Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of trigeminal ganglia of Brn3a knockout and wild type mice to determine the regulatory targets of transcription factor Brn3a.

ABSTRACT: Mice lacking the POU-domain transcription factor Brn3a exhibit marked defects in sensory axon growth and abnormal sensory apoptosis. We have determined the regulatory targets of Brn3a in the developing trigeminal ganglion using microarray analysis of Brn3a mutant mice. These results show that Brn3 mediates the coordinated expression of neurotransmitter systems, ion channels, structural components of axons and inter- and intracellular signaling systems. Loss of Brn3a also results in the ectopic expression of transcription factors normally detected in earlier developmental stages and in other areas of the nervous system. Target gene expression is normal in heterozygous mice, consistent with prior work showing that autoregulation by Brn3a results in gene dosage compensation. Detailed examination of the expression of several of these downstream genes reveals that the regulatory role of Brn3a in the trigeminal ganglion appears to be conserved in more posterior sensory ganglia but not in the CNS neurons that express this factor. Experiment Overall Design: Microarrays used to compare the patterns of gene expression in the trigeminal ganglia of Brn3a knockout and wild-type mice. Embryonic day 13.5 (E13.5) was chosen because at this point in development mutant mice exhibit major defects in sensory axon growth, but have yet to undergo the period of extensive sensory neuron death associated with later stages.

ORGANISM(S): Mus musculus

SUBMITTER: Eric Turner

PROVIDER: E-GEOD-3895 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Coordinated regulation of gene expression by Brn3a in developing sensory ganglia.

Eng S Raisa SR Lanier Jason J Fedtsova Natalia N Turner Eric E EE

Development (Cambridge, England) 20040714 16

Mice lacking the POU-domain transcription factor Brn3a exhibit marked defects in sensory axon growth and abnormal sensory apoptosis. We have determined the regulatory targets of Brn3a in the developing trigeminal ganglion using microarray analysis of Brn3a mutant mice. These results show that Brn3 mediates the coordinated expression of neurotransmitter systems, ion channels, structural components of axons and inter- and intracellular signaling systems. Loss of Brn3a also results in the ectopic e ...[more]

PMID: 15253936

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Project description:Using targeted gene deletion, we have firmly established that the Class IV POU domain transcription factor Brn-3.2 controls a developmental program regulating axon pathfinding in the mouse visual system. We have isolated and identified downstream gene targets of Brn-3.2, using Representational Difference Analysis (RDA), on cDNA populations derived from wildtype and Brn-3.2-/- retina at the appropriate embryonic stage. One of these candidate genes, the Homeodomain Interacting Protein Kinase 2 (HIPK2), is postulated to be a transcriptional coregulator, based on its in vitro interactions with repressor homeodomain proteins of the NK class, as well as other components of repressor complexes. HIPK2 has also been shown to be involved in post-translational modification of two major transcriptional regulators, p53 and CtBP. Expression of a dominant-negative form of HIPK2 in sensory neurons affects the innervation patterns of their target tissues, suggesting an axon pathfinding defect.,The aim of this project is to identify targets for the Homeodomain Interacting Protein Kinase 2 (HIPK2), that we isolated as a downstream gene target of the class IV POU domain transcription factor Brn-3.2, and to investigate their function in the Brn-3.2 dependent pathway regulating axon pathfinding. ,We hypothesize that the genes regulated by HIPK2 will play a critical role in axon pathfinding and that the results of this study will provide novel insights into the molecular mechanisms of axon pathfinding, and possibly neural plasticity and regeneration, and therefore, be of great interest to the field of neurobiology in general.,In order to uncover potential biological role(s) of HIPK2 in neural development, and particularly axon pathfinding, we generated transgenic mouse models for in vivo studies. Our preliminary observations in transgenic mice, indicate that a form of the molecule expected to act as a dominant-negative and designed to be expressed in sensory neurons, affects the innervation patterns of their target tissues, suggesting an axon pathfinding defect. We plan to take advantage of this model to identify genes regulated by HIPK2, and which are likely to be involved in axon pathfinding. To this end, we will compare gene expression profiles in sensory neurons isolated wild-type and transgenic mice. In our experimental paradigm, the trigeminal ganglion represents the ideal sensory structure in which to perform such an analysis: 1) High levels of transgene can be expressed in the trigeminal ganglion, as assessed by the expression of LacZ from the bicistronic construct which contains IRES-LacZ downstream of a dominant negative form of HIPK2. 2) The trigeminal ganglion can be dissected at embryonic day 13.5, when the phenotype is apparent, with ease and free of contamination from surrounding tissues. 3) The amounts of RNA that can be isolated from a single ganglion are in the range of 200-300 ng, which should be sufficient for microarray analysis following linear amplification of RNA. One series will be carried out with transgenic embryos and a control series will be carried out with wildtype littermates. Animals will be prepared and sacrificed by a standard protocol. Tissue will be rapidly dissected from E13.5 trigeminal ganglion, frozen in liquid nitrogen, and stored at -80C until RNA is extracted. RNA will be prepared using RNeasy Micro Kit. We will be providing 4 tissue samples for each of the wildtype and transgenic animals to mitigate any expression differences resulting from mouse to mouse variation.

Dataset Information

Transcription profiling of trigeminal ganglia of Brn3a knockout and wild type mice to determine the regulatory targets of transcription factor Brn3a.

Publications

Coordinated regulation of gene expression by Brn3a in developing sensory ganglia.

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