dual role is especially visible in humans with inherited Patched mutations. Such patients suffer from Gorlin, or nevoid basal cell carcinoma, syndrome and exhibit a variety of developmental defects accompanied by a predisposition to tumor formation. Activating mutations in Sonic Hedgehog and Smoothened lead to similar phenotypes as do loss-of function mutations in Patched. By means of transgenic and gene targeting technologies the respective mutations have been expressed in the mouse. Such mutant mouse strains exhibit many symptoms observed in humans. These strains are useful models to study the pathogenesis of several common human tumors and developmental defects. Furthermore they provide important tools to study the Sonic Hedgehog/Patched/Smoothened signaling at the molecular and biochemical level.

Overexpression of the human homologue of Drosophila patched (PTCH) in skin tumours: specificity for basal cell carcinoma.

The human homologue of the Drosophila segment polarity gene patched (PTCH) has been identified as the gene for the naevoid basal cell carcinoma (BCC) syndrome and has also been shown to be mutated in sporadic BCC. In order to elucidate the specificity of the PTCH abnormality in BCC, we examined normal skin and 12 BCC and 24 other types of tumour from Japanese patients for expression of the PTCH transcript by competitive reverse transcription-polymerase chain reaction, as mutational inactivation of PTCH leads to overexpression of the mutant transcript owing to failure of a negative feedback mechanism. We found a high level of PTCH expression in all 12 BCCs, while 23 of the other tumours and four specimens of normal skin showed no or weak expression of the gene, with the exception of one specimen from a patient with Bowen's disease which had high expression. These results indicate that the PTCH abnormality plays a critical role in the pathogenesis of BCC.

8 Somitogenesis in Zebrafish

Both genetic and embryological studies in the zebrafish, Danio rerio, have contributed to our general understanding of how somites form and differentiate. In the zebrafish, mutants have been isolated that have specific effects on virtually every aspect of somite development. The fss-type mutants, defining 5 genes, affect somite segmentation and epithelialization. The you-type mutants, comprising 7 genes, and mutants in another 13 genes defective in notochord formation, have somites with abnormal pattern and morphology. Eighteen genes have been identified that are required for the differentiation and maintenance of the somitic musculature, and 2 genes have been identified that are involved in the development of motoneurons that innervate the somitic musculature. The true utility of the zebrafish lies in the ability to combine genetic analysis with embryological experimentation. Such analysis of somite segmentation suggests that homologues of both the Drosophila pair-rule and segment polarity genes, her1 and Sonic hedge-hog, respectively, are involved generating periodicity during somitogenesis. The Sonic hedge-hog protein secreted from the notochord also induces the formation of specific muscle types including the slow muscle fibers which are initially induced in the medial somite and undergo a series of morphological transitions including migration through the somite to the lateral surface where they complete their differentiation. The role of the notochord in patterning the somite is also demonstrated by its involvement in regulating the permissiveness of the somite to the extension of axons of primary motoneurons.

Apical ectodermal ridge induction by the transplantation of En-1-overexpressing ectoderm in chick limb bud.

In the early chick embryo, the dorsal ventral (DV) boundary organizes the apical ectodermal ridge (AER) structure in the limb bud field. Here it is reported that Engrailed-1 (En-1), a homolog of the Drosophila segment polarity gene engrailed expressed in the ventral limb ectoderm, participates in AER formation at the DV boundary of the limb bud. Restricted ectopic expression of En-1 in the dorsal side of the limb bud by transplantation of En-1-overexpressing ectoderm induces ectopic AER at the boundary of En-1-positive and -negative cells. The results suggest that En-1 is involved in AER formation at the DV boundary of the limb bud.

Rhabdomyosarcomas and radiation hypersensitivity in a mouse model of Gorlin syndrome.

Gorlin (or nevoid basal cell carcinoma) syndrome is characterized by a variety of clinical problems including generalized overgrowth of the body, cysts, developmental abnormalities of the skeleton and a predisposition to benign and malignant tumors. The syndrome results from germline mutations of the human homolog of the drosophila segment polarity gene patched (ptc). Here we report that mice heterozygous for ptc develop many of the features characteristic of Gorlin syndrome and that they exhibit a high incidence of rhabdomyosarcomas (RMS), the most common soft-tissue sarcoma in children. The downstream signalling partner of ptc, gli1, was overexpressed in all RMSs analyzed, indicating that abnormal signalling of the ptc-gli1 pathway may be common for the various tumors associated with the syndrome. igf2, implicated in the formation of RMSs, was also overexpressed, suggesting cross-talk between the ptc and igf2 pathways in tumorigenesis. Developmental defects in Gorlin syndrome resemble those induced by ionizing radiation. We show that ptc heterozygous mice exhibit increased incidence of radiation-induced teratogenesis. This suggests a role for ptc in the response to ionizing radiation and provides a model for both the systemic (developmental) and stochastic (cancer) abnormalities observed in Gorlin syndrome.

Analysis of the human Sonic Hedgehog coding and promoter regions in sacral agenesis, triphalangeal thumb, and mirror polydactyly.

The human Sonic Hedgehog gene (SHH) is one of the vertebrate homologs related to the Drosophila segment polarity gene hedgehog. The entire coding and promoter region of the SHH gene, including 2 kb 5' of the transcriptional start site has been screened for mutations in families with autosomal dominant sacral agenesis and autosomal dominant triphalangeal thumb, two conditions previously known to be linked to 7q36. We have also studied the SHH gene in five families with mirror polydactyly associated with tibial hemimelia and in 51 unrelated patients with neural tube defects. Except for two sequence variants in exon 3, no mutations were found in these disease categories. OFF

Mutations in the human homologue of theDrosophila patched gene in esophageal squamous cell carcinoma

The human homologue (PTCH) of the Drosophila segment polarity gene patched has recently been identified as a tumor-suppressor gene for nevoid basal cell carcinoma syndrome and for sporadic basal cell carcinomas of the skin. We analyzed 30 esophageal squamous cell carcinomas (ESCC) for intrageneic mutations of the PTCH gene by polymerase chain reaction-single-strand conformation polymorphism analysis followed by DNA sequencing. We identified two somatic PTCH mutations (7%) in 30 ESCC. These were a nonsense mutation (CAG to TAG at codon 361) in exon 8 and a missense mutation (CAG to CTG, Gln to Leu at codon 816) in exon 14. These tumors exhibited loss of heterozygosity at the polymorphic site of the PTCH gene. These results indicate that inactivation of the PTCH gene via a two-hit mechanism occurs in a subset of ESCC.

Mutations in the human homologue of the Drosophila patched gene in esophageal squamous cell carcinoma

The human homologue (PTCH) of the Drosophila segment polarity gene patchedhas recently been identified as a tumor-suppressor gene for nevoid basal cell carcinoma syndrome and for sporadic basal cell carcinomas of the skin. We analyzed 30 esophageal squamous cell carcinomas (ESCC) for intrageneic mutations of the PTCH gene by polymerase chain reaction–single-strand conformation polymorphism analysis followed by DNA sequencing. We identified two somatic PTCH mutations (7%) in 30 ESCC. These were a nonsense mutation (CAG to TAG at codon 361) in exon 8 and a missense mutation (CAG to CTG, Gln to Leu at codon 816) in exon 14. These tumors exhibited loss of heterozygosity at the polymorphic site of the PTCH gene. These results indicate that inactivation of the PTCH gene via a two-hit mechanism occurs in a subset of ESCC. Genes Chromosomes Cancer 21:276–279, 1998. © 1998 Wiley-Liss, Inc.

No evidence for the H133Y mutation in SONIC HEDGEHOG in a collection of common tumour types.

The human homologue of the Drosophila segment polarity gene patched is mutated in the cancer predisposition syndrome naevoid basal cell carcinoma syndrome (NBCCS), as well as in several types of tumour associated with the disorder. It was recently reported that a single recurrent mutation in the SONIC HEDGEHOG gene, which encodes the PATCHED ligand, was found in one of 43 basal cell carcinomas (BCCs), one of 14 medulloblastomas and one of six breast carcinomas analysed (Oro et al., 1997). We have searched extensively for this same mutation in a large collection of BCCs, medulloblastomas and carcinomas of the breast, ovary and colorectum and have failed to detect the mutation in any sample analysed.

Expression of the Armadillo family member p120 cas 1B in Xenopus embryos affects head differentiation but not axis formation

The Armadillo family is formed by proteins which possess an Arm domain comprising multiple copies of a 42-amino-acid motif, the Arm repeat, initially described for the Drosophila segment polarity gene product Armadillo. The Arm domain serves in protein-protein interactions which are required for the family members Armadillo, beta-catenin and plakoglobin to mediate cell-cell adhesion and Wnt/Wingless signalling. Similarily, p120cas, the Arm domain containing src substrate, also binds to cadherins and becomes tyrosine phosphorylated in response to a variety of stimuli. However, a putative function of p120cas in adhesion or signalling has not yet been demonstrated. It has also not been shown until now that an Arm domain is a common signal transduction motif. Using Xenopus embryos we show by expression of murine p120cas1B (mp120cas1B) in ventral blastomeres that this catenin cannot replace beta-catenin function in dorsal axis formation. Thus, the presence of an Arm domain per se is not sufficient to activate the Wnt/Wg pathway. Indeed, injection of mp120cas1B into dorsal blastomeres led instead to delayed blastopore closure and posteriorized phenotypes with malformed head structures indicative of disturbed gastrulation movements. Because neither convergent extension behaviour nor adhesion to fibronectin was altered in the injected embryos we assume that mp120cas1B influences motility or orientation of migrating mesodermal cells.

The patched gene in development and cancer

The cloning of vertebrate homologues of the Drosophila segment polarity gene patched has led to confirmation of a role for the multipass transmembrane protein which it encodes as a receptor for secreted signalling proteins of the Hedgehog family. In addition, human patched has been identified as a tumour suppressor gene implicated in basal cell carcinomas and medullablastomas.

Zebrafish wnt11: pattern and regulation of the expression by the yolk cell and No tail activity

This study analyzed the spatial and temporal expression pattern of zebrafish wnt11 and the regulation of the expression during zebrafish early development, focusing on the interaction with the no tail (ntl) gene, a zebrafish orthologue of mouse Brachyury (T). Zygotic expression of wnt11 was first detected at the late blastula stage in the blastoderm margin, a presumptive mesoderm region. wnt11 expression coincided with mesoderm induction, and the expression was induced by mesoderm inducers such as the yolk cell (Mizuno, T., Yamaha, E., Wakahara, M., Kuroiwa, A., Takeda, H., 1996. Mesoderm induction in zebrafish. Nature 383, 131–132) or fibroblast growth factors (FGFs), indicating that, like ntl, wnt11 is one of the immediate-early genes in mesoderm induction. Initial expression domains of wnt11 and ntl overlapped, and these genes showed a similar response to mesoderm inducers. However, analysis of the ntl mutant embryos suggested that wnt11 and ntl are placed in distinct genetic pathways; the ntl mutation had no effect on wnt11 expression in the blastoderm margin. This was further supported by the result of RNA injection experiments showing that overexpression of Wnt11 did not affect ntl expression in the margin. Thus, wnt11 and ntl expression are induced and maintained independently in their initial phase of expression. In later stages, wnt11 was expressed in various organs, such as the somites, particularly in the developing notochord. Since no wnt gene has been reported to be expressed in the axial mesoderm, which is known to act as a signaling source that patterns the neural tube and somites, zebrafish wnt11 is the first wnt gene expressed in the notochord. Furthermore, in contrast to early expression, wnt11 expression in the notochord depended on Ntl activity. In the ntl mutant in which somite patterning is severely affected, wnt11 expression was completely lost, while another signaling molecule, sonic hedgehog is expressed in the mutant notochord precursor cells (Krauss, S., Concordet, J.-P., Ingham, P.W., 1993. A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos. Cell 75, 1431–1444). wnt11 expression in the somite also shows a characteristic pattern, correlated with the migration and differentiation of slow muscle precursors. These observations suggest a role for wnt11 in patterning the somites.

Zebrafish segmentation and pair‐rule patterning

Segmentation in the vertebrate embryo is evident within the paraxial mesoderm in the form of somites, which are repeated structures that give rise to the vertebrae and muscle of the trunk and tail. In the zebrafish, our genetic screen identified two groups of mutants that affect somite formation and pattern. Mutations of one class, the fss-type mutants, disrupt the formation of the anterior-posterior somite boundaries during somitogenesis. However, segmentation within the paraxial mesoderm is not completely eliminated in these mutants. Irregular somite boundaries form later during embryogenesis and, strikingly, the vertebrae are not fused. Here, we show that formation of the irregular somite boundaries in these mutants is dependent upon the activity of a second group of genes, the you-type genes, which include sonic you, the zebrafish homologue of the Drosophila segment polarity gene, sonic hedgehog. Further to characterize the defects caused by the fss-type mutations, we examined their effects on the expression of her1, a zebrafish homologue of the Drosophila pair-rule gene hairy. In wild-type embryos, her1 is expressed in a dynamic, repeating pattern, remarkably similar to that of its Drosophila and Tribolium counterparts, suggesting that a pair-rule mechanism also functions in the segmentation of the vertebrate paraxial mesoderm. We have found that the fss-type mutants have abnormal pair-rule patterning. Although a her1 mutant could not be identified, analysis of a double mutant that abolishes most her1 expression suggests that a her1 mutant may not display a pair-rule phenotype analogous to the hairy phenotype observed in Drosophila. Cumulatively, our data indicate that zebrafish homologues of both the Drosophila segment polarity genes and pair-rule genes are involved in segmenting the paraxial mesoderm. However, both the relationship between these two groups of genes within the genetic heirarchy governing segmentation and the precise roles that they play during segmentation likely differ significantly between the two organisms. Dev. Genet. 23:65–76, 1998. © 1998 Wiley-Liss, Inc.

Gorlin syndrome: Identification of 4 novel germ‐line mutations of the human patched (PTCH) gene

PTCH, the human homologue of the Drosophila segment polarity gene, patched, has been identified as the gene responsible for Gorlin or nevoid basal cell carcinoma syndrome (NBCCS). We report here the characterization of four novel mutations in the human PTCH gene in germ-line DNA from Gorlin patients. All mutations lead to truncation of the predicted protein product. Also included is a list of putative polymorphic nucleotide positions in the sequence covered by the published primers. Hum Mutat 11:480, 1998. © 1998 Wiley-Liss, Inc.