TMBIM4 affects left-right patterning via pluripotency exit during gastrulation

Abstract

Congenital heart disease (CHD) is the most prevalent congenital defect, but its underlying genetic and developmental mechanisms remain incompletely understood. Transmembrane BAX inhibitor motif-containing protein 4 (TMBIM4) has emerged as a candidate gene from genomic studies in CHD patients. Patients with deleterious genetic variation in TMBIM4 can exhibit cardiac heterotaxy, a type of left-right (LR) patterning defect characterized by abnormal cardiac asymmetry. Using Xenopus tropicalis, we investigated tmbim4's developmental roles and identified its critical function in LR patterning. tmbim4 depletion in Xenopus produced cardiac asymmetry defects which could be rescued by human and viral orthologs of the protein, reflecting remarkable evolutionary conservation. We identified gastrulation as a critical window for tmbim4 function. tmbim4 depletion impairs gastrulation, leading to abnormal pluripotency marker expression and delayed pluripotency exit. TMBIM4's underlying function is as a putative ion channel, and ion channels are emerging as key regulators of LR patterning and cell fate determination. Using sharp electrodes to measure membrane potential (V_m), tmbim4 depletion depolarized affected embryos. The application of choline, which we have previously shown recues depolarization of Xenopus embryos, rescued the gastrulation defects and pluripotency in tmbim4 depleted embryos. Interestingly, TMBIM4 has previously been localized to the Golgi, and therefore how it might affect V_m was unclear. We find evidence that TMBIM4 localizes to the plasma membrane as well as the Golgi suggesting that it may directly act to establish cellular V_m. Our results establish tmbim4 as a plausible CHD gene and offer the first study of tmbim4 in a developmental context.