Novel Tubgcp4 murine models of microcephaly and chorioretinopathy

Abstract

Purpose : Tubulin γ complex protein 4 (TUBGCP4) variants result in microcephaly, chorioretinopathy, and abnormal retinal vascular development in an autosomal recessive manner. TUBGCP4 is a component of the gamma-tubulin ring complex, which is necessary for microtubule nucleation in cells. Li et al. reported that homozygous Tubgcp4 knockout (KO) results in murine embryonic lethality, and that Tubgcp4^+/- mice are haplo-insufficient, which is not observed in humans. Thus, viable in vivo models of TUBGCP4 deficiency are needed to define its role in development.

Methods : Mice heterozygous for a novel CRISPR-generated Tubgcp4^KO allele were intercrossed embryos were harvested at multiple timepoints. A “gene trap with conditional potential” allele (Tubgcp4^cgt) with exon 6 flanked by loxP sites was also generated, which facilitates creation of a conditional Tubgcp4^fl “floxed” allele for tissue-specific Tubgcp4 deletion. A χ2 test was used to evaluate deviation from expected Mendelian ratios. Murine fundus photography, optical coherence tomography (OCT), and electroretinograms (ERG) were performed. Retinal flat mounts were stained with an endothelial marker.

Results : Intercrosses of Tubgcp4^KO/+ mice produced no Tubgcp4^KO/KO mice, demonstrating embryonic lethality (p<0.0005) with loss of Tubgcp4^KO/KO mice prior to embryonic day 9.5 (p<0.014). Tubgcp4^KO/+ embryos were morphologically indistinguishable from wildtype (WT) embryos. Tubgcp4^KO/+ and WT littermates were aged for 18 months to evaluate for susceptibility to retinal degeneration. There were no significant differences between these genotypes in ERG amplitudes or implicit times, retinal anatomy, or retinal thickness. Tubgcp4^KO/+ retinal flat mounts revealed no vascularization defect. Mice heterozygous for the Tubgcp4^cgt/+ allele were intercrossed. Embryonic lethality of Tubgcp4^cgt/cgt mice was also observed (p< 6.7x10-5), and heterozygous mice were observed in expected ratios. The Tubgcp4^cgt allele was therefore converted to the Tubgcp4^fl allele to enable tissue- or time-specific conditional KO mice, which are in generation.

Conclusions : Two novel models targeting Tubgcp4 (Tubgcp4^KO/KO and Tubgcp4^cgt/cgt) demonstrate embryonic lethality. Initial characterization of heterozygous mice did not reveal evidence of haploinsufficiency, in contrast to a prior report. The conditional Tubgcp4 KO mice should allow mechanistic studies of microcephaly and chorioretinopathy induced by TUBGCP4 loss.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.