Differential roles for 3-OSTs in the regulation of cilia length and motility.

As cells integrate molecular signals from their environment, cell surface receptors require modified proteoglycans for the robust activation of signaling pathways. Heparan sulfate proteoglycans (HSPGs) have long unbranched chains of repetitive disaccharide units that can be sulfated at specific positions by heparan sulfate O-sulfotransferase (OST) families. Here, we show that two members of the 3-OST family are required in distinct signaling pathways to control left-right (LR) patterning through control of Kupffer's vesicle (KV) cilia length and motility. 3-OST-5 functions in the fibroblast growth factor pathway to control cilia length via the ciliogenic transcription factors FoxJ1a and Rfx2. By contrast, a second 3-OST family member, 3-OST-6, does not regulate cilia length, but regulates cilia motility via kinesin motor molecule (Kif3b) expression and cilia arm dynein assembly. Thus, two 3-OST family members cell-autonomously control LR patterning through distinct pathways that regulate KV fluid flow. We propose that individual 3-OST isozymes create distinct modified domains or 'glycocodes' on cell surface proteoglycans, which in turn regulate the response to diverse cell signaling pathways.

Dand5 is involved in zebrafish tailbud cell movement.

During vertebrate development, symmetry breaking occurs in the left-right organizer (LRO). The transfer of asymmetric molecular information to the lateral plate mesoderm is essential for the precise patterning of asymmetric internal organs, such as the heart. However, at the same developmental time, it is crucial to maintain symmetry at the somite level for correct musculature and vertebrae specification. We demonstrate how left-right signals affect the behavior of zebrafish somite cell precursors by using live imaging and fate mapping studies in dand5 homozygous mutants compared to wildtype embryos. We describe a population of cells in the vicinity of the LRO, named Non-KV Sox17:GFP+ Tailbud Cells (NKSTCs), which migrate anteriorly and contribute to future somites. We show that NKSTCs originate in a cluster of cells aligned with the midline, posterior to the LRO, and leave that cluster in a left-right alternating manner, primarily from the left side. Fate mapping revealed that more NKSTCs integrated somites on the left side of the embryo. We then abolished the asymmetric cues from the LRO using dand5-/- mutant embryos and verified that NKSTCs no longer displayed asymmetric patterns. Cell exit from the posterior cluster became bilaterally synchronous in dand5-/- mutants. Our study revealed a new link between somite specification and Dand5 function. The gene dand5 is well known as the first asymmetric gene involved in vertebrate LR development. This study revealed a new link for Dand5 as a player in cell exit from the maturation zone into the presomitic mesoderm, affecting the expression patterns of myogenic factors and tail size.

Dynamics of cilia length in left-right development.

Reduction in the length of motile cilia in the zebrafish left-right organizer (LRO), also known as Kupffer's vesicle, has a large impact on left-right development. Here we demonstrate through genetic overexpression in zebrafish embryos and mathematical modelling that the impact of increased motile cilia length in embryonic LRO fluid flow is milder than that of short cilia. Through Arl13b overexpression, which increases cilia length without impacting cilia beat frequency, we show that the increase in cilium length is associated with a decrease in beat amplitude, resulting in similar flow strengths for Arl13b overexpression and wild-type (WT) embryos, which were not predicted by current theory. Longer cilia exhibit pronounced helical beat patterns and, consequently, lower beat amplitudes relative to WT, a result of an elastohydrodynamic shape transition. For long helical cilia, fluid dynamics modelling predicts a mild (approx. 12%) reduction in the torque exerted on the fluid relative to the WT, resulting in a proportional reduction in flow generation. This mild reduction is corroborated by experiments, providing a mechanism for the mild impact on organ situs.