LacZ Knock-in Mice Research Articles

Implication of nanos2-3′UTR in the expression and function of nanos2

Translational control of gene expression is an important component of the regulation of cellular differentiation and development. To elucidate the function of the 3′untranslated region (UTR) of the nanos2 gene in mice, we compared the phenotypes of lacZ knock-in mice with or without a native nanos2 3′UTR and found that this region of the nanos2 gene has a potential role during translational regulation in germ cells. The nanos2-3′UTR functions to repress the translation of mRNA in oocytes, but enhances the production of protein in the male gonads. To further understand the significance of the nanos2 3′UTR in vivo, we generated the mouse line nanos2 pA/pA, which lacks this region endogenously. In nanos2 −/pA mice, the number of germ cell-depleted seminiferous tubules was increased when compared with that of nanos2 pA/pA mice, indicating a dose-dependent defect in spermatogenesis. These results suggest that the level of nanos2 protein is critical for normal spermatogenesis, and that this pathway may be regulated through the nanos2-3′UTR. We found that the defects in nanos2 pA/pA and nanos2 −/pA mice were caused by apoptosis of gonocytes in the embryonic gonads and gonocyte/spermatogonia in neonatal testes. In addition, it was noted that the nanos2 expression was restricted to a particular subset of spermatogonia after birth, which indicates that nanos2 plays a role in the maintenance and differentiation of gonocytes/spermatogonia in neonatal testes.

Essential role of COUP-TFII in vascular formation

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) is a member of the orphan nuclear receptor superfamily. The spatiotemporal expression pattern of COUP-TFII suggests that it participates in mesenchymal-epithelial interactions required for organogenesis. The null mutants of COUP-TFII die around embryonic (E) day E10 due to defects in angiogenesis and heart formation. To further understand the biologic functions of COUP-TFII during development, LacZ knockin mice and floxed COUP-TFII mice were generated. 5-bomo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) staining of COUP-TFII-LacZ knockin mice shows high COUP-TFII expression in the endothelium of the vein but not in the artery. In contrast, COUP-TFII is highly expressed in the smooth muscle layer surrounding the artery. This differential expression pattern suggests that COUP-TFII may play a role in establishing arterial-venous identity. To further elucidate the functional role of COUP-TFII in the formation of endothelium of the vein, COUP-TFII was specifically knocked out in the endothelium using Tie-2 Cre mice. In mutant mice lacking COUP-TFII in the endothelium, the vein acquires arterial characteristics and ectopic expression of Notch signal pathway. In addition, NP-1, an upstream regulator of Notch signaling, is also up-regulated. Finally, ectopic formation of hematopieitic cell clusters (HCC) is observed in the mutant veins. Our results are consistent with the notion that cell fate determination of the vein is under genetic control rather than deriving from a default pathway as proposed previously. In addition, COUP-TFII is the first transcription factor identified that specifically marks the endothelium of the vein. Understanding the role of COUP-TFII during the vasculature development will provide insights into the molecular mechanism for the establishment of arterial-venous identity.

Cross-species characterization of the ALS2 gene and analysis of its pattern of expression in development and adulthood

Mutations in the ALS2 gene, which encodes alsin, cause autosomal recessive juvenile-onset amyotrophic lateral sclerosis (ALS2) and related conditions. Using both a novel monoclonal antibody and LacZ knock-in mice, we demonstrate that alsin is widely expressed in neurons of the CNS, including the cortex, brain stem and motor neurons of the spinal cord. Interestingly, the highest levels of alsin are found in the molecular layer of the cerebellum, a brain region not previously implicated in ALS2. During development, alsin is expressed by day E9.5, but CNS expression does not become predominant until early postnatal life. At the subcellular level, alsin is tightly associated with endosomal membranes and is likely to be part of a large protein complex that may include the actin cytoskeleton. ALS2 is present in primates, rodents, fish and flies, but not in the nematode worm or yeast, and is more highly conserved than expected among mammals. Additionally, the product of a second, widely expressed gene, ALS2 C-terminal like (ALS2CL), may subserve or modulate some of the functions of alsin as an activator of Rab and Rho GTPases.

NSCL-1 and NSCL-2 synergistically determine the fate of GnRH-1 neurons and control necdin gene expression.

To study the role of the bHLH genes NSCL-1 and NSCL-2 in the development of GnRH-1 neurons, we have generated compound mutant mice. Mutant animals die at birth and show a virtually complete absence of GnRH-1 neurons in the posterior parts of the brain at E18.5 and an aberrant morphology of the remaining GnRH-1 neurons in the anterior parts of the brain indicating that NSCL-1 and NSCL-2 might concomitantly control differentiation/migration of GnRH-1 neurons in a cell autonomous manner. To gain further insights into this process, we screened for NSCL target genes using DNA array hybridization and detected necdin, which is deleted in the human Prader-Willi syndrome phenotypically resembling the NSCL-2 mutation. Using chromatin immunoprecipitation and site-directed mutagenesis of the necdin promoter, we demonstrate that NSCLs together with additional cofactors directly control transcription of the necdin gene. NSCL-dependent control of necdin expression might be instrumental for proper neuronal cell differentiation and enable GnRH-1 neurons to migrate.

Angiopoietin-2 causes pericyte dropout in the normal retina: evidence for involvement in diabetic retinopathy.

Pericyte loss is an early pathologic feature of diabetic retinopathy, consistently present in retinae of diabetic humans and animals. Because pericyte recruitment and endothelial cell survival are controlled, in part, by the angiopoietin/Tie2 ligand/receptor system, we studied the expression of angiopoietin-2 and -1 in relation to the evolution of pericyte loss in diabetic rat retinae, using quantitative retinal morphometry, and in retinae from mice with heterozygous angiopoietin deficiency (Ang-2 LacZ knock-in mice). Finally, recombinant angiopoietin-2 was injected into eyes of nondiabetic rats, and pericyte numbers were quantitated in retinal capillaries. Angiopoietin-1 protein was present in the normal maturing retina and was upregulated 2.5-fold in diabetic retinae over 3 months of diabetes. In contrast, angiopoietin-2 protein was consistently upregulated more than 30-fold in the retinae of diabetic rats, preceding the onset of pericyte loss. Heterozygous angiopoietin-2 deficiency completely prevented diabetes-induced pericyte loss and reduced the number of acellular capillary segments. Injection of angiopoietin-2 into the eyes of normal rats induced a dose-dependent pericyte loss. These data show that upregulation of angiopoietin-2 plays a critical role in the loss of pericytes in the diabetic retina.

Runx1/AML1 hematopoietic transcription factor contributes to skeletal development in vivo.

The requirement of Runx2 (Cbfal/AML3), a runt homology domain transcription factor essential for bone formation and osteoblast differentiation, is well established. Although Runx2 is expressed in the developing embryo prior to ossification, yet in the absence of Runx2 initial formation of the skeleton is normal, suggesting a potential redundancy in function of Runx family members. Here we addressed expression of the hematopoietic family member Runx1 (AML1/Cbfa2) in relation to skeletal development using a LacZ knock-in mouse model (Runx1(lz/+)). The resulting fusion protein reflects Runx1 promoter activity in its native context. Our studies show that Runx1 is expressed by prechondrocytic tissue forming the cartilaginous anlagen in the embryo, resting zone chondrocytes, suture lines of the calvarium, and in periosteal and perichondral membranes of all bone. Runx1 continues to be expressed in these tissues in adult mice, but is absent in mature cartilage or mineralized bone. However, hyaline cartilage outside the bone environment (trachea, xiphoid tissues), and epithelium of many soft tissues (trachea, thyroid, lung, skin) also express Runx1. The robust expression of Runx1 in vivo in chondroblasts at sites of cartilage growth and in osteoblasts at sites of new bone formation, suggests that Runx1 expression may be related to osteochondroprogenitor cell differentiation. This observation is further supported by high expression of Runx1 in ex vivo cultures of marrow stromal cells and calvarial derived osteoblasts from Runx1(lz/+) mice. These data indicate that Runx1 may contribute to the early stages of skeletogenesis and continues to function in the progenitor cells of tissues that support bone formation in the adult.

Receptor protein tyrosine phosphatase mu expression as a marker for endothelial cell heterogeneity; analysis of RPTPmu gene expression using LacZ knock-in mice.

The receptor-like protein tyrosine phosphatase mu (RPTPmu) belongs to the subfamily of meprin, A5, RPTPmu (MAM) domain-containing RPTPs, which are thought to play an important role in cell-cell adhesion mediated processes. The current study was designed to examine the expression pattern of RPTPmu in mice. We have generated RPTPmu-LacZ knock-in mice that express the beta-galactosidase (LacZ) reporter gene under the control of the RPTPmu promoter. LacZ expression patterns were analysed in embryos and adult mice by whole mount LacZ staining. Analysis of beta-galactosidase activity of heterozygous embryos and adult tissues revealed RPTPmu expression in endothelial cells of arteries and capillaries. In contrast, expression was virtually absent in endothelial cells of veins and in fenestrated endothelial cells in the adult liver and spleen. Moreover, RPTPmu expression was found in endothelial cells from the endocardium and the aorta in embryos, but not in adult mice. In addition to heterogeneous expression in endothelial cells, RPTPmu expression was found in cardiac muscle cells but not in skeletal muscle cells or smooth muscle cells. Expression was also found in Type II pneumonocytes in the lung alveoli and in Purkinje cells and other neurons in the brain. The specific expression of RPTPmu in arterial endothelial cells and in cardiac myocytes suggests that RPTPmu may play a role in the regulation of cardiovascular functions.