斑馬魚Pax1a和Pax1b調控咽囊形態形成與角鰓軟骨發育之研究

Abstract

咽弓 (pharyngeal arches) 是由三個胚層所衍生的結構，且其組織間的分子相互作用是之後咽軟骨正常發育所必需的。然而，此過程的基礎機制尚未完全釐清。這篇論文主要報導斑馬魚Pax1a和Pax1b在咽囊形態形成和隨後的角鰓軟骨(ceratobranchial cartilage) 發育中具有重疊與不可或缺的功能。 pax1a和pax1b共同表現在咽囊中，運用增強子捕獲所產生的Tg（pax1b：eGFP）螢光魚進行延時攝影影像觀察進一步揭示了咽囊發育的連續性分節情形。由CRISPR-Cas9誘變產生的pax1a-/-; pax1b-/-雙突變斑馬魚胚胎的咽囊2-5無分節，囊狀外凸(outpocketings) 變小。受精後36小時（hpf）雙突變斑馬魚胚胎的咽囊2-5中也沒有fgf3，tbx1和edn1在內胚層表現。在受精後96或36小時的CRISPR突變魚胚胎與以反義嗎啉基(antisense morpholino)弱化斑馬魚pax1a和pax1b基因的胚胎中，觀察到角鰓軟骨1-4的消失以及在咽弓3-6中dlx2a和hand2表現的減少或缺乏。這些結果顯示斑馬魚Pax1a和Pax1b透過調節fgf3和tbx1的表現來調節咽囊的形態形成。此外，我們的數據支持一個假說:內胚層表現的Pax1a和Pax1b蛋白質透過Fgf3和Tbx-Edn1訊號傳導，經由調控dlx2a和hand2的表現來非自主性地調節角鰓軟骨的發育。

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 hours 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.