Absence of Basement Membranes after Targeting the LAMC1 Gene Results in Embryonic Lethality Due to Failure of Endoderm Differentiation

Abstract

The LAMC1 gene coding for the laminin γ1 subunit was targeted by homologous recombination in mouse embryonic stem cells. Mice heterozygous for the mutation had a normal phenotype and were fertile, whereas homozygous mutant embryos did not survive beyond day 5.5 post coitum. These embryos lacked basement membranes and although the blastocysts had expanded, primitive endoderm cells remained in the inner cell mass, and the parietal yolk sac did not develop. Cultured embryonic stem cells appeared normal after targeting both LAMC1 genes, but the embryoid bodies derived from them also lacked basement membranes, having disorganized extracellular deposits of the basement membrane proteins collagen IV and perlecan, and the cells failed to differentiate into stable myotubes. Secretion of the linking protein nidogen and a truncated laminin α1 subunit did occur, but these were not deposited in the extracellular matrix. These results show that the laminin γ1 subunit is necessary for laminin assembly and that laminin is in turn essential for the organization of other basement membrane components in vivo and in vitro. Surprisingly, basement membranes are not necessary for the formation of the first epithelium to develop during embryogenesis, but first become required for extra embryonic endoderm differentiation.

Cell and molecular regulation of the mouse blastocyst

“…Dab2 is a PTB domain containing adaptor protein that modulates cellular adhesiveness during megakaryocytic differentiation of the human chronic myeloid leukemic cell line K562 (Tseng et al, 2003) and transiently interacts with Integrin ␤1 to regulate its activation during induced epithelial to mesenchymal transition in normal murine mammary gland cells (Prunier and Howe, 2005). Moreover, mutation of LamC1 and Integrin␤1, genes known to modulate cell adhesiveness, causes similar phenotypes (Stephens et al, 1995;Smyth et al, 1999). These genes thus represent excellent candidates for regulating cell sorting since PE lineage progenitors form but do not segregate to the surface of the ICM in these mutants.…”

Cell biology of embryonic migration

“…(Costell et al, 1999) Tenascin C Extracellular matrix Total NC cells fail to disperse laterally (Tucker, 2001) Laminin γ1 Extracellular matrix Total Death at E5.5, lack of basement membranes (Smyth et al, 1999) Laminin β2 Extracellular matrix Total Postnatal death between P15-30, neuromuscular junctions and glomerular defects (Noakes et al, 1995a;Noakes et al, 1995b;Patton et al, 1997) Laminin α2 Extracellular matrix Total Death by 5 weeks postnatal, severe muscular dystrophy and peripheral neurophathy (Miyagoe et al, 1997) Laminin α2 (dy/dy) Extracellular matrix Spontaneous Adult lethality, severe muscular dystrophy and peripheral nerve dysmyelination (Patton et al, 1999;Patton et al, 1997) Laminin α3 Extracellular matrix Total Death at P2-3, epithelial adhesion defect (Ryan et al, 1999) Laminin α4 Extracellular matrix Total Transient microvascular defect with hemorrhages and misalignment of neuromuscular junctions (Patton et al, 2001;Thyboll et al, 2002) Laminin α5 Extracellular matrix Total Death at E14-E17, with placental vessel, neural (Miner et al, 1998;Miner and Li, 2000) Protein Function Type Phenotype Reference tube, limb, and kidney defects Fibronectin Extracellular matrix Total Death before E14.5, shortened anterior-posterior axes, deformed neural tubes, and defects in mesodermally derived tissues. (George et al, 1993) Collagen XVIII Extracellular matrix Total Eye abnormalities modeling Knoblock syndrome (Fukai et al, 2002) Extracellular matrix Total Basement membrane defects (Utriainen et al, 2004) Collagen XV Extracellular matrix Total Skeletal myopathy and cardiovascular defects (Eklund et al, 2001) betaglycan Extracellular matrix Total Embryonic lethality of heart and liver defects (Stenvers et al, 2003) Connexin 43 Cell-cell adhesion In vitro studies of cells from knockout mice…”

Extracellular matrix remodeling accompanies axial muscle development and morphogenesis in the mouse

“…They are formed by the combination of three chains, a, b, and g (Durbeej, 2010). Several laminins are essential for embryogenesis and their assembly is the first step of basement membrane formation and also a prerequisite for the incorporation of the other basement membrane components (Smyth et al, 1999;Miner and Yurchenco, 2004;Yurchenco and Patton, 2009;Durbeej, 2010). Laminin 111 and 511 are part of the basement membrane of the epithelial somites, dermomyotome, and myotome (Bajanca et al, 2006;Anderson et al, 2009;Thorsteinsdóttir et al, 2011).…”