Vision in insects: pathways possibly underlying neural adaptation and lateral inhibition.

Like horizontal cells in vertebrate retinas, horizontal amacrine cells beneath the insect eye intervene between receptors and interneurons at the first level of synapses. Synaptic arrangements between amacrines and interneurons that give rise to regular networks of axon collaterals may explain recent electrophysiological observations of lateral inhibition beneath the insect retina. Neural adaptation mechanisms acting on single retinotopic channels or assemblies of channels can also be referred to reciprocal relationships between receptors and first-order interneurons as well as to centrifugal cells from levels of so-called photopic receptor endings.

Persistent expression of genes of the enhancer of split complex suppresses neural development in Drosophila.

The segregation of neural and epidermal progenitors in Drosophila requires the activity of transcription factors encoded by the proneural genes and the genes of the E(SPL)-C. Persistent expression of two genes of the E(SPL)-C suppresses neural development. Embryos exhibit conspicuous central neural hypoplasia and lack sensory organs; imaginal sensory organs are also affected. Suppression of neural development is associated with suppression of the activity of proneural genes. DNA binding is not essential for this effect. Large cells with characteristics of neuroblasts segregate normally in embryos, but these cells fail to express various markers, and the segregated cells and/or their progeny eventually die. These findings indicate that proneural and E(spl) proteins exert antagonistic functions.

Defective neuroblast commitment in mutants of the achaete-scute complex and adjacent genes of D. melanogaster.

Loss of function mutations in genes of the achaete-scute complex (ASC) or in the gene vnd of D. melanogaster result in neural hypoplasia. Two types of defects contribute to the development of the neural hypoplasic phenotype: a lower than normal proportion of neuroblasts delaminate from the neuroectoderm, and there is abundant cell death in the neural primordium during later stages. In addition, we found that increasing the copy number of ASC wild-type alleles leads to effects opposite to those caused by their deletion. All of these results indicate that the function of these genes is required for the commitment of neuroectodermal cells as neuroblasts and that the loss of these genetic functions causes the cells either to take on an epidermal fate or to die.

Single amino acid substitutions in EGF-like elements of Notch and Delta modify Drosophila development and affect cell adhesion in vitro.

Notch locus EGF-like element mutations spl, altering eye development, and AxE2, affecting wing and sensilla development, are modified by mutations at Delta. It is shown that two allele-specific suppressors of spl involve single amino acid substitutions in the 4th (Dlsup5) and 9th (Dlsup4) EGF-like elements of the Delta protein. Cultured cells producing spl or AxE2 aggregate with cells producing wild-type Delta or Dlsup5 protein, and Dlsup5-producing cells adhere to cells producing wild-type Notch protein. However, spl,AxE2, and Dlsup5 are each defective in promoting these cell affinities, as none of the mutant proteins can compete with the corresponding wild-type proteins for formation of cell aggregates. Thus, widely separated EGF-like elements of Notch and Delta appear to participate in functional molecular interactions between the proteins. Dlsup5 does not improve adhesiveness of spl in vitro, so suppression in vivo may involve altered developmental signaling by spl-Dlsup5 complexes, rather than modified cell adhesion.

Asymmetic division: dynastic intricacies of neuroblast division.

The cytoplasmic determinants Numb and Prospero are distributed asymmetrically into the daughter cells of Drosophila neuroblasts. The proteins encoded by the genes inscuteable, staufen and miranda are involved in the localisation of Prospero.

Genetic Analysis of Delta, a Neurogenic Gene of Drosophila melanogaster.

We report here the results of a genetic analysis of the gene Delta (Dl) of Drosophila melanogaster. Dl has been mapped to the band 92A2, on the basis of two pieces of evidence: (1) this band is the common breakpoint of several chromosomal aberrations associated with Dl mutations and (2) recombination mapping of alleles of five different lethal complementation groups that are uncovered by Df( 3R)Dl(FX3) (breakpoints at 91F11; 92A3). Dl was found to map most distally of all five complementation groups. The analysis of a large number of Dl alleles demonstrates the considerable genetic and functional complexity of Dl. Three types of Dl alleles are distinguishable. Most alleles behave as amorphic or hypomorphic recessive embryonic lethal alleles, which in addition cause various defects in heterozygosity over the wild-type allele. The defects are due to haplo-insufficient expression of the locus and can be suppressed by a duplication of the wild-type allele. The second class is comprised of three alleles with antimorphic expression. The phenotype of these alleles can only be reduced, rather than suppressed, by a duplication of the wild-type allele. The third group is comprised of three visible, predominantly hypomorphic alleles with an antimorphic component of phenotypic expression. The pattern of interallelic complementation is complex. On the one hand, there is a group of hypomorphic, fully penetrant embryonic lethal alleles which complement each other. On the other hand, most alleles, including all amorphic alleles, are viable over the visible ones; alleles of antimorphic expression, however, are lethal over visible alleles. These results are compatible with a rather complex genetic organization of the Dl locus.

The Enhancer of split complex and adjacent genes in the 96F region of Drosophila melanogaster are required for segregation of neural and epidermal progenitor cells.

The Enhancer of split complex [E(spl)-C] of Drosophila melanogaster is located in the 96F region of the third chromosome and comprises at least seven structurally related genes, HLH-m delta, HLH-m gamma, HLH-m beta, HLH-m3, HLH-m5, HLH-m7 and E(spl). The functions of these genes are required during early neurogenesis to give neuroectodermal cells access to the epidermal pathway of development. Another gene in the 96F region, namely groucho, is also required for this process. However, groucho is not structurally related to, and appears to act independently of, the genes of the E(spl)-C; the possibility is discussed that groucho acts upstream to the E(spl)-C genes. Indirect evidence suggests that a neighboring transcription unit (m4) may also take part in the process. Of all these genes, only gro is essential; m4 is a dispensable gene, the deletion of which does not produce detectable morphogenetic abnormalities, and the genes of the E(spl)-C are to some extent redundant and can partially substitute for each other. This redundancy is probably due to the fact that the seven genes of the E(spl)-C encode highly conserved putative DNA-binding proteins of the bHLH family. The genes of the complex are interspersed among other genes which appear to be unrelated to the neuroepidermal lineage dichotomy.

Seven genes of the Enhancer of split complex of Drosophila melanogaster encode helix-loop-helix proteins.

Enhancer of split [E(spl)] is one of the neurogenic loci of Drosophila and, as such, is required for normal segregation of neural and epidermal cell progenitors. Genetic observations indicate that the E(spl) locus is in fact a gene complex comprising a cluster of related genes and that other genes of the region are also required for normal early neurogenesis. Three of the genes of the complex were known to encode helix-loop-helix (HLH) proteins and to be transcribed in nearly identical patterns. Here, we show that four other genes in the vicinity also encode HLH proteins and, during neuroblast segregation, three of them are expressed in the same pattern. We show by germ-line transformation that these three genes are also necessary to allow epidermal development of the neuroectodermal cells.

Genetic analysis of enhancer of split, a locus involved in neurogenesis in Drosophila melanogaster.

Enhancer of split (E(spl)), one of the neurogenic loci of Drosophila, is uncovered by the deletion Df(3R)E(spl)(R-B251) with breakpoints at 96F8 and 96F13. We describe here the results of a genetic analysis of this chromosomal interval. Thirty-one mutations in genes of this region were recovered during various programs of mutagenesis. In addition, we included the spontaneous mutations E(spl)(D) and groucho (gro), which are known to map to this region, in our study. These 33 mutations define four lethal complementation groups, one of which includes E(spl)(D) and gro. Mutations of the E(spl) group behave as complementing and noncomplementing pseudoalleles, defining different functions. Alleles are classified according to their complementation behavior in two different ways: with respect to their viability as heterozygotes with other lethal alleles and with respect to gro and to E(spl)(D). The phenotypes of these mutations and the pattern of heteroallelic complementation speak in favor of a considerable genetic complexity of the E(spl) locus.

Functional interactions of neurogenic genes of Drosophila melanogaster.

The neurogenic genes of Drosophila melanogaster are involved in the decision of ectodermal cells to take on a neural or an epidermal fate. We present evidence in support of the notion that six of the neurogenic genes are functionally related. We studied the phenotype of embryos lacking one of the neurogenic genes in the presence of an increased dosage of the wild-type allele of another neurogenic gene. Our analysis also included the Hairless locus, whose function is related to that of the neurogenic genes, as well as to many other genes. The effects observed were asymmetric in that triploidy for a given gene modified the phenotype of loss of the function of another gene, but triploidy of the latter gene did not modify the phenotype of loss of the function of the former gene. These asymmetries allowed us to establish a polarity of gene interactions, as well as to order the genes according to the assumed ability of some of them to modify the activity of others. In this sequence, almondex is the first link and Enhancer of split the last one. Our evidence suggests that the function of big brain is independent of the function of the other six. The consequences of this arrangement for the commitment of ectodermal cells are discussed.

A zebrafish homologue of the Drosophila neurogenic gene Notch and its pattern of transcription during early embryogenesis.

We describe here the primary structure of a zebrafish homologue of the Drosophila neurogenic gene Notch and its pattern of mRNA accumulation during embryogenesis. The gene produces a 8.5 kb transcript encoding a putative transmembrane protein with a high degree of sequence similarity to members of the Notch family, comprising 36 EGF-like repeats, three lin-12/Notch repeats, six cdc10/SWI6 repeats, OPA repeats and a PEST sequence. Transcription of the zebrafish Notch gene is spatially and temporally regulated. A high density of transcripts, most probably of maternal origin, can already be detected in the 2-cell stage. During pregastrulation stages, RNA is present in all cells. However, following gastrulation, transcripts accumulate in specific regions of the embryo following a rapidly changing pattern. In some of these regions, cell divisions take place at the time of Notch expression, in others processes of cell differentiation. This holds true for various mesodermal derivatives, such as the prospective notochord, and for different neural primordia, such as the neural plate and the brain vesicles. This pattern of transcript accumulation suggests a role for the zebrafish Notch homologue in processes of regionalization and cell diversification.

A zebrafish Id homologue and its pattern of expression during embryogenesis.

We describe the cloning, sequencing and pattern of transcript distribution during embryogenesis of a zebrafish Id homologue that we have called Id6. Transcription of the gene is spatially regulated, and its pattern of transcription shows considerable overlaps with those of other zebrafish genes with homology to Drosophila neurogenic genes, such as Notch and Delta. Since all these genes are coexpressed in particular cells, they may function together in a single genetic circuit in zebrafish as they do in Drosophila. A zebrafish homologue of Drosophila AS-C proteins can activate transcription of a CAT reporter gene by binding to an E-box in mouse 3T3 cells, either alone or in conjunction with ZfE12. The activation of transcription is inhibited in the presence of Id6. This indicates that the zebrafish gene described here is a genuine member of the Id family, and suggests that it may serve a function similar to that of the Drosophila gene emc and mammalian Ids during development.

Use of the Gal4-UAS technique for targeted gene expression in the zebrafish.

The most common way to analyze the function of cloned genes in zebrafish is to misexpress the gene product or an altered variant of it by mRNA injection. However, mRNA injection has several disadvantages. The GAL4-UAS system for targeted gene expression allows one to overcome some of these disadvantages. To test the GAL4-UAS system in zebrafish, we generated two different kinds of stable transgenic lines, carrying activator and effector constructs, respectively. In the activator lines the gene for the yeast transcriptional activator GAL4 is under the control of a given promoter, while in the effectors the gene of interest is fused to the sequence of the DNA-binding motif of GAL4 (UAS). Crosses of animals from the activator and effector lines show that effector genes are transcribed with the spatial pattern of the activators. This work smoothes the way for a novel method of misexpression of gene products in zebrafish in order to analyze the function of genes in developmental processes.

Overexpression of a zebrafish homologue of the Drosophila neurogenic gene Delta perturbs differentiation of primary neurons and somite development.

We describe here the isolation and characterization of a zebrafish Delta homologue (delta D). A PCR fragment was used to obtain overlapping cDNA clones encoding a protein of 717 amino acids with all characteristic features of proteins of this family, a signal peptide, a transmembrane domain, and an extracellular region comprising the DSL domain and eight EGF-like repeats. The gene is transcribed in a complex pattern in the developing nervous system as well as in the hypoblast. Overexpression of this gene following mRNA injections leads to a reduction in the number of islet-I positive cells, which are assumed to be primary neurons, and to various defects in the adaxial mesoderm, as well as in the somites and myotomes. This suggests that delta D, and the Notch signalling pathway are involved in the differentiation of primary neurons within the neural plate, as well as in somite development.

Genomic regions regulating early embryonic expression of the Drosophila neurogenic gene Delta.

To identify genomic regions required for transcriptional regulation of Delta during early embryogenesis, constructs carrying promoter and gene fusions to the lacZ gene were used for germ line transformation. Most cis-regulatory sequences are dispersed throughout 6.6 kb of genomic DNA, 5' of the transcription start site; the first intron contains an enhancer element that increases the amount of RNA produced in several organs. A region was defined which drives Delta-lacZ RNA expression in clusters of neuroectodermal cells preceding and during SI neuroblast segregation. This pattern is regulated by genes of the AS-C (achaete-scute complex). To identify regulatory regions necessary for normal function of Delta during neural-epidermal lineage segregation, five minigenes consisting of fragments of the 5' genomic DNA fused to a cDNA encoding the entire protein sequence were tested for their ability to rescue the neural hyperplasia caused by a deletion of the Delta locus. Regulatory sequences required for this function are differentially distributed throughout 9 kb of genomic DNA upstream of the transcription start site. The possible significance of these findings with respect to the function of Delta during lineage segregation is discussed.

The HLH domain of a zebrafish HE12 homologue can partially substitute for functions of the HLH domain of Drosophila DAUGHTERLESS.

We have identified a zebrafish homologue of the human E12 protein, which we have called ZFE12. It shows a high degree of identity to HE12 throughout its entire sequence, particularly in the bHLH domain (93%); amino acid sequence identity of the bHLH domain of ZFE12 to Drosophila DAUGHTERLESS is also very high (75%). During early embryogenesis, expression of ZfE12 is widespread. Following gastrulation, ZfE12 transcripts can be detected in all embryonic tissues with the exception of the notochord, although zones with relatively higher densities of transcripts are present in the developing brain and in the somites. To assay biological activities associated with the ZFE12 protein, two P-element constructs were made, each carrying a Drosophila daughterless gene that had been modified by replacing either the HLH domain or the entire C-terminus including the bHLH domain, by the equivalent domains of ZfE12. These constructs were used to transform flies and tested for their ability to rescue the daughterless mutant phenotype. Complete rescue of the neural phenotype and of the embryonic lethality was obtained. However, the daughterless function could not be completely restored. Although fertile flies transheterozygous for a hypomorphic and an amorphic mutation and carrying the construct encoding the zebrafish HLH domain emerged, they lacked various types of sensory organs on head and thorax and showed slight wing defects. Mutants carrying the second construct occasionally reached pupal stages but died subsequently. These data demonstrate that the HLH domain of ZFE12 can carry out most, but not all, functions performed by the corresponding region of the DAUGHTERLESS protein in Drosophila.

Lethal of scute requires overexpression of daughterless to elicit ectopic neuronal development during embryogenesis in Drosophila.

Classical genetics indicates that the achaete-scute gene complex (AS-C) of Drosophila promotes development of neural progenitor cells. To further analyze the function of proneural genes, we have studied the effects of Gal4-mediated expression of lethal of scute, a member of the AS-C, during embryogenesis. Expression of lethal of scute forces progenitor cells of larval internal sensory organs, which are normally committed to this fate independently of the activity of the AS-C, to take on features of external sensory organs. Supernumerary neural cells can be induced ectopically only if daughterless is overexpressed, either alone or together with lethal of scute: cells of the amnioserosa and the hindgut then express neuronal markers. Furthermore, cells of the proctodeal anlage, which normally lack neural competence, acquire the ability to develop as neuroblasts following transplantation into the neuroectoderm. We show here that activated Notch prevents the cells of the neuroectoderm from forming extra neural tissue when they express an excess of proneural proteins. Under the present conditions, lateral inhibition is thus dominant over the activity of proneural genes.

A recessive mutation leading to vertebral ankylosis in zebrafish is associated with amino acid alterations in the homologue of the human membrane-associated guanylate kinase DLG3.

We describe the characterization of the zebrafish homologue of the human gene DLG3. The zebrafish dlg3 gene encodes a membrane-associated guanylate kinase containing a single PDZ domain. This gene was cloned using a gene-trap construct inserted in the gene's first intron. The insertion co-segregates with a viable mutation called humpback (hmp), which leads to formation of ankylotic vertebrae in adult fishes. Insertion and mutation have both been mapped to chromosome 12, in a segment which is syntenic with region p12 to q12 of human chromosome 17. The hmp mutant phenotype, however, appears to be due to two point mutations in the guanylate kinase domain rather than to the transgene insertion itself. The results of this study are discussed in the light of the possible function of the guanylate kinase domain.

Distinct regulatory elements direct delta1 expression in the nervous system and paraxial mesoderm of transgenic mice.

The Delta1 gene encodes one of the Notch ligands in mice. Delta1 is expressed during early embryogenesis in a complex and dynamic pattern in the paraxial mesoderm and neuroectoderm, and is essential for normal somitogenesis and neuronal differentiation. Molecular components thought to act in response to ligand binding and Notch activation have been identified in different species. In contrast, little is known about the transcriptional regulation of Notch receptors and their ligands. As a first step to identify upstream factors regulating Delta1 expression in different tissues, we searched for cis-regulatory regions in the Delta1 promoter able to direct heterologous gene expression in a tissue specific manner in transgenic mice. Our results show that a 4.3 kb genomic DNA fragment of the Delta1 gene is sufficient in a lacZ reporter transgene to reproduce most aspects of Delta1 expression from the primitive streak stage to early organogenesis. Using a minimal Delta1 promoter we also show that this upstream region contains distinct regulatory modules that individually direct tissue-specific transgene expression in subdomains of the endogenous expression pattern. It appears that expression in the paraxial mesoderm depends on the interaction of multiple positive and negative regulatory elements. We also find that at least some regulatory sequences required for transgene expression in subdomains of the neural tube have been maintained during the evolution of mammals and teleost fish, suggesting that part of the regulatory network that controls expression of Delta genes may be conserved.

The genetics of the Drosophila achaete-scute gene complex: a historical appraisal.

The Drosophila achaete-scute complex consists of four genes encoding transcription factors of the bHLH family. Due to their intricate organization, these genes have occupied geneticists and developmental biologists for many years. Here, genetic studies on the complex are discussed from a historical point of view.