The V-ATPase accessory protein Atp6ap1b mediates dorsal forerunner cell proliferation and left–right asymmetry in zebrafish

Abstract

Asymmetric fluid flows generated by motile cilia in a transient ‘organ of asymmetry’ are involved in establishing the left-right (LR) body axis during embryonic development. The vacuolar-type H+-ATPase (V-ATPase) proton pump has been identified as an early factor in the LR pathway that functions prior to cilia, but the role(s) for V-ATPase activity are not fully understood. In the zebrafish embryo, the V-ATPase accessory protein Atp6ap1b is maternally supplied and expressed in dorsal forerunner cells (DFCs) that give rise to the ciliated organ of asymmetry called Kupffer’s vesicle (KV). V-ATPase accessory proteins modulate V-ATPase activity, but little is known about their functions in development. We investigated Atp6ap1b and V-ATPase in KV development using morpholinos, mutants and pharmacological inhibitors. Depletion of both maternal and zygotic atp6ap1b expression reduced KV organ size, altered cilia length and disrupted LR patterning of the embryo. Defects in other ciliated structures—neuromasts and olfactory placodes—suggested a broad role for Atp6ap1b during development of ciliated organs. V-ATPase inhibitor treatments reduced KV size and identified a window of development in which V-ATPase activity is required for proper LR asymmetry. Interfering with Atp6ap1b or V-ATPase function reduced the rate of DFC proliferation, which resulted in fewer ciliated cells incorporating into the KV organ. Analyses of pH and subcellular V-ATPase localizations suggested Atp6ap1b functions to localize the V-ATPase to the plasma membrane where it regulates proton flux and cytoplasmic pH. These results uncover a new role for the V-ATPase accessory protein Atp6ap1b in early development to maintain the proliferation rate of precursor cells needed to construct a ciliated KV organ capable of generating LR asymmetry.

Chemokine signaling links cell cycle progression and cilia formation for left-right symmetry breaking

“…To address this issue, we first examined the proliferation profile of DFC/KV cells by performing bromodeoxyuridine (BrdU) incorporation assays in Tg(sox17:GFP) embryos during gastrulation and early somite stages. Consistent with previous reports [16], approximately 70% of DFCs were positively stained with BrdU at the mid-gastrulation stage, whereas very few BrdU-positive cells were observed in the developing KV at the bud and the 6-somite stages (Fig 3A and 3B), suggesting that vigorous proliferation occurs in DFCs during epiboly stages and then declines at the end of gastrulation. Impressively, we found a dramatic decrease of the BrdU-positive DFC number in cxcr4a -deficient embryos at mid-gastrulation stage (Fig 3C and 3D), indicating a crucial requirement of cxcr4a in DFC proliferation.…”

“…In zebrafish embryos, DFCs undergo mitotic proliferation during epiboly and then exit the cell cycle, giving rise to epithelial cells that assemble cilia in the mature KV organ [16]. Cell cycle defects in DFCs are often accompanied by an alteration in KV cilia elongation, raising the issue of whether there exists a feasible link between the cell cycle and cilia formation [16]. In this study, our experiments resolve this issue by demonstrating that Cxcr4 signaling is required for DFC proliferation and KV ciliogenesis by promoting Cyclin D1 expression.…”

“…They then cluster and differentiate into polarized epithelial cells of KV [4,14,15]. Interestingly, DFC proliferation defects are often accompanied by impaired cilia elongation in KV [16], indicating a possible connection between cell-cycle events and KV cilia formation. However, the underlying mechanism remains poorly understood.…”

Cilia in vertebrate left–right patterning

“…It is possible that early developmental events generate LR positional information that can be used to guide ciliated cells as they develop into a functional LRO or to completely bypass the requirement for cilia [93,94]. Several connections have been made between molecules involved in early LR events and the development of the LRO: Syndecan 2 [95] and the vacuolar-type H þ -ATPase [88,96,97] are involved in the development of normal LRO morphogenesis and cilia formation in zebrafish, and H þ / K þ -ATPase activity [98] and serotonin [99] are needed for Wnt signalling pathways that control LRO development and ciliogenesis in frog embryos. One outstanding question is whether precursor cells receive laterality cues prior to forming the LRO.…”

Copy number variation as a genetic basis for heterotaxy and heterotaxy-spectrum congenital heart defects

“…Together with observations from yeast that PFK-1 is required to maintain activity of the HþV-ATPase proton pump and that even a catalytically inactive PFK-1 is capable of completing this function [31], a reasonable hypothesis is that PFKP knockdown may impact L -R patterning through loss of a functionally critical interaction with the Hþ-V-ATPase a-subunit. As recent zebrafish studies indicate a primary role for Hþ-V-ATPase in KV formation [68], it is possible that PFKP may impact L -R patterning in a similar manner. Interestingly, a rare duplication involving the gene ATP6V1G1, which encodes the G-subunit of Hþ-V-ATPAase, was also identified in a male heterotaxy patient in our cohort (Patient 41).…”