A Moving Source of Matrix Components Is Essential for De Novo Basement Membrane Formation

Yutaka Matsubayashi,Adam Louani,Anca Dragu,Besaiz J Sánchez-Sánchez,Eduardo Serna-Morales,Lawrence Yolland,Attila Gyoergy,Gema Vizcay,Roland A Fleck,John M Heddleston,Teng-Leong Chew,Daria E Siekhaus,Brian M Stramer

doi:10.1016/j.cub.2017.10.001

Abstract

SummaryThe basement membrane (BM) is a thin layer of extracellular matrix (ECM) beneath nearly all epithelial cell types that is critical for cellular and tissue function. It is composed of numerous components conserved among all bilaterians [1]; however, it is unknown how all of these components are generated and subsequently constructed to form a fully mature BM in the living animal. Although BM formation is thought to simply involve a process of self-assembly [2], this concept suffers from a number of logistical issues when considering its construction in vivo. First, incorporation of BM components appears to be hierarchical [3, 4, 5], yet it is unclear whether their production during embryogenesis must also be regulated in a temporal fashion. Second, many BM proteins are produced not only by the cells residing on the BM but also by surrounding cell types [6, 7, 8, 9], and it is unclear how large, possibly insoluble protein complexes [10] are delivered into the matrix. Here we exploit our ability to live image and genetically dissect de novo BM formation during Drosophila development. This reveals that there is a temporal hierarchy of BM protein production that is essential for proper component incorporation. Furthermore, we show that BM components require secretion by migrating macrophages (hemocytes) during their developmental dispersal, which is critical for embryogenesis. Indeed, hemocyte migration is essential to deliver a subset of ECM components evenly throughout the embryo. This reveals that de novo BM construction requires a combination of both production and distribution logistics allowing for the timely delivery of core components.

Highlights

We subsequently examined embryonic basement membrane (BM) protein production using endogenously tagged BM fly lines
We first analyzed the developmental profile of BM components from the Drosophila model organism Encyclopedia of DNA Elements project [11]
This revealed that, while Laminin mRNAs are observed early in development, extracellular matrix (ECM) components associated with a mature BM, such as Collagen IV (Vkg in Drosophila) and Perlecan (Trol in Drosophila), are expressed later (Figure S1A), suggesting that there is a temporal hierarchy of BM production during embryogenesis

Summary

Introduction

We subsequently examined embryonic BM protein production using endogenously tagged BM fly lines. We used homozygous viable GFP-protein traps in Collagen IV (Col IV) [12] and Perlecan (Perl) [13] as well as a recently generated line containing GFP-tagged Laminina (LanA) [14] This LanA-GFP is capable of biochemically interacting with other Laminin subunits to form a mature Laminin trimer [14], and it rescued LanA mutant embryos (Figure S1B). When expressed in a Lamininb (LanB1) mutant background, LanA levels were severely reduced (Figures S1C and S1D), suggesting that subunit trimerization is essential for Laminin production and secretion [15]. Using these GFP-tagged lines, we analyzed the dynamics of BM production by quantifying GFP intensity over time during development (Movie S1). We examined a second GFP-tagged construct of the sole Drosophila Lamininb isoform (LanB1), which was previously confirmed to be fully functional [14], and this revealed Laminin expression to occur prior to Col IV or Perl (Figure S1E)

Methods

Results

Conclusion