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Enhancer diversity and the control of a simple pattern of Drosophila CNS midline cell expression.

ABSTRACT: Transcriptional enhancers integrate information derived from transcription factor binding to control gene expression. One key question concerns the extent of trans- and cis-regulatory variation in how co-expressed genes are controlled. The Drosophila CNS midline cells constitute a group of neurons and glia in which expression changes can be readily characterized during specification and differentiation. Using a transgenic approach, we compare the cis-regulation of multiple genes expressed in the Drosophila CNS midline primordium cells, and show that while the expression patterns may appear alike, the target genes are not equivalent in how these common expression patterns are achieved. Some genes utilize a single enhancer that promotes expression in all midline cells, while others utilize multiple enhancers with distinct spatial, temporal, and quantitative contributions. Two regulators, Single-minded and Notch, play key roles in controlling early midline gene expression. While Single-minded is expected to control expression of most, if not all, midline primordium-expressed genes, the role of Notch in directly controlling midline transcription is unknown. Midline primordium expression of the rhomboid gene is dependent on cell signaling by the Notch signaling pathway. Mutational analysis of a rhomboid enhancer reveals at least 5 distinct types of functional cis-control elements, including a binding site for the Notch effector, Suppressor of Hairless. The results suggest a model in which Notch/Suppressor of Hairless levels are insufficient to activate rhomboid expression by itself, but does so in conjunction with additional factors, some of which, including Single-minded, provide midline specificity to Notch activation. Similarly, a midline glial enhancer from the argos gene, which is dependent on EGF/Spitz signaling, is directly regulated by contributions from both Pointed, the EGF transcriptional effector, and Single-minded. In contrast, midline primordium expression of other genes shows a strong dependence on Single-minded and varying combinations of additional transcription factors. Thus, Single-minded directly regulates midline primordium-expressed genes, but in some cases plays a primary role in directing target gene midline expression, and in others provides midline specificity to cell signaling inputs.

SUBMITTER: Pearson JC

PROVIDER: S-EPMC4104230 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Enhancer diversity and the control of a simple pattern of Drosophila CNS midline cell expression.

Pearson Joseph C JC Crews Stephen T ST

Developmental biology 20140520 2

Transcriptional enhancers integrate information derived from transcription factor binding to control gene expression. One key question concerns the extent of trans- and cis-regulatory variation in how co-expressed genes are controlled. The Drosophila CNS midline cells constitute a group of neurons and glia in which expression changes can be readily characterized during specification and differentiation. Using a transgenic approach, we compare the cis-regulation of multiple genes expressed in the ...[more]

PMID: 24854999

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Project description:The Drosophila Zelda transcription factor plays an important role in regulating transcription at the embryonic maternal-to-zygotic transition. However, expression of zelda continues throughout embryogenesis in cells including the developing CNS and trachea, but little is known about its post-blastoderm functions. In this paper, it is shown that zelda directly controls CNS midline and tracheal expression of the link (CG13333) gene, as well as link blastoderm expression. The link gene contains a 5' enhancer with multiple Zelda TAGteam binding sites that in vivo mutational studies show are required for link transcription. The link enhancer also has a binding site for the Single-minded:Tango and Trachealess:Tango bHLH-PAS proteins that also influences link midline and tracheal expression. These results provide an example of how a transcription factor (Single-minded or Trachealess) can interact with distinct co-regulatory proteins (Zelda or Sox/POU-homeodomain proteins) to control a similar pattern of expression of different target genes in a mechanistically different manner. While zelda and single-minded midline expression is well-conserved in Drosophila, midline expression of link is not well-conserved. Phylogenetic analysis of link expression suggests that ~60 million years ago, midline expression was nearly or completely absent, and first appeared in the melanogaster group (including D. melanogaster, D. yakuba, and D. erecta) >13 million years ago. The differences in expression are due, in part, to sequence polymorphisms in the link enhancer and likely due to altered binding of multiple transcription factors. Less than 6 million years ago, a second change occurred that resulted in high levels of expression in D. melanogaster. This change may be due to alterations in a putative Zelda binding site. Within the CNS, the zelda gene is alternatively spliced beginning at mid-embryogenesis into transcripts that encode a Zelda isoform missing three zinc fingers from the DNA binding domain. This may result in a protein with altered, possibly non-functional, DNA-binding properties. In summary, Zelda collaborates with bHLH-PAS proteins to directly regulate midline and tracheal expression of an evolutionary dynamic enhancer in the post-blastoderm embryo.

Project description:Neuron-glial interactions are crucial for growth, guidance and ensheathment of axons across species. In the Drosophila CNS midline, neuron-glial interactions underlie ensheathment of commissural axons by midline glial (MG) cells in a manner similar to mammalian oligodendrocytes. Although there has been some advance in the study of neuron-glial interactions and ensheathment of axons in the CNS midline, key aspects of axonal ensheathment are still not fully understood. One of the limitations has been the unavailability of MG membrane markers that could highlight the glial processes wrapping the axons. Previous studies have identified two key molecular players from the neuronal and glial cell types in the CNS midline. These are the neuronal transmembrane protein Neurexin IV (Nrx IV) and the membrane-anchored MG protein Wrapper, both of which interact in trans to mediate neuron-glial interactions and ensheathment of commissural axons. In the current study, we attempt to further our understanding of MG biology and try to overcome some of the technical difficulties posed by the lack of a robust MG driver that will specifically allow expression or knockdown of genes in MG. We report the generation of BAC transgenic flies of wrapper-GAL4 and demonstrate how these flies could be used as a genetic tool to understand MG biology. We have utilized the GAL4/UAS system to drive GFP-reporter lines (membrane-bound mCD8-GFP; microtubule-associated tau-GFP) and nuclear lacZ using wrapper-GAL4 to highlight the MG cells and/or their processes that surround and perform axonal ensheathment functions in the embryonic midline. We also describe the utility of the wrapper-GAL4 driver line to down-regulate known MG genes specifically in Wrapper-positive cells. Finally, we validate the functionality of the wrapper-GAL4 driver by rescue of wrapper mutant phenotypes and lethality. Together, these studies provide us with a versatile genetic tool to investigate MG functions and will aid in future investigations where genetic screens using wrapper-GAL4 could be designed to identify novel molecular players at the Drosophila midline and unravel key aspects of MG biology.

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Enhancer diversity and the control of a simple pattern of Drosophila CNS midline cell expression.

Publications

Enhancer diversity and the control of a simple pattern of Drosophila CNS midline cell expression.

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