Significant advances have been made in the application of genetics to probe the functions of basement membrane laminins. These studies have shown that different laminin subunits profoundly affect tissue morphogenesis, starting around the time of embryonic implantation and extending through organogenesis and into the postnatal period. Collectively they have revealed common functions that include the induction and maintenance of cell polarity, the establishment of barriers between tissue compartments, the organization of cells into tissues, and the protection of adherent cells from detachment-induced cell death, anoikis. Interpreted in light of what is known about laminin structure and self-assembly and binding activities, these advances have begun to provide insights into mechanisms of action. In this review we focus on the contributions of the laminins in invertebrate and vertebrate tissue morphogenesis.

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CD2-associated protein (CD2AP) is an 80-kilodalton protein that is critical for stabilizing contacts between T cells and antigen-presenting cells. In CD2AP-deficient mice, immune function was compromised, but the mice died at 6 to 7 weeks of age from renal failure. In the kidney, CD2AP was expressed primarily in glomerular epithelial cells. Knockout mice exhibited defects in epithelial cell foot processes, accompanied by mesangial cell hyperplasia and extracellular matrix deposition. Supporting a role for CD2AP in the specialized cell junction known as the slit diaphragm, CD2AP associated with nephrin, the primary component of the slit diaphragm.

Laminin trimers composed of α, β, and γ chains are major components of basal laminae (BLs) throughout the body. To date, three α chains (α1–3) have been shown to assemble into at least seven heterotrimers (called laminins 1–7). Genes encoding two additional α chains (α4 and α5) have been cloned, but little is known about their expression, and their protein products have not been identified. Here we generated antisera to recombinant α4 and α5 and used them to identify authentic proteins in tissue extracts. Immunoprecipitation and immunoblotting showed that α4 and α5 assemble into four novel laminin heterotrimers (laminins 8–11: α4β1γ1, α4β2γ1, α5β1γ1, and α5β2γ1, respectively). Using a panel of nucleotide and antibody probes, we surveyed the expression of α1-5 in murine tissues. All five chains were expressed in both embryos and adults, but each was distributed in a distinct pattern at both RNA and protein levels. Overall, α4 and α5 exhibited the broadest patterns of expression, while expression of α1 was the most restricted. Immunohistochemical analysis of kidney, lung, and heart showed that the α chains were confined to extracellular matrix and, with few exceptions, to BLs. All developing and adult BLs examined contained at least one α chain, all α chains were present in multiple BLs, and some BLs contained two or three α chains. Detailed analysis of developing kidney revealed that some individual BLs, including those of the tubule and glomerulus, changed in laminin chain composition as they matured, expressing up to three different α chains and two different β chains in an elaborate and dynamic progression. Interspecific backcross mapping of the five α chain genes revealed that they are distributed on four mouse chromosomes. Finally, we identified a novel full-length α3 isoform encoded by the Lama3 gene, which was previously believed to encode only truncated chains. Together, these results reveal remarkable diversity in BL composition and complexity in BL development.

Laminins, heterotrimers of α, β, and γ chains, are prominent constituents of basal laminae (BLs) throughout the body. Previous studies have shown that laminins affect both myogenesis and synaptogenesis in skeletal muscle. Here we have studied the distribution of the 10 known laminin chains in muscle and peripheral nerve, and assayed the ability of several heterotrimers to affect the outgrowth of motor axons. We show that cultured muscle cells express four different α chains (α1, α2, α4, and α5), and that developing muscles incorporate all four into BLs. The portion of the muscle's BL that occupies the synaptic cleft contains at least three α chains and two β chains, but each is regulated differently. Initially, the α2, α4, α5, and β1 chains are present both extrasynaptically and synaptically, whereas β2 is restricted to synaptic BL from its first appearance. As development proceeds, α2 remains broadly distributed, whereas α4 and α5 are lost from extrasynaptic BL and β1 from synaptic BL. In adults, α4 is restricted to primary synaptic clefts whereas α5 is present in both primary and secondary clefts. Thus, adult extrasynaptic BL is rich in laminin 2 (α2β1γ1), and synaptic BL contains laminins 4 (α2β2γ1), 9 (α4β2γ1), and 11 (α5β2γ1). Likewise, in cultured muscle cells, α2 and β1 are broadly distributed but α5 and β2 are concentrated at acetylcholine receptor–rich “hot spots,” even in the absence of nerves. The endoneurial and perineurial BLs of peripheral nerve also contain distinct laminin chains: α2, β1, γ1, and α4, α5, β2, γ1, respectively. Mutation of the laminin α2 or β2 genes in mice not only leads to loss of the respective chains in both nerve and muscle, but also to coordinate loss and compensatory upregulation of other chains. Notably, loss of β2 from synaptic BL in β2−/− “knockout” mice is accompanied by loss of α5, and decreased levels of α2 in dystrophic α2dy/dy mice are accompanied by compensatory retention of α4. Finally, we show that motor axons respond in distinct ways to different laminin heterotrimers: they grow freely between laminin 1 (α1β1γ1) and laminin 2, fail to cross from laminin 4 to laminin 1, and stop upon contacting laminin 11. The ability of laminin 11 to serve as a stop signal for growing axons explains, in part, axonal behaviors observed at developing and regenerating synapses in vivo.

Small-vessel diseases of the brain underlie 20 to 30 percent of ischemic strokes and a larger proportion of intracerebral hemorrhages. In this report, we show that a mutation in the mouse Col4a1 gene, encoding procollagen type IV alpha1, predisposes both newborn and adult mice to intracerebral hemorrhage. Surgical delivery of mutant mice alleviated birth-associated trauma and hemorrhage. We identified a COL4A1 mutation in a human family with small-vessel disease. We concluded that mutation of COL4A1 may cause a spectrum of cerebrovascular phenotypes and that persons with COL4A1 mutations may be predisposed to hemorrhage, especially after environmental stress.

Kidney disease affects over 20 million people in the United States alone. Although the causes of renal failure are diverse, the glomerular filtration barrier is often the target of injury. Dysregulation of VEGF expression within the glomerulus has been demonstrated in a wide range of primary and acquired renal diseases, although the significance of these changes is unknown. in the glomerulus, VEGF-A is highly expressed in podocytes that make up a major portion of the barrier between the blood and urinary spaces. in this paper, we show that glomerular-selective deletion or overexpression of VEGF-A leads to glomerular disease in mice. Podocyte-specific heterozygosity for VEGF-A resulted in renal disease by 2.5 weeks of age, characterized by proteinuria and endotheliosis, the renal lesion seen in preeclampsia. Homozygous deletion of VEGF-A in glomeruli resulted in perinatal lethality. Mutant kidneys failed to develop a filtration barrier due to defects in endothelial cell migration, differentiation, and survival. In contrast, podocyte-specific overexpression of the VEGF-164 isoform led to a striking collapsing glomerulopathy, the lesion seen in HIV-associated nephropathy. Our data demonstrate that tight regulation of VEGF-A signaling is critical for establishment and maintenance of the glomerular filtration barrier and strongly supports a pivotal role for VEGF-A in renal disease

Laminins are the major noncollagenous glycoproteins of all basal laminae (BLs). They are α/β/γ heterotrimers assembled from 10 known chains, and they subserve both structural and signaling roles. Previously described mutations in laminin chain genes result in diverse disorders that are manifested postnatally and therefore provide little insight into laminin's roles in embryonic development. Here, we show that the laminin α5 chain is required during embryogenesis. The α5 chain is present in virtually all BLs of early somite stage embryos and then becomes restricted to specific BLs as development proceeds, including those of the surface ectoderm and placental vasculature. BLs that lose α5 retain or acquire other α chains. Embryos lacking laminin α5 die late in embryogenesis. They exhibit multiple developmental defects, including failure of anterior neural tube closure (exencephaly), failure of digit septation (syndactyly), and dysmorphogenesis of the placental labyrinth. These defects are all attributable to defects in BLs that are α5 positive in controls and that appear ultrastructurally abnormal in its absence. Other laminin α chains accumulate in these BLs, but this compensation is apparently functionally inadequate. Our results identify new roles for laminins and BLs in diverse developmental processes.