Echinoid mutants exhibit neurogenic phenotypes and show synergistic interactions with the Notch signaling pathway

During neurogenesis in Drosophila, groups of ectodermal cells are endowed with the capacity to become neuronal precursors. The Notch signaling pathway is required to limit the neuronal potential to a single cell within each group. Loss of genes of the Notch signaling pathway results in a neurogenic phenotype: hyperplasia of the nervous system accompanied by a parallel loss of epidermis. Echinoid (Ed), a cell membrane associated Immunoglobulin C2-type protein, has previously been shown to be a negative regulator of the EGFR pathway during eye and wing vein development. Using in situ hybridization and antibody staining of whole-mount embryos, we show that Ed has a dynamic expression pattern during embryogenesis. Embryonic lethal alleles of ed reveal a role of Ed in restricting neurogenic potential during embryonic neurogenesis, and result in a phenotype similar to that of loss-of-function mutations of Notch signaling pathway genes. In this process Ed interacts closely with the Notch signaling pathway. Loss of ed suppresses the loss of neuronal elements caused by ectopic activation of the Notch signaling pathway. Using a temperature-sensitive allele of ed we show, furthermore, that Ed is required to suppress sensory bristles and for proper wing vein specification during adult development. In these processes also, ed acts in close concert with genes of the Notch signaling pathway. Thus the extra wing vein phenotype of ed is enhanced upon reduction of Delta (Dl) or Enhancer of split [E(spl)] proteins. Overexpression of the membrane-tethered extracellular region of Ed results in a dominant-negative phenotype. This phenotype is suppressed by overexpression of E(spl)m7 and enhanced by overexpression of Dl. Our work establishes a role of Ed during embryonic nervous system development, as well as adult sensory bristle specification and shows that Ed interacts synergistically with the Notch signaling pathway.     

A role for the Drosophila neurogenic genes in mesoderm differentiation

The neurogenic genes of Drosophila have long been known to regulate cell fate decisions in the developing ectoderm. In this paper we show that these genes also control mesoderm development. Embryonic cells that express the muscle-specific gene nautilus are overproduced in each of seven neurogenic mutants (Notch, Delta, Enhancer of split, big brain, mastermind, neuralized, and almondex), at the apparent expense of neighboring, nonexpressing mesodermal cells. The mesodermal defect does not appear to be a simple consequence of associated neural hypertrophy, suggesting that the neurogenic genes may function similarly and independently in establishing cell fates in both ectoderm and mesoderm. Altered patterns of beta 3-tubulin and myosin heavy chain gene expression in the mutants indicate a role for the neurogenic genes in development of most visceral and somatic muscles. We propose that the signal produced by the neurogenic genes is a general one, effective in both ectoderm and mesoderm.     

Isolation of a murine homologue of the Drosophila neuralized gene, a gene required for axonemal integrity in spermatozoa and terminal maturation of the mammary gland

The Drosophila neuralized gene shows genetic interactions with Notch, Enhancer of split, and other neurogenic genes and is thought to be involved in cell fate specification in the central nervous system and the mesoderm. In addition, a human homologue of the Drosophila neuralized gene has been described as a potential tumor suppressor gene in malignant astrocytomas. We have isolated a murine homologue of the Drosophila and human Neuralized genes and, in an effort to understand its physiological function, derived mice with a targeted deletion of this gene. Surprisingly, mice homozygous for the introduced mutation do not show aberrant cell fate specifications in the central nervous system or in the developing mesoderm. This is in contrast to mice with targeted deletions in other vertebrate homologues of neurogenic genes such as Notch, Delta, and Cbf-1. Male Neuralized null mice, however, are sterile due to a defect in axoneme organization in the spermatozoa that leads to highly compromised tail movement and sperm immotility. In addition, female Neuralized null animals are defective in the final stages of mammary gland maturation during pregnancy.     

Interaction of hairless, delta, enhancer of split and notch genes of Drosophila melanogaster as expressed in adult morphology

The interaction of three neurogenic loci viz. Delta, Enhancer of split and Notch, and a related gene, Hairless, of Drosophila melanogaster was investigated at the adult morphology level by measuring the effects of the mutations of the three other genes on the expression of the recessive lethal antimorphic Abruptex mutations of the Notch locus. The Abruptex mutations were also coupled in cis or trans with facet-glossy or split mutations of the Notch locus. In some of the experiments, the genotype of the fly was homozygous for either facet-glossy or split mutation or their wild type alleles but heterozygous for the Abruptex. Facet-glossy is located in a large intron of the locus, whereas split is located in the same exon as Abruptex. In all compounds studied, Delta suppressed the expression of Abruptex while Hairless and Enhancer of split enhanced it. The interactions of the four genes studied were allele specific, suggesting an interaction at the protein level. The comparison of the results presented in this study on the interaction of the neurogenic genes with other results on the same subject suggests that the interactions are similar in embryonic and imaginal development.     

Regulation of epithelial polarity by the E3 ubiquitin ligase Neuralized and the Bearded inhibitors in Drosophila

Understanding how epithelial polarity is established and regulated during tissue morphogenesis is a major issue. Here, we identify a regulatory mechanism important for mesoderm invagination, germ-band extension and transepithelial migration in the Drosophila melanogaster embryo. This mechanism involves the inhibition of the conserved E3 ubiquitin ligase Neuralized by proteins of the Bearded family. First, Bearded mutant embryos exhibited a loss of epithelial polarity associated with an early loss of the apical domain. Bearded regulated epithelial polarity by antagonizing neuralized. Second, repression of Bearded gene expression by Snail was required for the Snail-dependent disassembly of adherens junctions in the mesoderm. Third, neuralized was strictly required to promote the downregulation of the apical domain in the midgut epithelium and to facilitate the transepithelial migration of primordial germ cells across this epithelium. This function of Neuralized was independent of its known role in Notch signalling. Thus, Neuralized has two distinct functions in epithelial cell polarity and Notch signalling.     

crumbs encodes an EGF-like protein expressed on apical membranes of Drosophila epithelial cells and required for organization of epithelia

We describe the molecular characterization of the Drosophila gene crumbs, which encodes an integral membrane protein with 30 EGF-like repeats in the extracellular part and exhibits a striking expression pattern. The protein is exclusively localized on the apical membranes of epithelial cells and concentrated at the borders between cells. Mutations in crumbs lead to severe disruptions in the organization of ectodermally derived epithelia and in some cases to cell death in these tissues. The structure and the expression pattern of the protein and the phenotype of mutations indicate a function of crumbs during the development of epithelia, possibly for the establishment and/or maintenance of cell polarity.     

Cloning, chromosomal organization and expression analysis of Neurl, the mouse homolog of Drosophila melanogaster neuralized gene

The Drosophila neuralized (neur) gene belongs to the neurogenic group of genes involved in regulating cell-cell interactions required for neural precursor development. neur mutant phenotypes include strong overcommitment to neural fates at the expense of epidermal fates. The human neuralized homolog (NEURL) has been recently determined and found to map to chromosome 10q25.1 within the region frequently deleted in malignant astrocytomas. Because of its potential importance in developmental processes, we analyzed the structure of the mouse homolog, Neurl, and its expression pattern in embryonic tissues. Neurl activity is detected from early developmental stages in several tissues and organs including neural tissues, limbs, the skeletal system, sense organs and internal organs undergoing epithelial-mesenchymal interactions. Neurl encodes a polypeptide associated with the plasma membrane but also detected in the cytoplasm. Similarly to the Drosophila gene, mammalian neuralized may code for an important regulatory factor.     

Cytogenetic Definition and Morphogenetic Analysis of Delta, a Gene Affecting Neurogenesis in Drosophila Melanogaster

We have conducted a genetic analysis of a small interval of the third chromosome known to include Delta (Dl), a locus that affects the segregation of the ectoderm into neural and epidermal lineages during embryogenesis and the morphogenesis of some ectodermally derived structures, in Drosophila melanogaster. This analysis has led to the definition of seven independent complementation groups, one of which is Delta, within the interval extending from 91F6-13 to 92A2. Among the extant mutations in these seven loci, only mutations in Dl lead to the so-called neurogenic phenotype: hypertrophy of the nervous system and reduction of the epidermis. Combined cytogenetic and genetic analyses allow us to define absolute proximal (91F5-92A1) and distal (92A2) cytogenetic limits for the Dl locus. We have isolated hypomorphic and amorphic alleles of Dl and find that, for any given allele, there is an inverse correlation between neural hypertrophy and epidermal reduction in embryos and a direct correlation between the severity of embryonic phenotypes in mutant homozygotes and hemizygotes and the imaginal phenotype in heterozygous adults.     

Neurogenic and antineurogenic effects from modifications at the Notch locus

The best studied mutations at the Notch locus produce a neurogenic phenotype, with a massive overgrowth of the nervous system at the expense of epidermis. We report here that, in the development of the adult peripheral nervous system, the Abruptex alleles of Notch have the opposite phenotype, namely an underproduction of sensory organs or sensilla. This arises primarily not from an arrest of the lineages that produce sensilla, from the degeneration of sensillar cells, or from the transformation into neurons of cells that normally secrete the cuticular components of a sensillum (as can happen in Notch alleles). Rather, our evidence argues strongly that the sensillar mother cells never form. This implies that the Notch protein plays a role in the process that first generates a difference between sensillar mother cells and ordinary epidermal cells. The number of sensilla formed on the wing of flies carrying multiple doses of Notch+ is virtually the same as that of wild type, i.e. the Abruptex phenotype is not reproduced to any significant extent. This suggests that the single amino acid substitutions that occur in Abruptex mutants confer on the protein some functionally distinctive feature, possibly more powerful intermolecular binding or altered stability.     

Change of form of septate and gap junctions during development of the insect midgut

In insects, smooth septate junctions join cells derived from the embryonic midgut, and pleated septate junctions are found in all other tissues. Relatively little is known about either type of septate junction or the relationship between them, but they have been treated as two different junctions in the literature. The gap junctions which are associated with these septate junctions also differ. Crystalline gap junctions are found in the midgut, associated with smooth septate junctions, and irregular gap junctions are found in tissues where pleated septate junctions are located. We have examined the development of smooth septate junctions and crystalline gap junctions and the relationship between them, by studying the embryogenesis of the midgut in Manduca sexta (tobacco hornworm). At 56 hr of development (hatching is at 104 hr) pleated septate junctions and irregular gap junctions joined the midgut epithelial cells. At 65 hr, the septate junctions had disappeared, but gap junctions persisted. At 70 hr, smooth septate junctions had replaced the earlier pleated septate junctions and gap junctions associated with these smooth septate junctions were often of the crystalline form. In later embryos, the smooth septate junctions matured and enlarged, while all gap junctions became crystalline in form.     

Differential requirements for the neurogenic gene almondex during Drosophila melanogaster development

During early development, the neurogenic genes of Drosophila melanogaster are involved in the control of cell fates in the neurectoderm; almondex (amx) belongs to this category of genes. We have identified the amx locus and rescued the amx embryonic neurogenic phenotype with a 1.5 kb DNA fragment. Using a small deficiency, we generated a new amx mutant background called amx(m), which is a null allele. Besides the characteristic neurogenic maternal effect caused by loss of amx, amx(m) flies display a new imaginal phenotype resembling loss of function of Notch. We describe amx-induced misregulation of the Notch pathway target E(spl) m7 in embryos and genetic interactions between amx and Notch pathway mutants in adult flies. These data show that wildtype amx acts as a novel positive regulator of the Notch pathway and is required at different levels during development.     

A Genetic Analysis of Deltex and Its Interaction with the Notch Locus in Drosophila Melanogaster

During Drosophila development networks of genes control the developmental pathways that specify cell fates. The Notch gene is a well characterized member of some cell fate pathways, and several other genes belonging to these same pathways have been identified because they share a neurogenic null phenotype with Notch. However, it is unlikely that the neurogenic genes represent all of the genes in these pathways. The goal of this research was to use a genetic approach to identify and characterize one of the other genes that acts with Notch to specify cell fate. Mutant alleles of genes in the same pathway should have phenotypes similar to Notch alleles and should show phenotypic interactions with Notch alleles. With this approach we identified the deltex gene as a potential cell fate gene. An extensive phenotypic characterization of loss-of-function deltex phenotypes showed abnormalities (such as thick wing veins, double bristles and extra cone cells) that suggest that deltex is involved in cell fate decision processes. Phenotypic interactions between deltex and Notch as seen in double mutants showed that Notch and deltex do not code for duplicate functions and that the two genes function together in many different developing tissues. The results of these investigations lead to the conclusion that the deltex gene functions with the Notch gene in one or more developmental pathways to specify cell fate.     

The big brain gene of Drosophila functions to control the number of neuronal precursors in the peripheral nervous system.

big brain (bib) is one of the six known zygotic neurogenic genes involved in the decision of an ectodermal cell to take on the neurogenic or the epidermogenic cell fate. Previous studies suggest that bib functions in a pathway separate from the one involving Notch and other known neurogenic genes. For a better understanding of the bib function, it is essential first to characterize the mutant phenotype in detail. Our mutant analyses show that loss of bib function approximately doubles the number of neuronal precursors and their progeny cells in the embryonic peripheral nervous system. Mosaic studies reveal a hypertrophy of sensory bristles in bib mutant patches in adult flies. Our observations are compatible with a function of bib in specifying neuronal precursors of both the embryonic and adult sensory nervous system. This is in contrast to the function of Notch, which continues to be required at multiple stages of neural development subsequent to this initial determination event.     

Mutation in ankyrin repeats of the mouse Notch2 gene induces early embryonic lethality.

Notch family genes encode transmembrane proteins involved in cell-fate determination. Using gene targeting procedures, we disrupted the mouse Notch2 gene by replacing all but one of the ankyrin repeat sequences in the cytoplasmic domain with the E. coli (beta)-galactosidase gene. The mutant Notch2 gene encodes a 380 kDa Notch2-(beta)-gal fusion protein with (beta)-galactosidase activity. Notch2 homozygous mutant mice die prior to embryonic day 11.5, whereas heterozygotes show no apparent abnormalities and are fully viable. Analysis of Notch2 expression patterns, revealed by X-gal staining, demonstrated that the Notch2 gene is expressed in a wide variety of tissues including neuroepithelia, somites, optic vesicles, otic vesicles, and branchial arches, but not heart. Histological studies, including in situ nick end labeling procedures, showed earlier onset and higher incidence of apoptosis in homozygous mutant mice than in heterozygotes or wild type mice. Dying cells were particularly evident in neural tissues, where they were seen as early as embryonic day 9.5 in Notch2-deficient mice. Cells from Notch2 mutant mice attach and grow normally in culture, demonstrating that Notch2 deficiency does not interfere with cell proliferation and that expression of the Notch2-(beta)-gal fusion protein is not toxic per se. In contrast to Notch1-deficient mice, Notch2 mutant mice did not show disorganized somitogenesis, nor did they fail to properly regulate the expression of neurogenic genes such as Hes-5 or Mash1. In situ hybridization studies show no indication of altered Notch1 expression patterns in Notch2 mutant mice. The results indicate that Notch2 plays an essential role in postimplantation development in mice, probably in some aspect of cell specification and/or differentiation, and that the ankyrin repeats are indispensable for its function.     

Mutations of early neurogenesis inDrosophila

Summary Embryonic lethal mutations at the Notch locus are known to produce a conspicuous central nervous system hypertrophy accompanied by a hypotrophy of the epidermal sheath. We have studied several zygotic mutants belonging to four different autosomal complementation groups which produce the same phenotype. The embryonic development of the new mutants, as well as that of Notch, consists of an initial enlargement of the neurogenic region at the expenses of epidermal cell precursors. The possibility is discussed that these five loci are involved in the determination of neural and epidermal cell precursors.