Avian Primordial Germ Cells Contribute to and Interact With the Extracellular Matrix During Early Migration.

David J. Huss,Jennifer M. Singh,Mohit Dave,Jinhui Wang,Sasha Saias,Sevag Hamamah,James Eberwine,Rusty Lansford,Junhyong Kim

doi:10.3389/fcell.2019.00035

Abstract

During early avian development, primordial germ cells (PGC) are highly migratory, moving from the central area pellucida of the blastoderm to the anterior extra-embryonic germinal crescent. The PGCs soon move into the forming blood vessels by intravasation and travel in the circulatory system to the genital ridges where they participate in the organogenesis of the gonads. This complex cellular migration takes place in close association with a nascent extracellular matrix that matures in a precise spatio-temporal pattern. We first compiled a list of quail matrisome genes by bioinformatic screening of human matrisome orthologs. Next, we used single cell RNA-seq analysis (scRNAseq) to determine that PGCs express numerous ECM and ECM-associated genes in early embryos. The expression of select ECM transcripts and proteins in PGCs were verified by fluorescent in situ hybridization (FISH) and immunofluorescence (IF). Live imaging of transgenic quail embryos injected with fluorescent antibodies against fibronectin and laminin, showed that germinal crescent PGCs display rapid shape changes and morphological properties such as blebbing and filopodia while surrounded by, or in close contact with, an ECM fibril meshwork that is itself in constant motion. Injection of anti-β1 integrin CSAT antibodies resulted in a reduction of mature fibronectin and laminin fibril meshwork in the germinal crescent at HH4-5 but did not alter the active motility of the PGCs or their ability to populate the germinal crescent. These results suggest that integrin β1 receptors are important, but not required, for PGCs to successfully migrate during embryonic development, but instead play a vital role in ECM fibrillogenesis and assembly.

Highlights

The extracellular matrix (ECM) plays a vital role in the timing, speed and direction of embryonic cell movements (Dufour et al, 1988; Loganathan et al, 2016)
To elucidate the functional roles that integrin receptors play in primordial germ cells (PGC) movements, we introduced the cell substrate attachment antibody (CSAT) antibody, which blocks the ability of integrin β1 containing receptors to bind their ECM components including laminin and fibronectin (Drake and Little, 1991; Drake et al, 1991)
We have shown that the complexity of the ECM fibril meshwork in the germinal crescent increases from HH3 to HH6, which corresponds with the arrival and concentration of PGCs in this anterior area pellucida (AP)/area opaca (AO) border area (Figures 4–6; Critchley et al, 1979)

Summary

Introduction

The extracellular matrix (ECM) plays a vital role in the timing, speed and direction of embryonic cell movements (Dufour et al, 1988; Loganathan et al, 2016). In addition to its importance in cell migration, the ECM provides signaling cues that regulate cell behaviors and coordinate cell functions in tissue formation and homeostasis. The composition of the ECM, its three-dimensional organization and proteolytic renovations are critical factors in the microenvironmental signaling that regulates cell shape, motility, growth, survival, and differentiation. The ECM is known to play a dynamic role in shaping large-scale movements during early morphogenesis in Xenopus (Boucaut et al, 1990; Davidson et al, 2006), zebrafish (Latimer and Jessen, 2010), chick (Sanders, 1984), and quail (Zamir et al, 2006). In addition to changing cell behavior, the binding of integrins to the ECM has a profound effect upon the ECM itself by changing the rate and timing of fibrillogenesis, assembly and breakdown (Darribère et al, 1990; Danen et al, 2002; Leiss et al, 2008)

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