Golgb1 regulates protein glycosylation and is crucial for mammalian palate development

Abstract

Cleft palate is a common major birth defect for which currently known causes account for less than 30% of pathology in humans. In this study, we carried out mutagenesis screening in mice to identify new regulators of palatogenesis. Through genetic linkage mapping and whole exome sequencing, we identified a loss-of-function mutation in the Golgb1 gene that co-segregated with cleft palate in a new mutant mouse line. Golgb1 encodes a ubiquitously expressed large coiled-coil protein, known as giantin, that is localized at the Golgi membrane. Using CRISPR/Cas9-mediated genome editing, we generated and analyzed developmental defects in mice carrying additional Golgb1 loss-of-function mutations, which validated a critical requirement for Golgb1 in palate development. Through maxillary explant culture assays, we demonstrate that the Golgb1 mutant embryos have intrinsic defects in palatal shelf elevation. Just prior to the developmental stage of palatal shelf elevation in the wildtype littermates, Golgb1 mutant embryos exhibit increased cell density, reduced hyaluronan accumulation, and impaired protein glycosylation in the palatal mesenchyme. Together, these results demonstrate that, although it is a ubiquitously expressed Golgi-associated protein, Golgb1 has specific functions in protein glycosylation and tissue morphogenesis.

Golga5 is dispensable for mouse embryonic development and postnatal survival

“…Since Golga5 is structurally closely related to Golgb1 and since we recently showed that mice lacking Golgb1 survive to birth and exhibit specific craniofacial developmental defects (Lan et al 2016), we investigated whether the lack of obvious developmental defects in Golga5 -deficient mice is due to functional complementation by Golgb1. We crossed Golga5 E3-4del/E3-4del homozygous female mice with Golgb1 E13-25bp/+ male mice, which are heterozygous for a 25-bp deletion in exon-13 of the Golgb1 gene that causes complete loss of Golgb1 function (Lan et al 2016).…”

“…We crossed Golga5 E3-4del/E3-4del homozygous female mice with Golgb1 E13-25bp/+ male mice, which are heterozygous for a 25-bp deletion in exon-13 of the Golgb1 gene that causes complete loss of Golgb1 function (Lan et al 2016). We then crossed Golga5 E3-4del/+ Golgb1 E13-25bp/+ double heterozygous progeny with Golga5 E3-4del/E3-4del homozygous mice and found that the Golga5 E3-4del/E3-4del Golgb1 E13-25bp/+ mice (n=15) survive postnatally without obvious developmental abnormality and behaved similarly as the Golga5 E3-4del/E3-4del and Golga5 E3-4del/+ Golgb1 E13-25bp/+ littermates.…”

“…The target sequences were subcloned into the PX459 [pSpCas9(BB)-2A-Puro] mammalian Cas9-expression vector (Addgene #48139) and tested by transfection into cultured immortalized mouse embryonic kidney (MK4) cells as described previously (Lan et al 2016). Both sgRNAs were synthesized in vitro , combined with humanized Cas9 mRNAs and co-injected into zygotes from FVB/NJ inbred mice at the Cincinnati Children’s Hospital Medical Center Transgenic Animal and Genome Editing Facility.…”

“…Structurally, Golga5 is most similar to Golgb1, containing a coiled-coil cytoplasmic domain and a C-terminal transmembrane domain that anchors to the Golgi membrane (Bascom et al 1999). Whereas studies of Golgb1 -deficient rat and mouse models showed that Golgb1 plays crucial roles in craniofacial and skeletal development (Katayama et al 2011; Lan et al 2016), the physiological functions of Golga5 are unknown. We report here the generation and analysis of Golga5 -deficent mice.…”

Deficiency of RDH10 causes cleft secondary palate in mouse.

“…Freshly dissected maxillary explants of E13.5 embryos were cultured as described previously by Lan et al (Lan, Zhang et al 2016). …”