Distinct functions of the laminin β LN domain and collagen IV during cardiac extracellular matrix formation and stabilization of alary muscle attachments revealed by EMS mutagenesis in Drosophila

Abstract

BackgroundThe Drosophila heart (dorsal vessel) is a relatively simple tubular organ that serves as a model for several aspects of cardiogenesis. Cardiac morphogenesis, proper heart function and stability require structural components whose identity and ways of assembly are only partially understood. Structural components are also needed to connect the myocardial tube with neighboring cells such as pericardial cells and specialized muscle fibers, the so-called alary muscles.ResultsUsing an EMS mutagenesis screen for cardiac and muscular abnormalities in Drosophila embryos we obtained multiple mutants for two genetically interacting complementation groups that showed similar alary muscle and pericardial cell detachment phenotypes. The molecular lesions underlying these defects were identified as domain-specific point mutations in LamininB1 and Cg25C, encoding the extracellular matrix (ECM) components laminin β and collagen IV α1, respectively. Of particular interest within the LamininB1 group are certain hypomorphic mutants that feature prominent defects in cardiac morphogenesis and cardiac ECM layer formation, but in contrast to amorphic mutants, only mild defects in other tissues. All of these alleles carry clustered missense mutations in the laminin LN domain. The identified Cg25C mutants display weaker and largely temperature-sensitive phenotypes that result from glycine substitutions in different Gly-X-Y repeats of the triple helix-forming domain. While initial basement membrane assembly is not abolished in Cg25C mutants, incorporation of perlecan is impaired and intracellular accumulation of perlecan as well as the collagen IV α2 chain is detected during late embryogenesis.ConclusionsAssembly of the cardiac ECM depends primarily on laminin, whereas collagen IV is needed for stabilization. Our data underscore the importance of a correctly assembled ECM particularly for the development of cardiac tissues and their lateral connections. The mutational analysis suggests that the β6/β3/β8 interface of the laminin β LN domain is highly critical for formation of contiguous cardiac ECM layers. Certain mutations in the collagen IV triple helix-forming domain may exert a semi-dominant effect leading to an overall weakening of ECM structures as well as intracellular accumulation of collagen and other molecules, thus paralleling observations made in other organisms and in connection with collagen-related diseases.

Integrating Activities of Laminins that Drive Basement Membrane Assembly and Function

“…A forward genetic screen of Drosophila led to identification of hypomorphic point-specific mutations in the b1-LN domain that cause prominent defects in cardiac basement membrane generation and cardiac morphogenesis (Hollfelder, Frasch, & Reim, 2014). The distribution of defective laminin in the basement membranes around the myocardial tube lacked continuity and instead appeared as a series of disconnected dots.…”

An Org-1–Tup transcriptional cascade reveals different types of alary muscles connecting internal organs in Drosophila

“…4E-E″). Muscle PC into FC divisions do not occur in Regulator of cyclin A1 (Rca1) mutant embryos, and Rca1 mutations were obtained in an EMS screen utilizing org-1-RFP as a marker for abnormal development of AMs [and LO1/VT1/SBM muscles (Hollfelder et al, 2014)]. Triple staining of Rca1 embryos showed that, in the absence of division, the AM PC remains Org-1 positive and Zfh1 negative (Fig.…”

“…Projections and three-dimensional reconstructions were created using ImageJ (NIH) and Volocity (PerkinElmer) software, respectively. Time-lapse imaging of embryos carrying GFP and RFP reporter genes was performed as previously described (Hollfelder et al, 2014).…”

Org-1-Dependent Lineage Reprogramming Generates the Ventral Longitudinal Musculature of the Drosophila Heart

“…To determine whether org-1 is also involved in the establishment of the adult AMs, we probed larvae, pupae, and adults for Org-1 protein expression as well as for the reporters org-1-HN18-RFP (org-1-RFP) and org-1-HN39-nGFP (org-1-nGFP), which recapitulate somatic muscle expression of org-1 in the embryo [13,14]. We detected Org-1 expression and enhancer activity only in anterior AMs of wandering third instar larva and in early pupal stages ( Figures 1A and 1A 0 ).…”