Abstract
ABSTRACTThe sodium osmotic gradient is necessary for the initiation of brain ventricle inflation, but a previous study predicted that organic and inorganic osmolytes play equivalently important roles in osmotic homeostasis in astrocytes. To test whether organic osmoregulation also plays a role in brain ventricle inflation, the core component for volume-regulated anion and organic osmolyte channel, lrrc8a, was investigated in the zebrafish model. RT-PCR and whole-mount in situ hybridization indicated that both genes were ubiquitously expressed through to 12 hpf, and around the ventricular layer of neural tubes and the cardiogenic region at 24 hpf. Knocking down either one lrrc8a paralog with morpholino oligos resulted in abnormalities in circulation at 32 hpf. Morpholino oligos or CRISPR interference against either paralog led to smaller brain ventricles at 24 hpf. Either lrrc8aa or lrrc8ab mRNA rescued the phenotypic penetrance in both lrrc8aa and lrrc8ab morphants. Supplementation of taurine in the E3 medium and overexpression csad mRNA also rescued lrrc8aa and lrrc8ab morphants. Our results indicate that the two zebrafish lrrc8a paralogs are maternal message genes and are ubiquitously expressed in early embryos. The two genes play redundant roles in the expansion of brain ventricles and the circulatory system and taurine contributes to brain ventricle expansion via the volume-regulated anion and organic osmolyte channels.
Highlights
The regulation and homeostasis of osmolality is a critical mechanism for all active cells as biological activities are generally in an aqueous environment with various organic and inorganic solutes
Zebrafish has two lrrc8a genes We confirmed that there were two genes in the zebrafish genome with protein coding sequences similar to human leucine-rich repeat containing 8A (LRRC8A) and these have been annotated as lrrc8aa (ENSDART00000148138) and lrrc8ab (ENSDART0000144732)
Despite the similar amino acid sequences of zebrafish Lrrc8aa and Lrrc8ab compared to mammalian LRRC8A, only lrrc8ab has a similar genomic structure to human LRRC8A (Fig. 1A)
Summary
The regulation and homeostasis of osmolality is a critical mechanism for all active cells as biological activities are generally in an aqueous environment with various organic and inorganic solutes. Received 30 September 2019; Accepted 23 December 2019 outwardly rectifying Cl− current and an RVD when cells were exposed to hypotonic solutions (Cahalan and Lewis, 1988; Hazama and Okada, 1988). Later, organic osmolytes such as taurine were considered accountable for at least half of the total RVD, and a putative volume-sensitive organic osmolyte/anion channel (VSOAC) was proposed for this activity (Garcia-Romeu et al, 1991; Jackson and Strange, 1993; Strange and Jackson, 1995). Due to the pharmacological similarity and controversial experimental results, the question of whether VRAC is identical to VSOAC was once a highly debated issue (Díaz et al, 1993; Kirk and Kirk, 1994; Lambert and Hoffmann, 1994; Sanchez-Olea et al, 1995; Shennan et al, 1994)
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