Optimizing Genomic Methods for Mapping and Identification of Candidate Variants in ENU Mutagenesis Screens Using Inbred Mice

Positional cloning of ENU-induced mutations has traditionally relied on analysis of polymorphic variation between two strains. In contrast, the application of whole-genome sequencing (WGS) has enabled gene discovery in mutant lines maintained on an inbred genetic background. This approach utilizes genetic variation derived from ENU-induced variants for mapping and reduces the likelihood of phenotypic variation, making it an ideal method for genetic modifier screening. Here, we describe the results of such a screen, wherein we determined the minimal number of mutant genomic DNA samples to include in our analyses and improved the sensitivity of our screen by individually barcoding each genomic DNA library. We present several unique cases to illustrate this approach’s efficacy, including the discovery of two distinct mutations that generate essentially identical mutant phenotypes, the ascertainment of a non-ENU-induced candidate variant through homozygosity mapping, and an approach for the identification of putative dominant genetic modifiers

A novel loss-of-function mutation in Npr2 clarifies primary role in female reproduction and reveals a potential therapy for acromesomelic dysplasia, Maroteaux type

We discovered a new spontaneous mutant allele of Npr2 named peewee (pwe) that exhibits severe disproportionate dwarfism and female infertility. The pwe phenotype is caused by a four base-pair deletion in exon 3 that generates a premature stop codon at codon 313 (L313X). The Npr2(pwe/pwe) mouse is a model for the human skeletal dysplasia acromesomelic dysplasia, Maroteaux type (AMDM). We conducted a thorough analysis of the female reproductive tract and report that the primary cause of Npr2(pwe/pwe) female infertility is premature oocyte meiotic resumption, while the pituitary and uterus appear to be normal. Npr2 is expressed in chondrocytes and osteoblasts. We determined that the loss of Npr2 causes a reduction in the hypertrophic and proliferative zones of the growth plate, but mineralization of skeletal elements is normal. Mutant tibiae have increased levels of the activated form of ERK1/2, consistent with the idea that natriuretic peptide receptor type 2 (NPR2) signaling inhibits the activation of the MEK/ERK mitogen activated protein kinase pathway. Treatment of fetal tibiae explants with mitogen activated protein kinase 1 and 2 inhibitors U0126 and PD325901 rescues the Npr2(pwe/pwe) growth defect, providing a promising foundation for skeletal dysplasia therapeutics

LINE-1 Mediated Insertion into Poc1a (Protein of Centriole 1 A) Causes Growth Insufficiency and Male Infertility in Mice

Skeletal dysplasias are a common, genetically heterogeneous cause of short stature that can result from disruptions in many cellular processes. We report the identification of the lesion responsible for skeletal dysplasia and male infertility in the spontaneous, recessive mouse mutant chagun. We determined that Poc1a, encoding protein of the centriole 1a, is disrupted by the insertion of a processed Cenpw cDNA, which is flanked by target site duplications, suggestive of a LINE-1 retrotransposon-mediated event. Mutant fibroblasts have impaired cilia formation and multipolar spindles. Male infertility is caused by defective spermatogenesis early in meiosis and progressive germ cell loss. Spermatogonial stem cell transplantation studies revealed that Poc1a is essential for normal function of both Sertoli cells and germ cells. The proliferative zone of the growth plate is small and disorganized because chondrocytes fail to re-align after cell division and undergo increased apoptosis. Poc1a and several other genes associated with centrosome function can affect the skeleton and lead to skeletal dysplasias and primordial dwarfisms. This mouse mutant reveals how centrosome dysfunction contributes to defects in skeletal growth and male infertility

<i>Poc1a</i>^cha/cha males exhibit progressive germ cell loss.

<p>Paraffin sections of testes tissues collected from wild type and <i>Poc1a</i>^cha/cha mice at the indicated ages were stained with Periodic acid-Schiff reagent. At P1 and P7 <i>Poc1a</i>^cha/cha tubules resemble wild type seminiferous tubules, but from P14 through 12 wks, the <i>Poc1a</i>^cha/cha tubules are obviously abnormal. (N = 3 animals of each genotype at each age, scale bar = 50 μm).</p

The <i>Poc1a</i>^cha/cha long bone growth plates become disorganized.

<p>A. Standard hematoxylin and eosin histological staining was carried out on paraffin sections from P0 and P15 tibial growth plates. Scale bar = 50 μm. B. Immunohistochemistry using a primary antibody against the Golgi marker GM130 or acetylated tubulin counterstained with DAPI in wild type and mutant P15 tibia sections. Scale bar = 25 μm. C. Immunohistochemistry for primary antibodies against the proliferation marker Ki67 (i) or cell death marker TUNEL (ii), with quantification of the number of TUNEL-positive cells per field of view in wild type and <i>chagun</i> tibia (iii). Scale bar = 100 μm.</p

A <i>Poc1a</i> BAC transgene rescues <i>chagun</i>, and a null allele phenocopies <i>chagun</i>.

<p>A. Female and male mice of the indicated genotypes were weighed at 6 wks of age. N = 5-19/group. B. Adult male animals of the indicated genotypes were analyzed for testis morphology (0.8X objective) and histology. Testis sections were stained with hematoxylin and eosin. Scale bar = 100 μm. C. Bones were collected from <i>Poc1a</i>^+/+ and <i>Poc1a</i>^-/- animals at P21 and testes from P40 animals. Samples were fixed, sectioned and stained with hematoxylin and eosin. Scale bar = 100 μm.</p

POC1A is expressed in the growth plate and seminiferous tubules.

<p>A. POC1A immunostaining of sections from postnatal day 14 (P14) wild type and <i>Poc1a</i>^cha/cha mutant tibial growth plates reveals strongest expression of POC1A (magenta) in the proliferative zone. B. POC1A immunostaining was carried out on testis sections from 3 mo old wild type and <i>Poc1a</i>^cha/cha mutants. C. POC1A staining was also performed on testis sections from P21 wild type and <i>Poc1a</i> knockout mice. Scale bars as indicated.</p