Transcriptional activation domains of the single-minded bHLH protein are required for CNS midline cell development.
ABSTRACT The single-minded gene functions as a master developmental regulator within the midline cell lineage of the embryonic central nervous system of Drosophila melanogaster. Genetic experiments suggest that Single-minded can function as a transcriptional activator. Regions of the Single-minded protein were fused to the DNA binding domain of the mammalian transcription factor Sp1 and shown to activate transcription from a reporter gene linked to Sp1 binding sites. Three independent activation domains were identified in the carboxy terminal region of Single-minded that include areas rich in serine, threonine, glutamine and proline residues. Germ line transformation experiments indicate that the carboxy terminal activation domains, the PAS dimerization domain, and the putative DNA binding basic domain of Single-minded are required for expression of CNS midline genes in vivo. These results define in vivo a functional activation domain within Single-minded and suggest a model in which Single-minded activates transcription through a direct interaction with promoter elements of CNS midline genes.
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ABSTRACT: Developmental regulatory proteins are commonly utilized in multiple cell types throughout development. The Drosophila single-minded (sim) gene acts as master regulator of embryonic CNS midline cell development and transcription. However, it is also expressed in the brain during larval development. In this paper, we demonstrate that sim is expressed in three clusters of anterior central brain neurons: DAMv1/2, BAmas1/2, and TRdm and in three clusters of posterior central brain neurons: a subset of DPM neurons, and two previously unidentified clusters, which we term PLSC and PSC. In addition, sim is expressed in the lamina and medulla of the optic lobes. MARCM studies confirm that sim is expressed at high levels in neurons but is low or absent in neuroblasts (NBs) and ganglion mother cell (GMC) precursors. In the anterior brain, sim(+) neurons are detected in 1st and 2nd instar larvae but rapidly increase in number during the 3rd instar stage. To understand the regulation of sim brain transcription, 12 fragments encompassing 5'-flanking, intronic, and 3'-flanking regions were tested for the presence of enhancers that drive brain expression of a reporter gene. Three of these fragments drove expression in sim(+) brain cells, including all sim(+) neuronal clusters in the central brain and optic lobes. One fragment upstream of sim is autoregulatory and is expressed in all sim(+) brain cells. One intronic fragment drives expression in only the PSC and laminar neurons. Another downstream intronic fragment drives expression in all sim(+) brain neurons, except the PSC and lamina. Thus, together these two enhancers drive expression in all sim(+) brain neurons. Sequence analysis of existing sim mutant alleles identified three likely null alleles to utilize in MARCM experiments to examine sim brain function. Mutant clones of DAMv1/2 neurons revealed a consistent axonal fasciculation defect. Thus, unlike the embryonic roles of sim that control CNS midline neuron and glial formation and differentiation, postembryonic sim, instead, controls aspects of axon guidance in the brain. This resembles the roles of vertebrate sim that have an early role in neuronal migration and a later role in axonogenesis.Gene Expression Patterns 09/2011; 11(8):533-46. · 1.64 Impact Factor
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ABSTRACT: Basic helix-loop-helix (bHLH) transcription factors play critical roles in the regulation of a wide range of developmental processes in higher organisms and have been identified in more than 20 organisms. Mosquitoes are important vectors of certain human diseases. In this study, Aedes aegypti, Anopheles gambiae str. PEST and Culex quinquefasciatus genomes were found to encode 55, 55 and 57 bHLH genes, respectively. Further phylogenetic analyses and OrthoDB and Kyoto encyclopedia of genes and genomes orthology database searches led us to define orthology for all the identified mosquito bHLHs successfully. This provides useful information with which to update annotations to 40 Ae. aegypti, 55 An. gambiae and 38 C. quinquefasciatus bHLH genes in VectorBase. The mosquito lineage has more bHLH genes in the Atonal, neurogenin (Ngn) and Hes-related with YRPW motif (Hey) families than do other insect species, suggesting that mosquitoes have evolved to be more sensitive to vibration, light and chemicals. Mosquito bHLH genes generally have higher evolutionary rates than other insect species. However, no pervasive positive selection occurred in the evolution of insect bHLH genes. Only episodic positive selection was found to affect evolution of bHLH genes in 11 families. Besides, coding regions of several Ae. aegypti bHLH motifs have unusually long introns in which multiple copies of transposable elements have been identified. These data provide a solid basis for further studies on structures and functions of bHLH proteins in the regulation of mosquito development and for prevention and control of mosquito-mediated human diseases.Insect Molecular Biology 08/2013; · 3.04 Impact Factor
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ABSTRACT: In the CNS neurons are highly sensitive to the availability of oxygen. In conditions where oxygen availability is decreased neuronal function can be altered, leading to injury and cell death. Hypoxia has been implicated in a number of central nervous system pathologies including stroke, head trauma, and neurodegenerative diseases. Cellular responses to oxygen deprivation are complex and result in activation of short- and long-term mechanisms to conserve energy and protect cells. Failure of synaptic transmission can be observed within minutes following this hypoxia. The acute effects of hypoxia on synaptic transmission are primarily mediated by altering ion fluxes across membranes, presynaptic effects of adenosine and other actions at glutamatergic receptors. A more long-term feature of the response of neurons to hypoxia is the activation of transcription factors such as hypoxia inducible factor. The activation of hypoxia inducible factor is governed by a family of dioxygenases called hypoxia inducible factor prolyl 4 hydroxylases (PHDs). Under hypoxic conditions, PHD activity is inhibited, thereby allowing hypoxia inducible factor to accumulate and translocate to the nucleus, where it binds to the hypoxia-responsive element sequences of target gene promoters. Inhibition of PHD activity stabilizes hypoxia inducible factor and other proteins thus acting as a neuroprotective agent. This review will focus on the response of neuronal cells to hypoxia inducible factor and its targets, including the prolyl hydroxylases. We also present evidence for acute effects of PHD inhibition on synaptic transmission and plasticity in the hippocampus. This article is protected by copyright. All rights reserved.Acta Physiologica 05/2013; · 4.38 Impact Factor