Oroshigane, a New Segment Polarity Gene of Drosophila Melanogaster, Functions in Hedgehog Signal Transduction

Here we describe a new segment polarity gene of Drosophila melanogaster, oroshigane (oro). Identified as a dominant enhancer of Bar (B), oro is also recessive embryonic lethal, and homozygous oro embryos show variable substitution of naked cuticle with denticles. These patterns are distinctly similar to those of hedgehog (hh) and wingless (wg) embryos, which indicates that oro functions in determining embryonic segment polarity. Evidence that oro function is involved in Hh signal transduction during embryogenesis is provided by its genetic interactions with the segment polarity genes patched (ptc) and fused (fu). Furthermore, ptc(IN) is a dominant suppressor of the oro embryonic lethal phenotype, suggesting a close and dose-dependent relationship between oro and ptc in Hh signal transduction. oro function is also required in imaginal development. The oro(1) allele significantly reduces decapentaplegic (dpp), but not hh, expression in the eye imaginal disc. Furthermore, oro enhances the fu(1) wing phenotype in a dominant manner. Based upon the interactions of oro with hh, ptc, and fu, we propose that the oro gene plays important roles in Hh signal transduction.     

The torpedo (DER) receptor tyrosine kinase is required at multiple times during Drosophila embryogenesis.

The torpedo (DER) gene of Drosophila, which encodes a receptor tyrosine kinase of the EGF receptor subfamily, is essential for oogenesis, embryogenesis and imaginal disc development. To gain insight into the nature of the signals transduced by the torpedo product, we have characterized the gene's loss-of-function phenotype in the embryo. Through the induction of germline clones, we provide a genetic demonstration that maternal torpedo product does not contribute to zygotic development. Thus, the embryonic lethal phenotypes examined accurately reflect the consequences of eliminating all gene activity from the zygote. Temperature-shift experiments with the conditional allele topIF26 show that torpedo is required at two distinct times during embryonic development: the gene is first needed for germband retraction and for the production of anterior, posterior and ventral cuticle, then later for the secretion of ventral denticles. Since denticle formation can be severely disrupted in topIF26 animals without affecting cuticle production, the early and late requirements for torpedo appear to be functionally unrelated. torpedo, therefore, is required at multiple times in the development of the ventral epidermis, and may transduce qualitatively different signals. Since the early requirement for torpedo correlates with the first visible defect in embryonic development, increased cell death in the amnioserosa, cephalic ectoderm and ventral epidermis, the abnormalities in cuticle production and germband shortening seen in the mutant may be secondary consequences of a primary defect in cell viability. Given that the onset of cell death in torpedo embryos is not preceded by any obvious defects in mitogenesis, the establishment of cell identities or the maintenance of gene expression, it is possible that torpedo transduces a signal necessary for cell survival per se during early embryogenesis. During late embryogenesis, torpedo may mediate the reception of a second signal which regulates ventral epidermal cell differentiation.     

Developmental Genetics of the 2e-F Region of the Drosophila X Chromosome: A Region Rich in "Developmentally Important" Genes

We have analyzed the 2E1-3A1 area of the X chromosome with special attention to loci related to embryogenesis. Published maps indicate that this chromosomal segment contains ten bands. Our genetic analysis has identified 11 complementation groups: one recessive visible (prune), two female steriles and eight lethals. One of the female sterile loci is fs(1)k10 for which homozygous females produce both egg chambers and embryos with a dorsalized morphology. The second female sterile is the paternally rescuable fs(1)pecanex in which unrescued embryos have a hypertrophic nervous system. Of the eight lethal complementation groups two are recessive embryonic lethals: hemizygous giant (gt) embryos possess segmental defects, and hemizygous crooked neck (crn) embryos exhibit a twisted phenotype. Analysis of these mutations in the female germ line indicates that gt does not show a maternal effect, whereas normal activity of crn is required for germ cell viability. Analysis of the maternal effect in germ line clones of the remaining six recessive lethal complementation groups indicates that four are required for germ cell viability and one produces ambiguous results for survival of the germ cells. The remaining, l(1)pole hole, is a recessive early pupal lethal in which embryos derived from germ line clones and lacking wild-type gene activity exhibit the "torso" or "pole hole" phenotype.     

R-spondin2 expression in the apical ectodermal ridge is essential for outgrowth and patterning in mouse limb development

Mouse R-spondin2 (Rspo2) is a member of the R-spondin protein family, which is characterized by furin-like cysteine-rich domains and a thrombospondin type 1 repeat. R-spondin is a secreted molecule that activates Wnt/ β -catenin signaling. Rspo2 -deficient mice were generated to investigate the function of mouse Rspo2 during embryonic development. The homozygous mutant forelimb showed defects in distal phalanges and nail structures, and the digits were anomalous in shape. The homozygous mutant hindlimb showed more severe malformations, including lack of digits and zeugopod components. Rspo2 is expressed in the apical ectodermal ridge (AER) of the developing limb. Fgf8 expression in the AER was significantly lower in the homozygous mutant forelimb than in the wild-type forelimb and it was disturbed along the dorsoventral axis. In the homozygous mutant hindlimb, Fgf8 and Fgf4 expression in the posterior AER and Sonic hedgehog expression in the zone of polarizing activity (ZPA) were reduced. The homozygous mutant hindlimb also showed expansion of Wnt7a expression in the dorsal ectoderm toward the ventral side. This study shows that Rspo2 is critical for maintenance of the AER and for growth and patterning in limb development.     

Multiple functions of segment polarity genes in Drosophila

l(1)dishevelled (l(1)dsh) is a late zygotic lethal mutation that exhibits a rescuable maternal effect lethal phenotype. l(1)dsh/Y embryos, derived from females possessing a homozygous l(1)dsh germline clone, exhibit a segment polarity embryonic phenotype. Analysis of the development of these embryos indicates: (1) that segmental boundaries do not form although the correct number of tracheal pits is formed; (2) that pockets of cell death occur between the tracheal pits; and (3) that engrailed expression becomes abnormal during germ band shortening. We propose that, in the absence of both maternal and zygotic expression of l(1)dsh+, cells from each posterior compartment die. Subsequently, cells from the anterior compartment must rearrange their positional values to generate the segment polarity phenotype. We have compared the phenotype of five other segment polarity loci: four embryonic lethals [l(1)armadillo, l(2)gooseberry, l(2)wingless, and l(3)hedgehog]; and the late zygotic lethal, l(1)fused. Only l(2)wingless embryos exhibit early segmentation defects similar to those found in l(1)dsh/Y embryos derived from homozygous germline clones. In contrast, segmentation is essentially normal in l(1)armadillo, l(2)gooseberry, l(3)hedgehog, and l(1)fused embryos. The respective maternal and zygotic contribution and the roles of the segment polarity loci for the patterning of the embryo and the adult are discussed.     

Developmental regulation of EVF-1, a novel non-coding RNA transcribed upstream of the mouse Dlx6 gene

We previously reported that sonic hedgehog (Shh) induces the differentiation of rat ventral forebrain neurons expressing a novel marker, EVF-1 [Development 125 (1998) 5079]. In this report, we show that EVF-1 is a novel, developmentally regulated, non-coding RNA, with no homology to other known non-coding RNA sequences. Sequence analysis, in vitro translation, and comparison of the rat and mouse EVF-1 sequences suggest that EVF-1 contains no protein coding regions. Chromosomal location indicates that EVF-1 maps adjacent to the Dlx6 gene on mouse chromosome 6. RNA in situ hybridization of the embryonic rat forebrain shows that EVF-1 is expressed by immature neurons in the subventricular zone and its expression decreases during forebrain development. Whole mount in situ hybridization shows that EVF-1 is expressed at high levels in the branchial arches, ventral forebrain, olfactory bulb, and limbs. EVF-1 expression is linked to Shh and the Dlx family of proteins, genes with a demonstrated importance to ventral forebrain and craniofacial development.     

orthodenticle activity is required for the development of medial structures in the larval and adult epidermis of Drosophila.

Lethal alleles of orthodenticle (= otd) cause abnormalities in the embryonic head that reflect an early role in anterior pattern formation. In addition, otd activity is required for the development of the larval and adult epidermis. Clonal analysis of both viable and lethal alleles shows that the adult requirement for otd is restricted to medial regions of certain discs. When otd activity is reduced or removed, some medial precursor cells produce bristles and cuticle characteristic of more lateral structures. Similar medial defects are observed in the larval epidermis of embryos homozygous for lethal otd alleles. Antibodies to otd recognize a nuclear protein found at high levels in the medial region of the eye antennal discs, the leg discs, the genital discs and along the ventral midline of the ventral epidermis of the embryo. These results suggest that the otd gene product is required to specify medial cell fates in both the larval and adult epidermis.     

Autonomous requirements for the segment polarity gene armadillo during Drosophila embryogenesis

Embryos hemizygous for armadillo produce a "segment polarity" phenotype in which the naked posterior two-thirds of each segment is replaced by denticles with reversed polarity. Small patches of homozygous arm cells induced by mitotic recombination also form such denticles, indicating that the changes in cellular fate observed in homozygous arm embryos are autonomous at the level of single cells. Clonally derived arm patches do not, however, show the characteristic arm polarity reversals, arguing that this feature of the phenotype depends on cell interactions in fully mutant embryos. Few, if any, clones were found in the posterior-most regions of the naked cuticle, and none were found in the posterior compartments of the thorax.     

The segment polarity phenotype of Drosophila involves differential tendencies toward transformation and cell death

The segment polarity genes of Drosophila are required for intrasegmental organization, as revealed by their abnormal cuticular morphology in mutant embryos. Lesions in most of these loci result in a similar cuticular phenotype, in which the normally naked, posterior region of the segment is covered to varying degrees by ectopic denticles. A temperature-sensitive allele of armadillo, which allows us to vary the level of arm+ activity, generates this entire range of phenotypes, suggesting that these genes affect a common pathway. Previous work with a strong allele of arm revealed the locus to be cell-autonomous, in that small homozygous epidermal clones secreted denticles. We have conducted a similar clonal analysis at all levels of arm+ activity. This shows a differential tendency toward cell transformation and cell death within the segment. Antibodies to segmentation gene-fusion products show that the cell death is primarily in the most posterior region of the segment. We suggest that differential cell respecification, resulting in transformation or death, is involved in generating the segment polarity phenotype.     

Retinaldehyde dehydrogenase 2 (RALDH2)-mediated retinoic acid synthesis regulates early mouse embryonic forebrain development by controlling FGF and sonic hedgehog signaling

Although retinoic acid (RA) has been implicated as one of the diffusible signals regulating forebrain development, patterning of the forebrain has not been analyzed in detail in knockout mouse mutants deficient in embryonic RA synthesis. We show that the retinaldehyde dehydrogenase 2 (RALDH2) enzyme is responsible for RA synthesis in the mouse craniofacial region and forebrain between the 8- and 15-somite stages. Raldh2-/- knockout embryos exhibit defective morphogenesis of various forebrain derivatives, including the ventral diencephalon, the optic and telencephalic vesicles. These defects are preceded by regionally decreased cell proliferation in the neuroepithelium, correlating with abnormally low D-cyclin gene expression. Increases in cell death also contribute to the morphological deficiencies at later stages. Molecular analyses reveal abnormally low levels of FGF signaling in the craniofacial region, and impaired sonic hedgehog signaling in the ventral diencephalon. Expression levels of several regulators of diencephalic, telencephalic and optic development therefore cannot be maintained. These results unveil crucial roles of RA during early mouse forebrain development, which may involve the regulation of the expansion of neural progenitor cells through a crosstalk with FGF and sonic hedgehog signaling pathways.     

RacGap50C negatively regulates wingless pathway activity during Drosophila embryonic development

The Wingless (Wg)/Wnt signal transduction pathway directs a variety of cell fate decisions in developing animal embryos. Despite the identification of many Wg pathway components to date, it is still not clear how these elements work together to generate cellular identities. In the ventral epidermis of Drosophila embryos, Wg specifies cells to secrete a characteristic pattern of denticles and naked cuticle that decorate the larval cuticle at the end of embryonic development. We have used the Drosophila ventral epidermis as our assay system in a series of genetic screens to identify new components involved in Wg signaling. Two mutant lines that modify wg-mediated epidermal patterning represent the first loss-of-function mutations in the RacGap50C gene. These mutations on their own cause increased stabilization of Armadillo and cuticle pattern disruptions that include replacement of ventral denticles with naked cuticle, which suggests that the mutant embryos suffer from ectopic Wg pathway activation. In addition, RacGap50C mutations interact genetically with naked cuticle and Axin, known negative regulators of the Wg pathway. These phenotypes suggest that the RacGap50C gene product participates in the negative regulation of Wg pathway activity.     

Regulation of pancreas development by hedgehog signaling

Pancreas organogenesis is regulated by the interaction of distinct signaling pathways that promote or restrict morphogenesis and cell differentiation. Previous work has shown that activin, a TGF(beta+) signaling molecule, permits pancreas development by repressing expression of Sonic hedgehog (Shh), a member of the hedgehog family of signaling molecules that antagonize pancreas development. Here we show that Indian hedgehog (Ihh), another hedgehog family member, and Patched 1 (Ptc1), a receptor and negative regulator of hedgehog activity, are expressed in pancreatic tissue. Targeted inactivation of Ihh in mice allows ectopic branching of ventral pancreatic tissue resulting in an annulus that encircles the duodenum, a phenotype frequently observed in humans suffering from a rare disorder known as annular pancreas. Shh(-)(/)(-) and Shh(-)(/)(-) Ihh(+/)(-) mutants have a threefold increase in pancreas mass, and a fourfold increase in pancreatic endocrine cell numbers. In contrast, mutations in Ptc1 reduce pancreas gene expression and impair glucose homeostasis. Thus, islet cell, pancreatic mass and pancreatic morphogenesis are regulated by hedgehog signaling molecules expressed within and adjacent to the embryonic pancreas. Defects in hedgehog signaling may lead to congenital pancreatic malformations and glucose intolerance.     

Temporal requirement for hedgehog signaling in ventral telencephalic patterning

Hedgehog signaling is required for multiple aspects of brain development, including growth, the establishment of both dorsal and ventral midline patterning and the generation of specific cell types such as oligodendrocytes and interneurons. To identify more precisely when during development hedgehog signaling mediates these events, we directed the removal of hedgehog signaling within the brain by embryonic day 9 of development, using a FoxG1(Cre) driver line to mediate the removal of a conditional smoothened null allele. We observed a loss of ventral telencephalic patterning that appears to result from an initial lack of specification of these structures rather than by changes in proliferation or cell death. A further consequence of the removal of smoothened in these mice is the near absence of both oligodendrocytes and interneurons. Surprisingly, the dorsal midline appears to be patterned normally in these mutants. Together with previous analyses, the present results demonstrate that hedgehog signaling in the period between E9.0 and E12 is essential for the patterning of ventral regions and the generation of cell types that are thought to largely arise from them.     

Gamma tropomyosin gene products are required for embryonic development

The actin filament system is essential for many cellular functions, including shape, motility, cytokinesis, intracellular trafficking, and tissue organization. Tropomyosins (Tms) are rod-like components of most actin filaments that differentially affect their stability and flexibility. The Tm gene family consists of four genes, alphaTm, betaTm, gammaTm (Tm5 NM, where "NM" indicates "nonmuscle"), and deltaTm (Tm4). Multiple isoforms of the Tm family are generated by alternative splicing of three of these genes, and their expression is highly regulated. Extensive spatial and temporal sorting of Tm isoforms into different cellular compartments has been shown to occur in several cell types. We have addressed the function of the low-molecular-weight Tms encoded by the gammaTm gene by eliminating the corresponding amino-terminal coding sequences from this gene. Heterozygous mice were generated, and subsequent intercrossing of the F1 pups did not result in any viable homozygous knockouts. Genotype analysis of day 2.5 morulae also failed to detect any homozygous knockouts. We have failed in our attempts to delete the second allele and generate in vitro double-knockout cells, although 51 clones displayed homologous recombination back into the originally targeted locus. We therefore conclude that low-molecular-weight products from the gammaTm gene are essential for both embryonic development and cell survival.     

Requirement for tumor suppressor Apc in the morphogenesis of anterior and ventral mouse embryo

Tumor suppressor Apc (adenomatous polyposis coli) is implicated in the Wnt signaling pathway that is involved in the early embryonic development and tumorigenesis in vertebrates. While the heterozygous null mutant mice develop intestinal polyps, the homozygous embryos die before gastrulation. To investigate the role of Apc in later embryonic development, we constructed a novel hypomorphic Apc allele whose expression was attenuated by approximately 80%. In the hypomorphic Apc homozygous ES cells, reduction in Apc expression caused beta-catenin accumulation and Wnt signaling activation. The homozygous mutant mouse embryos survived 3 days longer than the null mutant embryos. Interestingly, they showed anterior truncation, partial axis duplication, and defective ventral morphogenesis. To determine the tissues where Apc functions for anterior and ventral morphogenesis, we constructed chimeric embryos whose epiblast was derived predominantly from the Apc hypomorphic homozygous cells but the visceral endoderm was from the wild type. Although these chimeric embryos still showed some anterior defects, their ventral morphogenesis was rescued. In addition, marker studies indicated that the axial mesendoderm was also defective in the homozygous embryos. Our results provide genetic evidence that expression of Apc at the normal level is essential for both anterior and ventral development, in the epiblast derivatives and visceral endoderm.     

A genetic and developmental analysis of mutations in the Deformed locus in Drosophila melanogaster

Individuals expressing recessive mutations in the Deformed (Dfd) locus of Drosophila melanogaster were examined for embryonic and adult defects. Mutant embryos were examined in both scanning electron microscope and light microscope preparations. The adult Dfd recessive mutant phenotype was assessed in somatic clones and in survivors homozygous for hypomorphic alleles of the gene. The time of Dfd+ action was determined by studying a temperature conditional allele. Dfd+ is required in three embryonic cephalic segments to form a normal head. Mutant embryos of Dfd display defects in derivatives of the maxillary segment, of the mandibular segment, and of some more anterior segments. In the adult fly, defects are seen in the posterior aspect of the head when the gene is mutant. A transformation from head to thoracic-like tissue is seen dorsally and a deletion of structures is seen ventrally. Shift studies utilizing a temperature conditional allele have shown that the gene product is necessary during at least two periods of development, during embryonic segmentation and head involution and during the late larval and pupal stages. From these studies we conclude that Dfd is a homeotic gene necessary for proper specification of both the embryonic and the adult head.     

Edd, the murine hyperplastic disc gene, is essential for yolk sac vascularization and chorioallantoic fusion

EDD is the mammalian ortholog of the Drosophila melanogaster hyperplastic disc gene (hyd), which is critical for cell proliferation and differentiation in flies through regulation of hedgehog and decapentaplegic signaling. Amplification and overexpression of EDD occurs frequently in several cancers, including those of the breast and ovary, and truncating mutations of EDD are also observed in gastric and colon cancer with microsatellite instability. EDD has E3 ubiquitin ligase activity, is involved in regulation of the DNA damage response, and may control hedgehog signaling, but a definitive biological role has yet to be established. To investigate the role of Edd in vivo, gene targeting was used to generate Edd knockout (Edd(Delta/Delta)) mice. While heterozygous mice had normal development and fertility, no viable Edd-deficient embryos were observed beyond E10.5, with delayed growth and development evident from E8.5 onward. Failed yolk sac and allantoic vascular development, along with defective chorioallantoic fusion, were the primary effects of Edd deficiency. These extraembryonic defects presumably compromised fetal-maternal circulation and hence efficient exchange of nutrients and oxygen between the embryo and maternal environment, leading to a general failure of embryonic cell proliferation and widespread apoptosis. Hence, Edd has an essential role in extraembryonic development.