Segmentation of the embryonic vertebrate hindbrain into rhombomeres (r) ensures proper spatial positioning of hindbrain derivatives (e.g. reticulospinal interneurons and motornuclei of the cranial nerves) and is therefore essential for normal neurological activity. This segmentation process is regulated by a growing number of genes, but the function of many of these genes, as well as the regulatory relationships among them, remains unclear. We have identified 12 novel genes expressed in the hindbrain and we hypothesize that these genes act in a regulatory network controlling rhombomere formation. We have developed two aims to test our hypothesis: First we will determine the function of several novel hindbrain genes in rhombomere 4/5 formation. We are focusing particularly on 3-4 genes that we predict are involved in cell sorting at rhombomere boundaries. We will use loss of function approaches (morpholino-mediated knock-down, zinc-finger nuclease-mediated targeted deletions), as well as misexpression approaches (mRNA injections, GAL4:UAS transgenesis), to determine the function of these genes. Second, we will delineate transcription regulatory pathways controlling formation of rhombomere 4/5. Many known r4/r5 genes encode transcription factors, but it is not clear which genes they regulate. We have generated antisera to several of these transcription factors and will use chromatin immunoprecipitation (ChIP) assays to identify direct regulatory relationships among genes acting in r4/r5. We will take both a candidate approach, where we test binding of a specific transcription factor to a predicted target promoter, and a global approach, where we design a hindbrain-promoter tiling-array that will permit identification of all hindbrain promoters bound by a given transcription factor. Our experiments are important because the developing hindbrain is sensitive to disruptions by a variety of factors (e.g. environmental toxins, infectious agents and genetic conditions) that give rise to a range of birth defects - motor control problems such as ataxia, cognitive defects such as autism and craniofacial defects. A better understanding of hindbrain formation will therefore be applicable to a broad set of biological processes and human disease conditions. Our experiments also make novel use of several techniques - ChIP, GAL4:UAS transgenics - in zebrafish.

Public Health Relevance

The proposed experiments are aimed at understanding hindbrain formation during embryogenesis. The embryonic hindbrain (and associated neural crest cells) gives rise to many essential structures - sensory ganglia and branchiomotor neurons of the nervous system, as well as bone, cartilage and muscle of the head. The developing hindbrain is sensitive to disruptions by a variety of factors (e.g. environmental toxins, infectious agents and genetic conditions) that give rise to a range of birth defects - motor control problems such as ataxia, cognitive defects such as autism and craniofacial defects. In addition, the genes studied in this proposal regulate other aspects of neural development (e.g. dorsoventral patterning of the neural tube), and other aspects of embryogenesis (e.g. hematopoiesis). The results from our proposed experiments will therefore be applicable to a broad set of biological processes and human disease conditions.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD065081-03
Application #
8442315
Study Section
Neurogenesis and Cell Fate Study Section (NCF)
Program Officer
Henken, Deborah B
Project Start
2011-02-05
Project End
2016-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
3
Fiscal Year
2013
Total Cost
$331,736
Indirect Cost
$130,073
Name
University of Massachusetts Medical School Worcester
Department
Biochemistry
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
Ladam, Franck; Sagerström, Charles G (2014) Functional analysis of hox genes in zebrafish. Methods Mol Biol 1196:133-44
Weicksel, Steven E; Gupta, Ankit; Zannino, Denise A et al. (2014) Targeted germ line disruptions reveal general and species-specific roles for paralog group 1 hox genes in zebrafish. BMC Dev Biol 14:25
Ladam, Franck; Sagerstrom, Charles G (2014) Hox regulation of transcription: more complex(es). Dev Dyn 243:4-15
Zannino, Denise A; Downes, Gerald B; Sagerström, Charles G (2014) prdm12b specifies the p1 progenitor domain and reveals a role for V1 interneurons in swim movements. Dev Biol 390:247-60
Choe, Seong-Kyu; Nakamura, Mako; Ladam, Franck et al. (2012) A Gal4/UAS system for conditional transgene expression in rhombomere 4 of the zebrafish hindbrain. Dev Dyn 241:1125-32