José A. Campos-Ortega

On the phenotype and development of mutants of early neurogenesis inDrosophila melanogaster

SummaryThe central nervous system (CNS) ofDrosophila develops from precursor cells called neuroblasts. Neuroblasts segregate in early embryogenesis from an apparantly undifferentiated ectoderm and move into the embryo, whereas most of the remaining ectodermal cells continue development as epidermal cell precursors. Segregation of neuroblasts occurs within a region called the neurogenic field. We are interested in understanding how the genome ofDrosophila controls the parcelling of the ectoderm into epidermal and neural territories. We describe here mutations belonging to seven complementation groups which effect an abnormal neurogenesis. The phenotypes produced by these mutations are similar. Essential features of these phenotypes are a conspicuous hypertrophy of the CNS accompanied by epidermal defects; the remaining organs and tissues of the mutants are apparently unaffected. The study of mutant phenotype development strongly suggests this phenotype to be due to misrouting into the neural pathway of development of ectodermal cells which in the wildtype would have given rise to epidermal cells, i.e. to an initial enlargement of the neurogenic region at the expense of the epidermogenic region. These observations indicate that the seven genetic loci revealed by the mutations described in this study contribute to control the neurogenic field. The present results suggest that in wildtype development neurogenic genes are supressed within all derivatives of the mesoderm and endoderm and some derivatives of the ectoderm, and conditionally expressed in the remaining ectoderm. The organisation of the neurogenic field in the wildtype is discussed.

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.

Early neurogenesis in wild-typeDrosophila melanogaster

SummaryThis paper deals with morphological aspects of early neurogenesis inDrosophila, in particular with the segregation of neuroblasts from the neurogenic region of the ectoderm and the pattern formed by those wells within both the germ band and the procephalic lobe. The neurogenic ectoderm was found to contain neural precursors intermingled with epidermal precursors, extending from the midline up to the primordia of the tracheal tree along the germ band and laterodorsally in the procephalic lobe. Germ band neuroblasts segregate from the neurogenic ectoderm during a period of several hours according to characteristic spatial and temporal patterns. During the first half of the segregation process the pattern of germ band neuroblasts was found to be the same in different animals in both spatial arrangement and number of cells; this permitted the identification of individual neuroblasts from different embryos. Later in development several difficulties were encountered which precluded an exact description of the neuroblast pattern. The constitution of the neurogenic region is discussed in relation to the phenotype of mutants affecting neurogenesis.

The neurogenic gene Delta of Drosophila melanogaster is expressed in neurogenic territories and encodes a putative transmembrane protein with EGF-like repeats

The decision of an ectodermal cell to take on a neural or an epidermal fate depends on its interactions with the neighbouring cells. In Drosophila melanogaster, the available evidence suggests that a regulatory signal necessary for epidermal commitment is built by the products of the so‐called neurogenic genes. We have cloned 180 kb of genomic DNA surrounding the neurogenic gene Delta (Dl). Restriction fragment‐length polymorphisms were mapped to a region of 25 kb. These 25 kb of DNA are assumed to contain essential parts, or all, of the Dl gene. Northern blots detect two developmentally regulated transcripts, of 5.4 and 4.6 kb, which are associated with the region where the mutants map. Serveral cDNA clones were recovered from embryonic cDNA libraries by homology to the 25 kb of genomic DNA. The complete sequence of a cDNA clone containing an insert of 4.73 kb was determined. The conceptual translation of the longest open reading frame yields a protein of 880 amino acids. This protein displays characteristics of a membrane protein, with intercellular, transmembrane and extracellular domains. The extracellular domain contains a tandem array of nine EGF‐like repeats. In in situ hybridizations to tissue sections, transcripts homologous to Dl are detected in all territories with neurogenic abilities, e.g. the neurogenic ectoderm and the primordia of the sensory organs. Initially all cells of these neurogenic territories express Dl, but later on transcription of Dl becomes restricted to the cells that have adopted the neural fate. The topological specificity in the transcription of Dl corresponds to the one expected for a regulatory signal that mediates epidermal commitment.

Closely related transcripts encoded by the neurogenic gene complex enhancer of split of Drosophila melanogaster.

Genetic evidence suggests that E(spl), one of the neurogenic loci of Drosophila, is a gene complex comprising an as yet incompletely established number of transcription units. In order to correlate the various transcription units with E(spl) functions, wild‐type flies were transformed with genomic DNA encoding the transcription unit m8 from the mutant E(spl)D, which was known to be altered in embryos carrying this mutant allele. Transformants show the same dominant enhancement of the spl phenotype as E(spl)D itself. Since m8 has a virtually identical pattern of expression as m4, m5 and m7, we have determined the sequence of these four transcripts. The deduced protein products of m5, m7 and m8 exhibit extensive sequence homology with each other. All three encode a sequence similar to one of the conserved domains of representatives of the vertebrate myc gene family which is also present in the deduced protein sequences of the Drosophila achaete‐scute gene complex. Sequence analysis of the m8 transcription unit in the E(spl)D mutation revealed several DNA lesions. One of the lesions is a deletion in the region upstream of the transcription start site. Another lesion is a deletion in the coding region that leads to a shorter protein which, in addition, differs in its carboxy‐terminal end from the wild‐type protein by the presence of nine amino acids.

Fate-mapping in wild-typeDrosophila melanogaster

SummaryThe pattern of cell proliferation and cell movements inDrosophila embryogenesis has been analysed with the aim of constructing a blastoderm fate map. Post-blastoderm cell proliferation starts at gastrulation and ends around the stage of germ band shortening. Three mitotic waves affect the embryonic cells according to a constant spatio-temporal pattern. For any of these waves mitotic activity starts at well-defined loci, which have been called mitotic centres. During the first and second mitotic waves all cells undergo mitosis, except for those of the amnioserosa, which do not proliferate at all. The third wave spares most of the ectodermal cells. Neuroblasts, progenitors of epidermal sensilla and germ line cells show their own, different pattern of proliferation.

Proliferation pattern and early differentiation of the optic lobes in Drosophila melanogaster

SummaryThe larval and early pupal development of the optic lobes in Drosophila is described qualitatively and quantitatively using [3H]thymidine autoradiography on 2-μm plastic sections. The optic lobes develop from 30–40 precursor cells present in each hemisphere of the freshly hatched larva. During the first and second larval instars, these cells develop to neuroblasts arranged in two epithelial optic anlagen. In the third larval instar and in the early pupa these neuroblasts generate the cells of the imaginal optic lobes at discrete proliferation zones, which can be correlated with individual visual neuropils.The different neuropils as well as the repetitive elements of each neuropil are generated in a defined temporal sequence. Cells of the medulla are the first to become postmitotic with the onset of the third larval instar, followed by cells of the lobula complex and finally of the lamina at about the middle of the third instar. The elements of each neuropil connected to the most posterior part of the retina are generated first, elements corresponding to the most anterior retina are generated last.The proliferation pattern of neuroblasts into ganglion mother cells and ganglion cells is likely to include equal as well as unequal divisions of neuroblasts, followed by one or two generations of ganglion mother cells. For the lamina the proliferation pattern and its temporal coordination with the differentiation of the retina are shown.

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.

Genetic mechanisms of early neurogenesis inDrosophila melanogaster

The neurogenic ectoderm ofDrosophila melanogaster consists of the ventral neuroectoderm and the procephalic neuroectoderm. It is hypothesized that epidermal and central neural progenitor cells separate from each other in three steps: conference on the neuroectodermal cells the capability of producing neural or epidermal progenies, separation of the two classes of progenitor cells, and specification of particular types of neuroblasts and epidermoblasts. Separation of neuroblasts and epidermoblasts in controlled by proneural and neurogenic genes.Delta andNotch serve as mediators of direct protein-protein interactions. E(spl)-C inhibits neurogenesis, creating epidermal cells. The achaete-scute complex (AS-C) controls the commitment of nonoverlapping populations of neuroblasts and leads the development of neuroectodermal cells as neuroblasts.

Cellular interactions during early neurogenesis of Drosophila melanogaster

Abstract How are diverse cell types generated from initially homogeneous precursor cells? This question is at the heart of developmental biology, and has been studied extensively in insects because of their relatively small number of embryonic cells and because, in the case of Drosophila , they are amenable to genetic analysis. During neurogenesis in Drosophila , the progenitor cells of the neurogenic ectoderm give rise to two cell types: neural and epidermal. This developmental switch is not genetically hard-wired, but depends instead on cell-cell communication. These interactions are determined by the products of the neurogenic genes, which seem to provide a molecular signal chain that leads to this neural/epidermal developmental decision.