Jacqueline Mudd

Compositional and structural requirements for laminin and basement membranes during mouse embryo implantation and gastrulation

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 lamininα 1 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 Reicherts 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 Reicherts 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.

Quantitative Trait Loci Influence Renal Disease Progression in a Mouse Model of Alport Syndrome

Alport syndrome is a human hereditary glomerulonephritis which results in end-stage renal failure (ESRF) in most cases. It is caused by mutations in any one of the collagen alpha3(IV), alpha4(IV), or alpha5(IV) chain genes (COL4A3-COL4A5). Patients carrying identical mutations can exhibit very different disease courses, suggesting that other genes or the environment influence disease progression. We previously generated a knockout mouse model of Alport syndrome by mutating Col4a3. Here, we show that genetic background strongly influences the timing of onset of disease and rate of progression to ESRF in these mice. On the 129X1/SvJ background, Col4a3 -/- mice reached ESRF at approximately 66 days of age, while on the C57BL/6J background, the mean age at ESRF was 194 days of age. This suggests the existence of modifier genes that influence disease progression. A detailed histopathological analysis revealed that glomerular basement membrane lesions typical of Alport syndrome were significantly more frequent in homozygotes on the 129X1/SvJ background than on the C57BL/6J background as early as two weeks of age, suggesting that modifier genes act by influencing glomerular basement membrane structure. Additional data indicated that differential physiological responses to basement membrane splitting also underlie the differences in disease progression. We attempted to map the modifier genes as quantitative trait loci (QTLs) using age at ESRF as the quantitative trait. Genome scans were performed on mice at the two extremes in a cohort of mutant F1 x C57BL/6J backcross mice. Analysis with Map Manager QT revealed QTLs linked to markers on chromosomes 9 and 16. A more detailed understanding of how these QTLs act could lead to new approaches for therapy in diverse renal diseases.

Genetic heterogeneity of induced pluripotent stem cells: Results from 24 clones derived from a single C57BL/6 mouse

Induced pluripotent stem cells (iPSCs) have tremendous potential as a tool for disease modeling, drug testing, and other applications. Since the generation of iPSCs “captures” the genetic history of the individual cell that was reprogrammed, iPSC clones (even those derived from the same individual) would be expected to demonstrate genetic heterogeneity. To assess the degree of genetic heterogeneity, and to determine whether some cells are more genetically “fit” for reprogramming, we performed exome sequencing on 24 mouse iPSC clones derived from skin fibroblasts obtained from two different sites of the same 8-week-old C57BL/6J male mouse. While no differences in the coding regions were detected in the two parental fibroblast pools, each clone had a unique genetic signature with a wide range of heterogeneity observed among the individual clones: a total of 383 iPSC variants were validated for the 24 clones (mean 16.0/clone, range 0–45). Since these variants were all present in the vast majority of the cells in each clone (variant allele frequencies of 40–60% for heterozygous variants), they most likely preexisted in the individual cells that were reprogrammed, rather than being acquired during reprogramming or cell passaging. We then tested whether this genetic heterogeneity had functional consequences for hematopoietic development by generating hematopoietic progenitors in vitro and enumerating colony forming units (CFUs). While there was a range of hematopoietic potentials among the 24 clones, only one clone failed to differentiate into hematopoietic cells; however, it was able to form a teratoma, proving its pluripotent nature. Further, no specific association was found between the mutational spectrum and the hematopoietic potential of each iPSC clone. These data clearly highlight the genetic heterogeneity present within individual fibroblasts that is captured by iPSC generation, and suggest that most of the changes are random, and functionally benign.