Zebrafish Pax1a and Pax1b are required for pharyngeal pouch morphogenesis and ceratobranchial cartilage development.

Pharyngeal arches are derived from all three germ layers and molecular interactions among the tissue types are required for proper development of subsequent pharyngeal cartilages; however, the mechanisms underlying this process are not fully described. Here we report that in zebrafish, Pax1a and Pax1b have overlapping and essential functions in pharyngeal pouch morphogenesis and subsequent ceratobranchial cartilage development. Both pax1a and pax1b are co-expressed in pharyngeal pouches, and time-lapse imaging of a novel Tg(pax1b:eGFP) enhancer trap line further revealed the sequential segmental development of pharyngeal pouches. Zebrafish pax1a-/-; pax1b-/- double mutant embryos generated by CRISPR-Cas9 mutagenesis exhibit unsegmented pharyngeal pouches 2-5 with small outpocketings. Endodermal expression of fgf3, tbx1 and edn1 is also absent in pharyngeal pouches 2-5 at 36 h post fertilization (hpf). Loss of ceratobranchial cartilage 1-4 and reduced or absent expression of dlx2a and hand2 in the pharyngeal arches 3-6 are observed in CRISPR mutant and morphant embryos that are deficient in both zebrafish pax1a and pax1b at 96 or 36 hpf. These results suggest that zebrafish Pax1a and Pax1b both regulate pharyngeal pouch morphogenesis by modulating expression of fgf3 and tbx1. Furthermore, our data support a model wherein endodermal Pax1a and Pax1b act through Fgf3 and Tbx-Edn1 signaling to non-autonomously regulate the development of ceratobranchial cartilage via expression of dlx2a and hand2.

Phosphatidylinositol-5-phosphate 4-kinase gamma accumulates at the spindle pole and prevents microtubule depolymerization.

BACKGROUND: A previous screen of a human kinase and phosphatase shRNA library to select genes that mediate arsenite induction of spindle abnormalities resulted in the identification of phosphatidylinositol-5-phosphate 4-kinase type-2 gamma (PIP4KIIγ), a phosphatidylinositol 4,5-bisphosphate (PIP2)-synthesizing enzyme. In this study, we explored how PIP4KIIγ regulates the assembly of mitotic spindles. RESULTS: PIP4KIIγ accumulates at the spindle pole before anaphase, and is required for the assembly of functional bipolar spindles. Depletion of PIP4KIIγ enhanced the spindle pole accumulation of mitotic centromere-associated kinesin (MCAK), a microtubule (MT)-depolymerizing kinesin, and resulted in a less stable spindle pole-associated MT. Depletion of MCAK can ameliorate PIP4KIIγ depletion-induced spindle abnormalities. In addition, PIP2 binds to polo-like kinase (PLK1) and reduces PLK1-mediated phosphorylation of MCAK. These results indicate that PIP4KIIγ and PIP2 may negatively regulate the MT depolymerization activity of MCAK by reducing PLK1-mediated phosphorylation of MCAK. Consequently, depletion of PLK1 has been shown to counteract the PIP4KIIγ depletion-induced instability of spindle pole-associated MT and cell resistance to arsenite. CONCLUSIONS: Our current results imply that PIP4KIIγ may restrain MT depolymerization at the spindle pole through attenuating PLK1-mediated activation of MCAK before anaphase onset.