Laminins are components of all basement membranes and have well demonstrated roles in diverse developmental processes, from the peri-implantation period onwards. Laminin 1 (α1β1γ1) is a major laminin found at early stages of embryogenesis in both embryonic and extraembryonic basement membranes. The laminin γ1 chain has been shown by targeted mutation to be required for endodermal differentiation and formation of basement membranes; Lamc1-/- embryos die within a day of implantation. We report the generation of mice lacking lamininα1 and laminin β1, the remaining two laminin 1 chains. Mutagenic insertions in both Lama1 and Lamb1 were obtained in a secretory gene trap screen. Lamb1-/- embryos are similar to Lamc1-/- embryos in that they lack basement membranes and do not survive beyond embryonic day (E) 5.5. However, in Lama1-/- embryos, the embryonic basement membrane forms,the embryonic ectoderm cavitates and the parietal endoderm differentiates,apparently because laminin 10 (α5β1γ1) partially compensates for the absent laminin 1. However, such compensation did not occur for Reichert's membrane, which was absent, and the embryos died by E7. Overexpression of laminin α5 from a transgene improved the phenotype of Lama1-/- embryos to the point that they initiated gastrulation, but this overexpression did not rescue Reichert's membrane, and trophoblast cells did not form blood sinuses. These data suggest that both the molecular composition and the integrity of basement membranes are crucial for early developmental events.

Restrictive dermopathy (RD) is a lethal human genetic disorder characterized by very tight, thin, easily eroded skin, rocker bottom feet, and joint contractures. This disease was recently reported to be associated with a single heterozygous mutation in ZMPSTE24 and hypothesized to be a digenic disorder (Navarro et al, Lamin A and ZMPSTE24 (FACE-1) defects cause nuclear disorganization and identify restrictive dermopathy as a lethal neonatal laminopathy. Hum Mol Genet 13:2493-2503, 2004). ZMPSTE24 encodes an enzyme necessary for the correct processing and maturation of lamin A, an intermediate filament component of the nuclear envelope. Here we present four unrelated patients with homozygous mutations in ZMPSTE24 and a fifth patient with compound heterozygous mutations in ZMPSTE24. Two of the three different mutations we found are novel, and all are single base insertions that result in messenger RNA frameshifts. As a consequence of the presumed lack of ZMPSTE24 activity, prelamin A, the unprocessed toxic form of lamin A, was detected in the nuclei of both cultured cells and tissue from RD patients, but not in control nuclei. Abnormally aggregated lamin A/C was also observed. These results indicate that RD is an autosomal recessive laminopathy caused by inactivating ZMPSTE24 mutations that result in defective processing and nuclear accumulation of prelamin A.

Discs-large homolog 1 (DLGH1) is a mouse ortholog of the Drosophila discs-large (DLG) tumor suppressor protein, a founding member of the PDZ and MAGUK protein families. DLG proteins play important roles in regulating cell proliferation, epithelial cell polarity, and synapse formation and function. Here, we generated a null allele of Dlgh1 and studied its role in urogenital development. Dlgh1 ؊/؊ mice developed severe urinary tract abnormalities, including congenital hydronephrosis, which is the leading cause of renal failure in infants and children. DLGH1 is expressed in the developing ureter; in its absence, the stromal cells that normally lie between the urothelial and smooth muscle layers were missing. Moreover, in ureteric smooth muscle, the circular smooth muscle cells were misaligned in a longitudinal orientation. These abnormalities in the ureter led to severely impaired ureteric peristalsis. Similar smooth muscle defects are observed frequently in patients with ureteropelvic junction obstruction, a common form of hydronephrosis. Our results suggest that (i) besides its well documented role in regulating epithelial polarity, Dlgh1 also regulates smooth muscle orientation, and (ii) human DLG1 mutations may contribute to hereditary forms of hydronephrosis.SAP97 ͉ kidney ͉ postsynaptic density-95/discs-large/zonula occludens-1 ͉ urogenital ͉ sonic hedgehog

Hutchinson-Gilford progeria syndrome (HGPS) is a rare precocious aging syndrome caused by mutations in LMNA that lead to synthesis of a mutant form of prelamin A, generally called progerin, that cannot be processed to mature lamin A. Most HGPS patients have a recurrent heterozygous de novo mutation in exon 11 of LMNA, c.1824C>T/p.G608G; this synonymous mutation activates a nearby cryptic splice donor site, resulting in synthesis of the mutant prelamin A, progerin, which lacks 50 amino acids within the carboxyl-terminal domain. Abnormal splicing is incomplete, so the mutant allele produces some normally-spliced transcripts. Nevertheless, the synthesis of progerin is sufficient to cause misshapen nuclei in cultured cells and severe disease phenotypes in affected patients. Here we present two patients with extraordinarily severe forms of progeria caused by unusual mutations in LMNA. One had a splice site mutation (c.1968+1G>A; or IVS11+1G>A), and the other had a novel synonymous coding region mutation (c.1821G>A/p.V607V). Both mutations caused very frequent use of the same exon 11 splice donor site that is activated in typical HGPS patients. As a consequence, the ratios of progerin mRNA and protein to wild-type were higher than in typical HGPS patients. Fibroblasts from both patients exhibited nuclear shape abnormalities typical of HGPS, and cells treated with a protein farnesyltransferase inhibitor exhibited fewer misshapen nuclei. Thus, farnesyltransferase inhibitors may prove to be useful even when progerin expression levels are higher than those in typical HGPS patients.

Pierson syndrome is a recently defined disease usually lethal within the first postnatal months and caused by mutations in the gene encoding lamininβ2 (LAMB2). The hallmarks of Pierson syndrome are congenital nephrotic syndrome accompanied by ocular abnormalities, including microcoria(small pupils), with muscular and neurological developmental defects also present. Lamb2-/- mice are a model for Pierson syndrome;they exhibit defects in the kidney glomerular barrier, in the development and organization of the neuromuscular junction, and in the retina. Lamb2-/- mice fail to thrive and die very small at 3 weeks of age, but to what extent the kidney and neuromuscular defects each contribute to this severe phenotype has been obscure, though highly relevant to understanding Pierson syndrome. To investigate this, we generated transgenic mouse lines expressing rat laminin β2 either in muscle or in glomerular epithelial cells (podocytes) and crossed them onto the Lamb2-/- background. Rat β2 was confined in skeletal muscle to synapses and myotendinous junctions, and in kidney to the glomerular basement membrane. In transgenic Lamb2-/- mice, β2 deposition in only glomeruli prevented proteinuria but did not ameliorate the severe phenotype. By contrast, β2 expression in only muscle restored synaptic architecture and led to greatly improved health, but the mice died from kidney disease at 1 month. Rescue of both glomeruli and synapses was associated with normal weight gain, fertility and lifespan. We conclude that muscle defects in Lamb2-/- mice are responsible for the severe failure to thrive phenotype, and that renal replacement therapy alone will be an inadequate treatment for Pierson syndrome.