Gbx2 Hypomorph Mice with Cranial Nerve V, VII, and VIII as well as Craniofacial and Cardiac Abnormalities

Abstract

BackgroundThe Gastrulation‐brain‐homeobox (Gbx) transcription factor family member, Gbx2, regulates diverse developmental processes. Gene inactivation studies in mice have shown that expression of Gbx2 is required for the correct formation of rhombomeres r1‐r3. Importantly, loss of Gbx2 function in mouse embryos (Gbx2−/−), results in aberrant neural crest cell (NCC) patterning leading to defects in neural crest (NC) derivatives and to abnormalities in the central nervous system, craniofacial, and cardiovascular components. Cranial NCCs contribute to neuronal development, migrate together with mesodermal cells early during embryological development, and give rise to craniofacial bones and connective tissue of cranial muscles. Cardiac NCCs are a subpopulation of the cranial NCCs and are needed for proper cardiovascular development.Gbx2 hypomorph mice (Gbx2neo/neo) express 6–10% of wildtype mice Gbx2 expression and present with an overall milder phenotype than Gbx2−/− mice. Our previous data show that NC‐derived sensory neurons and axons constituting the trigeminal mandibular branch (cranial nerve V, CNV), which innervate the muscles of mastication, are absent in Gbx2neo embryos. However, other cranial nerves and the effect on craniofacial muscles and cardiovascular structures have not yet been investigated.HypothesisIn Gbx2neo mice, we expect deficiencies in craniofacial (branchiomeric) muscles as well as in the heart due to NCC contributions to head muscle connective tissue and to regulatory mechanism during heart development, e.g., outflow tract septation. Specifically, we expect less muscle mass in head and heart musculature, decreased attachment areas of craniofacial muscles to the head skeleton, as well as mild cardiac defects. We do not expect the defects to be as pronounced as in Gbx2−/− mice.MethodsWe dissected Gbx2neo mice under the dissection microscope to confirm our predictions about the morphology of head and heart musculature. Additionally, we started performing in situ hybridization and RT‐PCR to confirm the candidate genes identified through our ChIP‐Seq analysis are direct targets of GBX2.Results & DiscussionGbx2neo mice display an overall reduced muscle mass and a milder phenotype than to Gbx2−/− mice. Our data confirm previous observations regarding the absence of NC‐derived sensory neurons and axons constituting the mandibular division of the trigeminal nerve (CNV). Additionally, effected are the maxillary division of CNV (absent), as well as the facial‐acoustic ganglia (CNVII and CNVIII; smaller). The latter indicate abnormal development of NCCs deriving from more posterior rhombomeres than previously reported. Furthermore, we show that genes required for the migration and survival of NCCs (e.g., Robo1, Slit3, Nrp1), and vascular morphogenesis (e.g., Tgfα), are direct targets of GBX2.ConclusionThe Gbx2neo mice are a well‐suited model to study the NC influence on central nervous system, and craniofacial and cardiac development. Additionally, studying the effect of gene expression reduction on downstream targets of GBX2 will elucidate gene regulatory mechanisms between different embryonic tissues, i.e., NC and CPF mesoderm.