Dorsoventral patterning in the Drosophila central nervous system: the vnd homeobox gene specifies ventral column identity

The Drosophila CNS develops from three columns of neuroectodermal cells along the dorsoventral (DV) axis: ventral, intermediate, and dorsal. In this and the accompanying paper, we investigate the role of two homeobox genes, vnd and ind, in establishing ventral and intermediate cell fates within the Drosophila CNS. During early neurogenesis, Vnd protein is restricted to ventral column neuroectoderm and neuroblasts; later it is detected in a complex pattern of neurons. We use molecular markers that distinguish ventral, intermediate, and dorsal column neuroectoderm and neuroblasts, and a cell lineage marker for selected neuroblasts, to show that loss of vnd transforms ventral into intermediate column identity and that specific ventral neuroblasts fail to form. Conversely, ectopic vnd produces an intermediate to ventral column transformation. Thus, vnd is necessary and sufficient to induce ventral fates and repress intermediate fates within the Drosophila CNS. Vertebrate homologs of vnd (Nkx2.1 and 2.2) are similarly expressed in the ventral CNS, raising the possibility that DV patterning within the CNS is evolutionarily conserved.     

Site-directed mutations in the vnd/NK-2 homeodomain. Basis of variations in structure and sequence-specific DNA binding

Secondary structures, DNA binding properties, and thermal denaturation behavior of six site-directed mutant homeodomains encoded by the vnd/NK-2 gene from Drosophila melanogaster are described. Three single site H52R, Y54M, and T56W mutations, two double site H52R/T56W and Y54M/T56W mutations, and one triple site H52R/Y54M/T56W mutation were investigated. These positions were chosen based on their variability across homeodomains displaying differences in secondary structure and DNA binding specificity. Multidimensional NMR, electrophoretic mobility shift assays, and circular dichroism spectropolarimetry studies were carried out on recombinant 80-amino acid residue proteins containing the homeodomain. Position 56, but more importantly position 56 in combination with position 52, plays an important role in determining the length of the recognition helix. The H52R mutation alone does not affect the length of this helix but does increase the thermal stability. Introduction of site mutations at positions 52 and 56 in vnd/NK-2 does not modify their high affinity binding to the 18-base pair DNA fragment containing the vnd/NK-2 consensus binding sequence, CAAGTG. Site mutations involving position 54 (Y54M, Y54M/T56W, and H52R/Y54M/T56W) all show a decrease of 1 order of magnitude in their binding affinity. The roles in structure and sequence specificity of individual atom-atom interactions are described.     

white+ transgene insertions presenting a dorsal/ventral pattern define a single cluster of homeobox genes that is silenced by the polycomb-group proteins in Drosophila melanogaster

We used the white gene as an enhancer trap and reporter of chromatin structure. We collected white+ transgene insertions presenting a peculiar pigmentation pattern in the eye: white expression is restricted to the dorsal half of the eye, with a clear-cut dorsal/ventral (D/V) border. This D/V pattern is stable and heritable, indicating that phenotypic expression of the white reporter reflects positional information in the developing eye. Localization of these transgenes led us to identify a unique genomic region encompassing 140 kb in 69D1-3 subject to this D/V effect. This region contains at least three closely related homeobox-containing genes that are constituents of the iroquois complex (IRO-C). IRO-C genes are coordinately regulated and implicated in similar developmental processes. Expression of these genes in the eye is regulated by the products of the Polycomb-group (Pc-G) and trithorax-group (trx-G) genes but is not modified by classical modifiers of position-effect variegation. Our results, together with the report of a Pc-G binding site in 69D, suggest that we have identified a novel cluster of target genes for the Pc-G and trx-G products. We thus propose that ventral silencing of the whole IRO-C in the eye occurs at the level of chromatin structure in a manner similar to that of the homeotic gene complexes, perhaps by local compaction of the region into a heterochromatin-like structure involving the Pc-G products.     

Alteration of hormone levels in transgenic tobacco plants overexpressing the rice homeobox gene OSH1

In the search for an endogenous ligand of the orphan G protein-coupled receptor APJ, the presence of the ligand in various tissue extracts was examined by measuring the increase in extracellular acidification rate of the cells expressing the APJ receptor as a specific signal induced by the interaction of the receptor and ligand. By monitoring this activity, we isolated an APJ receptor ligand, designated apelin, from bovine stomach extracts. The structures of bovine and human apelin preproproteins were deduced from the sequences of the corresponding cDNAs. The preproproteins consisted of 77 amino acid residues, and the apelin sequence was encoded in the C-terminal regions. Synthetic peptides derived from the C-terminal amino acid sequence of bovine preproapelin were capable of specifically promoting the acidification rate in the cells expressing the APJ receptor in a range from 10(-7) to 10(-10) M, indicating that apelin is an endogenous ligand for the APJ receptor.     

A unique variant of a homeobox gene related to Drosophila cut is expressed in mouse testis

The wild-type gene encoding granule-bound starch synthase (GBSS) is capable of both complementing the amylose-free (amf) potato mutant and inhibiting the endogenous GBSS gene expression in wild-type potato. Co-suppression of the endogenous GBSS gene, easily visualised by staining the starch with iodine, occurred when the full-size GBSS sequence (genomic), GBSS cDNA or even the mutant amf allele were introduced into the wild-type potato. Conversely, introduction of the GBSS promoter sequence alone, did not result in co-suppression in the 80 analysed transformants. Neither the orientation of the GBSS gene with respect to kanamycin resistance nor the presence of an enhancer influenced the frequency of plants showing a co-suppression phenotype. After crossing a partially complemented amf mutant with a homozygous wild-type plant, the F1 offspring segregated into plant phenotypes with normal and decreased expression of the GBSS gene. This decreased expression correlated with the presence of a linked block of five T-DNA inserts which was previously shown to be correlated with partial complementation of the amf mutant. This crossing experiment indicates that co-suppression can cause inhibition of gene expression of both inserted and endogenous wild-type GBSS genes. The frequency of partially complemented amf plants was equal to the frequency of co-suppressed wild types when a construct, with an enhancer in front of the GBSS promoter, was used (pWAM 101E). This might suggest that partial complementation of the amf genotype caused by unstable expression of the transgene can be overcome by inserting an enhancer in front of the GBSS promoter.     

High level expression of the homeobox gene HB24 in a human T-cell line confers the ability to form tumors in nude mice

Epidermal growth factor (EGF)-induced c-fos and c-jun expression is strongly suppressed in microgravity. We investigate here whether this is due to inhibition of processes occurring during the initiation of EGF-induced signal transduction. For this purpose, EGF-induced receptor clustering is used as a marker. The lateral distribution of EGF receptors is directly visualized at an ultrastructural level by the label-fracture method. Quantification of the receptor distributions shows that EGF-induced receptor redistribution is similar under normal and microgravity conditions. This suggests that microgravity influences EGF-induced signal transduction downstream of EGF binding and EGF receptor redistribution, but upstream of early gene expression in human A431 cells.     

The role of the msh homeobox gene during Drosophila neurogenesis: implication for the dorsoventral specification of the neuroectoderm

Development of the Drosophila central nervous system begins with the delamination of neural and glial precursors, called neuroblasts, from the neuroectoderm. An early and important step in the generation of neural diversity is the specification of individual neuroblasts according to their position. In this study, we describe the genetic analysis of the msh gene which is likely to play a role in this process. The msh/Msx genes are one of the most highly conserved families of homeobox genes. During vertebrate spinal cord development, Msx genes (Msx1-3) are regionally expressed in the dorsal portion of the developing neuroectoderm. Similarly in Drosophila, msh is expressed in two longitudinal bands that correspond to the dorsal half of the neuroectoderm, and subsequently in many dorsal neuroblasts and their progeny. We showed that Drosophila msh loss-of-function mutations led to cell fate alterations of neuroblasts formed in the dorsal aspect of the neuroectoderm, including a possible dorsal-to-ventral fate switch. Conversely, ectopic expression of msh in the entire neuroectoderm severely disrupted the proper development of the midline and ventral neuroblasts. The results provide the first in vivo evidence for the role of the msh/Msx genes in neural development, and support the notion that they may perform phylogenetically conserved functions in the dorsoventral patterning of the neuroectoderm.     

Dorsoventral patterning in the Drosophila central nervous system: the intermediate neuroblasts defective homeobox gene specifies intermediate column identity

One of the first steps in neurogenesis is the diversification of cells along the dorsoventral axis. In Drosophila the central nervous system develops from three longitudinal columns of cells: ventral cells that express the vnd/nk2 homeobox gene, intermediate cells, and dorsal cells that express the msh homeobox gene. Here we describe a new Drosophila homeobox gene, intermediate neuroblasts defective (ind), which is expressed specifically in the intermediate column cells. ind is essential for intermediate column development: Null mutants have a transformation of intermediate to dorsal column neuroectoderm fate, and only 10% of the intermediate column neuroblasts develop. The establishment of dorsoventral column identity involves negative regulation: Vnd represses ind in the ventral column, whereas ind represses msh in the intermediate column. Vertebrate genes closely related to vnd (Nkx2.1 and Nkx2.2), ind (Gsh1 and Gsh2), and msh (Msx1 and Msx3) are expressed in corresponding ventral, intermediate, and dorsal domains during vertebrate neurogenesis, raising the possibility that dorsoventral patterning within the central nervous system is evolutionarily conserved.     

Isolation of a Drosophila homolog of the vertebrate homeobox gene Rx and its possible role in brain and eye development

Vertebrate and invertebrate eye development require the activity of several evolutionarily conserved genes. Among these the Pax-6 genes play a major role in the genetic control of eye development. Mutations in Pax-6 genes affect eye development in humans, mice, and Drosophila, and misexpression of Pax-6 genes in Drosophila can induce ectopic eyes. Here we report the identification of a paired-like homeobox gene, DRx, which is also conserved from flies to vertebrates. Highly conserved domains in the Drosophila protein are the octapeptide, the identical homeodomain, the carboxyl-terminal OAR domain, and a newly identified Rx domain. DRx is expressed in the embryo in the procephalic region and in the clypeolabrum from stage 8 on and later in the brain and the central nervous system. Compared with eyeless, the DRx expression in the embryo starts earlier, similar to the pattern in vertebrates, where Rx expression precedes Pax-6 expression. Because the vertebrate Rx genes have a function during brain and eye development, it was proposed that DRx has a similar function. The DRx expression pattern argues for a conserved function at least during brain development, but we could not detect any expression in the embryonic eye primordia or in the larval eye imaginal discs. Therefore DRx could be considered as a homolog of vertebrate Rx genes. The Rx genes might be involved in brain patterning processes and specify eye fields in different phyla.     

P-Element insertion at the polyhomeotic gene leads to formation of a novel chimeric protein that negatively regulates yellow gene expression in P-element-induced alleles of Drosophila melanogaster

Oxidative stress is implicated in neuronal apoptosis that occurs in physiological settings and in neurodegenerative disorders. Superoxide anion radical, produced during mitochondrial respiration, is involved in the generation of several potentially damaging reactive oxygen species including peroxynitrite. To examine directly the role of superoxide and peroxynitrite in neuronal apoptosis, we generated neural cell lines and transgenic mice that overexpress human mitochondrial manganese superoxide dismutase (MnSOD). In cultured pheochromocytoma PC6 cells, overexpression of mitochondria-localized MnSOD prevented apoptosis induced by Fe2+, amyloid beta-peptide (Abeta), and nitric oxide-generating agents. Accumulations of peroxynitrite, nitrated proteins, and the membrane lipid peroxidation product 4-hydroxynonenal (HNE) after exposure to the apoptotic insults were markedly attenuated in cells expressing MnSOD. Glutathione peroxidase activity levels were increased in cells overexpressing MnSOD, suggesting a compensatory response to increased H2O2 levels. The peroxynitrite scavenger uric acid and the antioxidants propyl gallate and glutathione prevented apoptosis induced by each apoptotic insult, suggesting central roles for peroxynitrite and membrane lipid peroxidation in oxidative stress-induced apoptosis. Apoptotic insults decreased mitochondrial transmembrane potential and energy charge in control cells but not in cells overexpressing MnSOD, and cyclosporin A and caspase inhibitors protected cells against apoptosis, demonstrating roles for mitochondrial alterations and caspase activation in the apoptotic process. Membrane lipid peroxidation, protein nitration, and neuronal death after focal cerebral ischemia were significantly reduced in transgenic mice overexpressing human MnSOD. The data suggest that mitochondrial superoxide accumulation and consequent peroxynitrite production and mitochondrial dysfunction play pivotal roles in neuronal apoptosis induced by diverse insults in cell culture and in vivo.     

Hox-type and non-Hox homeobox gene sequences in genomic DNA of the sea urchin Holopneustes purpurescens

As a preliminary step in an analysis of Hox gene expression and radial body plan specification in sea urchin development, we amplified partial homeobox sequences in H. purpurescens by PCR using degenerate primers. The primers, HoxE and HoxF (Pendleton et al., 1993), spanned a highly conserved region of 82 nucleotides encompassing amino acids 21-47 of the homeodomain. Seven Hox-type homeobox sequences and two non-Hox homeobox sequences were identified. The seven Hox-type sequences were placed provisionally in Hox paralogous groups, one in paralogous group 3, three in paralogous groups 6-8 and three in paralogous groups 9 13. The non-Hox sequences had similarities with Xlox and Gbx homeobox genes.     

The mithramycin gene cluster of Streptomyces argillaceus contains a positive regulatory gene and two repeated DNA sequences that are located at both ends of the cluster

Sequencing of a 4.3-kb DNA region from the chromosome of Streptomyces argillaceus, a mithramycin producer, revealed the presence of two open reading frames (ORFs). The first one (orfA) codes for a protein that resembles several transport proteins. The second one (mtmR) codes for a protein similar to positive regulators involved in antibiotic biosynthesis (DnrI, SnoA, ActII-orf4, CcaR, and RedD) belonging to the Streptomyces antibiotic regulatory protein (SARP) family. Both ORFs are separated by a 1.9-kb, apparently noncoding region. Replacement of the mtmR region by an antibiotic resistance cassette completely abolished mithramycin biosynthesis. Expression of mtmR in a high-copy-number vector in S. argillaceus caused a 16-fold increase in mithramycin production. The mtmR gene restored actinorhodin production in Streptomyces coelicolor JF1 mutant, in which the actinorhodin-specific activator ActII-orf4 is inactive, and also stimulated actinorhodin production by Streptomyces lividans TK21. A 241-bp region located 1.9 kb upstream of mtmR was found to be repeated approximately 50 kb downstream of mtmR at the other end of the mithramycin gene cluster. A model to explain a possible route for the acquisition of the mithramycin gene cluster by S. argillaceus is proposed.