Friday, May 9, 2025
5/9/2025
Role of Gsx2 in hindbrain neurogenesis - Project Summary. Central apnea in newborns results from dysregulation of neuronal breathing circuitry. In sudden infant death syndrome (SIDS), these circuits can function apparently normally at birth but fail at some point in the first year. It is imperative to better understand the molecular and genetic requirements of developing neuronal breathing circuitry to create neuronal targeted therapies for SIDS. Mice with homozygous null mutations in the homeobox transcription factor (TF) gene Gsx2 die within hours of birth after becoming cyanotic and ceasing to breathe. Our preliminary data demonstrate that Gsx2 is expressed in hindbrain neural progenitors of the “dA3” domain which give rise to dorsal respiratory group (dRG) glutamatergic neurons in the nucleus tractus solitarius (nTS) and their associated catecholaminergic neurons of the A1/C1 and A2/C2 groups. At birth, the Gsx2-null mutants show a nearly complete loss of glutamatergic nTS neurons and A1/C1, A2/C2 catecholamine neurons, consistent with their lack of viability. Furthermore, Gsx2 mutant dA3 progenitors exhibit a loss of the bHLH TF Ascl1, despite that Ascl1 mutants only phenocopy the complete loss of A1/C1, A2/C2 neurons suggesting that Gsx2 regulates other TFs for nTS neurogenesis. Functional analysis shows that Gsx2 null mice aspirate milk when fed and display fewer apneas than their control littermates, consistent with the nTS regulating beneficial apneas and gating between swallowing and breathing for airway protection. The central hypothesis for this proposal is that Gsx2 directly regulates Ascl1 to induce differentiation of early-born A1/C1, A2/C2 catecholamine neurons and glutamatergic nTS neurons, but additional direct target TF genes are required for the normal generation of glutamatergic nTS neurons. To test this hypothesis, 2 aims are proposed. In Aim 1a, Gsx2 will be knocked out during early (E9.5) and late (E11.5) dA3 nTS neurogenesis using a tamoxifen-inducible Olig3- CreERT2 mouse and conditional Gsx2Flx allele to assess the neuronal alterations and effect on swallowing and breathing. In Aim 1b, E10.5 dorsocaudal hindbrains containing Gsx2+ progenitors will be dissected, RNA-seq performed on Gsx2-nulls, and CUT&RUN performed on Gsx2Flag/Flag animals to identify direct transcriptional targets of Gsx2 in dA3 progenitors. In Aim 2a, Ascl1 will be conditionally re-expressed (with tetO-Ascl1) in the Gsx2-null background, and in Aim 2b, Ascl1 will be conditionally knocked out (with Ascl1Flx) in dA3 progenitors in both cases using Gsx2e-CIE to determine if Ascl1 is a) sufficient, and b) required for differentiation of dA3- derived A1/C1, A2/C2 neurons in breathing, airway protection, and survival. Successful completion of these aims will decipher the temporal requirements of Gsx2 in dA3-derived dRG neurogenesis as well as the gene regulatory network (GRN) controlled by Gsx2 during hindbrain neurogenesis, contributing to our understanding of central control of breathing. The training received at Cincinnati Children’s Medical Center under Drs. Kenneth Campbell (neural development) and Steven Crone (breathing physiology) will help the applicant build a novel hindbrain respiration control research program in the Campbell lab and transition into an independent investigator.
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