Invasion by Matrix Metalloproteinase-Expressing Cells Is Important for Primitive Streak Formation in Early Chick Blastoderm

Abstract

Epiblast cells in the early chick embryo differentiate to form all three germ layers through ingression of cells at the primitive streak across the basement membrane that underlies the epiblast. We tested the idea that degradation of the extracellular matrix components by matrix metalloproteinase(s) (MMPs) is involved in this process. Epiblast cells and primitive streak cells were dissociated into single cells and seeded onto a reconstituted basement membrane gel in vitro. Following overnight culture, approximately half the cells made holes in the substratum by dissolving the gel matrix. This invasive phenomenon was reproduced in vitro even when the cells were cultured upside down using a hanging culture system. We detected gelatinase activity in the culture supernatants from both prestreak epiblast cells and primitive streak cells. Pro-MMP-2 was detected in the culture media of the prestreak/streak cells as a 72-kDa band by gelatin zymography. In RT-PCR experiments, mRNAs for MMP-2, membrane-type (MT)3-MMP and MMP-11(stromelysin-3) were expressed in the epiblast cells before and during primitive streak formation. Injection of GM 6001 or other MMP inhibitors into the subgerminal cavity of the embryo inhibited the formation of the primitive streak and/or the primitive groove in more than 82% of the injected embryos. On the other hand, injection of a negative control compound instead of GM 6001 did not cause substantial inhibition. These results suggest that MMPs are involved in the enzymatic degradation of the basement membrane underlying the epiblast and are thus important for the ingression of mesendodermal cells along the primitive streak.

Collective Epithelial and Mesenchymal Cell Migration During Gastrulation

“…1B ). Furthermore HNK1 positive epiblast and hypoblast cells show invasive behavior on extracellular matrix gels and secrete different metalloproteases [42]. Finally, complement mediated deletion of HNK1 positive cells has been shown to inhibit streak formation [43].…”

Breaking down barriers: the evolution of cell invasion

“…There is also a striking conservation of the regulatory circuit that activates matrix metalloproteinases (MMPs) during BM invasion programs across the Metazoa, as the AP-1 transcription factor (TF), FOS, regulates the expression of MMPs across a wide range of cell types and contexts [58], including C. elegans AC invasion [59] and placentation in deciduate mammals [60,61]. It is unclear if FOS is required for the MMP-mediated cell invasive contexts of Drosophila wing disc eversion [31], sea urchin EMT [17], vertebrate gastrulation [62,63] or neural crest delamination [23,64]. However, FOS family members are not always involved in activating MMP expression during invasion, suggesting that evolution has shaped the transcriptional networks mediating the deployment of proteases.…”

Matrix metalloproteinase function in non-mammalian model organisms

“…The chicken genome also includes at least two TIMP genes (91) and a novel simple hemopexin domain containing MMP, MMP22 (50). One of the most described chicken MMPs during development is MMP2, shown to play a role in neural streak ingression, cornea formation, cell migration during embryogenesis and skeletal and heart development (90,(92)(93)(94). MMP2 is a gelatinase that in mammals is proteolytically activated by MMP14 (17).…”

“…MMP2 expression begins in the epiblast cells in the area where the primitive streak forms starting at day 2 of development. MMP11 and MMP16 are also found in the primitive streak and injection of MMP inhibitors produced embryos where primitive streak cells failed to ingress (94). MMP2, MMP11 and MMP16 have a variety of ECM substrates and it is thought that these MMPs break down the ECM within the primitive streak to allow cells to ingress (94).…”

“…Chicken MMP2 is also expressed in the forming heart (94), in the limb buds where the tendons form (92), in branchial arches (92), and in the developing long bones (90); however, the role of MMP2 in these tissues is unknown. MMP14, MMP13, and MMP16 are also expressed in the forming skeletal tissue indicating they may regulate development of the skeleton (90).…”