Comparative gene expression analysis of genital tubercle development reveals a putative appendicular Wnt7 network for the epidermal differentiation.

Here we describe the first detailed catalog of gene expression in the developing lower urinary tract (LUT), including epithelial and mesenchymal portions of the developing bladder, urogenital sinus, urethra, and genital tubercle (GT) at E13 and E14. Top compartment-specific genes implicated by the microarray data were validated using whole-mount in situ hybridization (ISH) over the entire LUT. To demonstrate the potential of this resource to implicate developmentally critical features, we focused on gene expression patterns and pathways in the sexually indeterminate, androgen-independent GT. GT expression patterns reinforced the proposed similarities between development of GT, limb, and craniofacial prominences. Comparison of spatial expression patterns predicted a network of Wnt7a-associated GT-enriched epithelial genes, including Gjb2, Dsc3, Krt5, and Sostdc1. Known from other contexts, these genes are associated with normal epidermal differentiation, with disruptions in Dsc3 and Gjb2 showing palmo-plantar keratoderma in the limb. We propose that this gene network contributes to normal foreskin, scrotum, and labial development. As several of these genes are known to be regulated by, or contain cis elements responsive to retinoic acid, estrogen, or androgen, this implicates this pathway in the later androgen-dependent development of the GT.

Urogenital tract expression of enhanced green fluorescent protein in transgenic mice driven by a smooth muscle gamma-actin promoter.

PURPOSE: Our understanding of urogenital tract development and its response to disease or injury is hindered by complex interactions between epithelial and mesenchymal cells, and the difficulties in studying either component in isolation. We investigated whether transgenic mice could be generated to express enhanced green fluorescent protein (EGFP) in smooth muscle cells (SMCs) and whether such cells could then be purified using flow cytometric sorting to isolate RNA to be used in future gene expression assays. MATERIALS AND METHODS: A 13.7 kb mouse smooth muscle gamma-actin promoter fragment was ligated to an EGFP reporter gene and microinjected into male mouse pronuclei. Adult transgenic mice were sacrificed and urogenital tissues were removed for histological and immunohistochemical studies. In other animals conditions were determined for dissociating bladder cells and the subsequent purification of bladder SMCs by sorting. RESULTS: Six lines of transgenic mice were generated (transgene copy numbers 1 to 30). EGFP was expressed in all smooth muscle beds examined except those associated with small blood vessels. EGFP levels appeared to correlate with transgene copy number. Histological and immunohistochemical analysis confirmed that reporter gene expression was restricted to SMCs of all tissues examined. Parameters for generating bladder cell suspensions were established and EGFP labeled bladder SMCs were identified by flow cytometric analysis. CONCLUSIONS: Several lines of transgenic mice have been generated in which SMCs of urogenital tissues have been labeled with EGFP and pure populations of SMCs have been obtained. The methods established for the rapid dissociation and purification of bladder SMCs should minimize degradative changes. These approaches may enable us to address issues involving bladder SMC development and differentiation as well as the response to injury and disease by performing transcriptome wide analyses on purified SMC populations.

Cellular disorganization and extensive apoptosis in the developing heart of mice that lack cardiac muscle alpha-actin: apparent cause of perinatal death.

Mice that lack cardiac muscle alpha-actin die during the perinatal period. Approximately 56% of mice that are homozygous null (-/-) for a functional cardiac alpha-actin gene do not survive to term, and the remainder generally die within 2 wk of birth. We found that there were neither morphologic differences nor differences in the extent of apoptosis between the mutant and normal hearts on embryonic day (E) 12 and E14 of development. However, apoptosis was greater in the hearts of homozygous null mice on E17 and postnatal day 1 when compared with wild-type hearts. The antiapoptotic factor Bcl-x/(L) was localized in regions adjacent to where apoptosis was detected. The distribution patterns of the apoptosis triggering protein p53 were similar to those of apoptotic cells. The growth of the prenatal and postnatal hearts of the cardiac alpha-actin-deficient mice was retarded, and the cytoplasmic filaments were disorganized. Although apoptotic cells were observed in both the atria and ventricles in the hearts of the homozygous null animals, the frequency was greater in the ventricles than in the atria. Our results indicate that the functional and structural disturbances in the mice with a homozygous lack of cardiac alpha-actin seem to be due to disorganized development of acto-myosin filaments in the affected cardiomyocytes. Other actin isoforms cannot compensate for the lack of cardiac alpha-actin, and this seems to induce apoptosis in defective cardiac myocytes, which are not able to cope with the increased workload in the perinatal phase.