Architectural delineation and molecular identification of extracellular matrix in ascidian embryos and larvae

Abstract

The extracellular matrix (ECM) not only provides essential physical scaffolding for cellular constituents but also initiates crucial biochemical and biomechanical cues that are required for tissue morphogenesis. In this study, we utilized wheat germ agglutinin (WGA) staining to characterize the ECM architecture in ascidian embryos and larvae. The results showed three distinct populations of ECM presenting in Ciona embryogenesis: the outer layer localized at the surface of embryo, an inner layer of notochord sheath and the apical ECM secreted by the notochord. To further elucidate the precise structure of Ciona embryonic ECM, we employed scanning and transmission electron microscopy, and found that the outer membrane was relatively thick with short fibres, whereas the ECM layer in notochord sheath was not as thick as the outer membrane but more regular arranged; the lumen between notochord cells was hydrostatic and sticky. Then, we used the RNA sequencing data from the embryos and larvae of Ciona savignyi to identify ECM genes and acquire their expression patterns. We identified 115 unigenes as 67 ECM genes, and 77 unigenes showed dynamic expression changes between different stages. Our results reveal the architecture, molecular composition and dynamic expression profile of ECM in ascidian embryogenesis, and may increase understanding of the function of the ECM in chordate development.

Keywords: Ciona; Collagen; Extracellular matrix; RNA sequencing.

Fig. 1.

ECM architecture of Ciona embryos as shown by WGA labelling. (A,A′) Global view of WGA-stained embryos at gastrula stage. The red asterisks indicate notochord cells. (B,B′) Global view of WGA-stained embryos at tailbud stage. There are two distinct layers of ECM: the outer layer (white arrowheads) at the apical surface of epithelia, and an inner layer (red arrowheads) of sheath surrounding the notochord. (C,C′) Magnified tail part of WGA-stained early embryos. The yellow arrowheads in C and C′ indicate the weaker layer localized at the basement membrane of epithelia. (D-D″) Magnified view of the ECM structure in later stage embryos. The red asterisks indicate the apical lumen, which cannot be stained by WGA in later stage embryos. Scale bars: 100 µm in A′ and B′; 10 µm in C′ and D″.

Fig. 2.

ECM structures of Ciona embryos as shown by electron microscopy. (A) SEM image of the body surface of tailbud stage embryos. The yellow arrow indicates separated short fibril structures. (B) Cross-section of a fragment of a tail part scanned by SEM. The red arrow indicates notochord sheath. (C) Longitudinal view of the tail part from tailbud embryos, as shown by SEM. The red arrow indicates linear notochord cells; the green arrow indicates the flank muscle cells. (D) The body surface of tailbud stage embryos, as shown by TEM. The bi-directional arrow indicates the thickness of the outer membrane. (E) Longitudinal section of the tail part, including the notochord and muscle cells. The red arrow indicates the fibrillar ECM in the notochord sheath. The red dashed line outlines the cell boundaries. (F) Cross section of the tail part including the notochord cells and central lumen. The red asterisks indicate low-density lumen. Inset shows a magnified view of the apical lumen. Scale bars: 10 µm in A and B; 100 µm in C; 500 nm in D and E; 1 µm in F. epi, epidermis cell; mus, muscle cells; no, notochord; no1, notochord cell 1; no2, notochord cell 2.

Fig. 3.

Expression profiles of selected ECM genes from qPCR and RNA-seq. Three developmental stages are shown in the x-axis. Data are mean±s.e.m. relative expression levels. RNA-Seq results are shown in black; qPCR results are shown in red. The expression profiles of the RNA-seq and qPCR data were consistent.

Fig. 4.

Phylogenetic tree analysis of selected collagens and their expression patterns. (A) The phylogenetic tree of collagens. The putative fibrillar collagens of C. savignyi are shown in red; the putative nonfibrillar collagens of C. savignyi are shown in blue. (B-J) Whole-mount in situ hybridization of c119589g1 [collagen alpha-1 (II)], c120533g1 [collagen alpha-1 (V)], c115706g1 [collagen alpha-1 (VI))] and c118728g1 [collagen alpha-1 (XXVIII)]. The red arrowheads indicate the signals in the different staged embryos. Scale bars: 100 µm.