Zmym4 is required for early cranial gene expression and craniofacial cartilage formation

Abstract

Introduction: The Six1 transcription factor plays important roles in the development of cranial sensory organs, and point mutations underlie craniofacial birth defects. Because Six1’s transcriptional activity can be modulated by interacting proteins, we previously screened for candidate interactors and identified zinc-finger MYM-containing protein 4 (Zmym4) by its inclusion of a few domains with a bona fide cofactor, Sine oculis binding protein (Sobp). Although Zmym4 has been implicated in regulating early brain development and certain cancers, its role in craniofacial development has not previously been described.Methods: We used co-immunoprecipitation and luciferase-reporter assays in cultured cells to test interactions between Zmym4 and Six1. We used knock-down and overexpression of Zmym4 in embryos to test for its effects on early ectodermal gene expression, neural crest migration and craniofacial cartilage formation.Results: We found no evidence that Zmym4 physically or transcriptionally interacts with Six1 in cultured cells. Nonetheless, knockdown of endogenous Zmym4 in embryos resulted in altered early cranial gene expression, including those expressed in the neural border, neural plate, neural crest and preplacodal ectoderm. Experimentally increasing Zmym4 levels had minor effects on neural border or neural plate genes, but altered the expression of neural crest and preplacodal genes. At larval stages, genes expressed in the otic vesicle and branchial arches showed reduced expression in Zmym4 morphants. Although we did not detect defects in neural crest migration into the branchial arches, loss of Zmym4 resulted in aberrant morphology of several craniofacial cartilages.Discussion: Although Zmym4 does not appear to function as a Six1 transcriptional cofactor, it plays an important role in regulating the expression of embryonic cranial genes in tissues critical for normal craniofacial development.

Bop1 is required to establish precursor domains of craniofacial tissues

“…Thus, in the majority of cases when loss of Bop1 expands the neural plate domain it is at the expense of neural crest and placodes. This result was also found with knockdown of other Six1 cofactors (Jourdeuil et al, 2023;Neilson et al, 2017;Neilson et al, 2020;Tavares et al, 2021), and is consistent with the observation that ectopic expression of neural plate genes represses neural crest, placode and epidermal genes (Klein et al, 2022). We therefore conclude that Bop1 is among those genes required for apportioning the early embryonic ectoderm into the domains required for proper development of the cranial central (neural plate) and peripheral (neural crest, placodes) nervous systems.…”

“…By adding additional markers, we confirm their observations that neural plate gene expression domains are broader and neural crest gene domains are reduced. In addition, as we found by reducing the expression of other putative Six1-interacting proteins (Jourdeuil et al, 2023;Neilson et al, 2017;Neilson et al, 2020;Tavares et al, 2021), we show that placode and epidermal gene domains also were reduced, ultimately leading to otic vesicle defects at larval stages. We also demonstrate that the cranial cartilage defects shown by Gärtner et al (2022) are likely due to defects in chondrogenesis gene expression rather than neural crest migration into the branchial arches.…”

“…Briefly, one cell of the embryo was injected at the 2-cell stage with either 200 pg of mbGFP mRNA alone or in combination with Bop1 MOs. At stage 15, explants of fluorescently labeled cranial neural were dissected, placed singly into a 96-well coated with 20 μg/mL of fibronectin, and cultured at 18 C in Danilchik medium containing 50 μg/mL gentamicin for about 10-11 h, as described in Jourdeuil et al (2023). The surface area occupied by the explant at the beginning and at the end of the culture were determined and the ratio of these areas (end/beginning) were calculated and normalized to the ratio obtained from control explants and compared using a two-tailed, paired Student's t-test.…”