Expression Pattern of the LacZ Reporter in Secretogranin III Gene-trapped Mice

Secretogranin III (SgIII) is a granin protein involved in secretory granule formation in peptide-hormone-producing endocrine cells. In this study, we analyzed the expression of the LacZ reporter in the SgIII knockout mice produced by gene trapping ( SgIII-gtKO) for the purpose of comprehensively clarifying the expression patterns of SgIII at the cell and tissue levels. In the endocrine tissues of SgIII-gtKO mice, LacZ expression was observed in the pituitary gland, adrenal medulla, and pancreatic islets, where SgIII expression has been canonically revealed. LacZ expression was extensively observed in brain regions, especially in the cerebral cortex, hippocampus, hypothalamic nuclei, cerebellum, and spinal cord. In peripheral nervous tissues, LacZ expression was observed in the retina, optic nerve, and trigeminal ganglion. LacZ expression was particularly prominent in astrocytes, in addition to neurons and ependymal cells. In the cerebellum, at least four cell types expressed SgIII under basal conditions. The expression of SgIII in the glioma cell lines C6 and RGC-6 was enhanced by excitatory glutamate treatment. It also became clear that the expression level of SgIII varied among neuron and astrocyte subtypes. These results suggest that SgIII is involved in glial cell function, in addition to neuroendocrine functions, in the nervous system:

Localization of Glucagon-Like Peptide-2 Receptor Expression in the Mouse

Glucagon-like peptide-2 (GLP-2), secreted from enteroendocrine cells, attenuates gut motility, enhances barrier function, and augments nutrient absorption, actions mediated by a single GLP-2 receptor (GLP-2R). Despite extensive analyses, the precise distribution and cellular localization of GLP-2R expression remains controversial, confounded by the lack of suitable GLP-2R antisera. Here, we reassessed murine Glp2r expression using regular and real-time quantitative PCR (qPCR), in situ hybridization (ISH), and a Glp2rLacZ reporter mouse. Glp2r mRNA expression was detected from the stomach to the rectum and most abundant in the jejunum. Glp2r transcripts were also detected in cerebral cortex, mesenteric lymph nodes, gallbladder, urinary bladder, and mesenteric fat. Surprisingly, Glp2r mRNA was found in testis by qPCR at levels similar to jejunum. However, the testis Glp2r transcripts, detected by different primer pairs and qPCR, lacked 5′ mRNA coding sequences, and only a minute proportion of them corresponded to full-length Glp2r mRNA. Within the gut, Glp2r-driven LacZ expression was localized to enteric neurons and lamina propria stromal cells, findings confirmed by ISH analysis of the endogenous Glp2r mRNA. Unexpectedly, vascular Glp2rLacZ expression was localized to mesenteric veins and not arteries. Moreover, mesenteric fat Glp2rLacZ expression was detected within blood vessels and not adipocytes. Reporter LacZ expression was not detected in all tissues expressing an endogenous Glp2r transcript, such as gallbladder, urinary bladder, and mesenteric lymph nodes. Collectively, these findings extend our understanding of the cellular domains of Glp2r expression and highlight limitations inherent in application of commonly used technologies to infer analysis of gene expression.

Abstract 463: The Key Regulatory Elements for SM22 Transcription

Gene transcription is controlled by an array of transcriptional regulatory elements. SM22 gene regulation has been widely used to characterize the molecular mechanisms of smooth muscle cell (SMC) phenotypic modulation during cardiovascular development and in vascular diseases. Our previous studies show that the proximal CArG box (CArGnear) in the SM22 promoter is required for its transcription in arterial smooth muscle cells (SMC). However, the role of the CArG box in visceral SMCs has not yet been explored. Here we aim to determine the role of CArG boxes in regulating S M22 transcription in both vascular and visceral SMCs. Using bacterial chromosome (BAC) recombineering, we knock-in a lacZ reporter into a SM22 BAC to trace SM22 transcriptional activities in transgenic mice. Anatomic/histology analyses show that the lacZ expression patterns in the BAC transgenic mice recapitulate that of the endogenous SM22 transcription during embryogenesis and in adult . Similar to the endogenous SM22 regulation, the expression of lacZ is highly sensitive to vascular remodeling: carotid injury abolishes lacZ expression in the arterial wall. Using seamless BAC recombineering mutagenesis, we generate mutations in the proximal and/or distal CArG box in the SM22-lacZ-BAC. Consistent with our previous results, we find that mutating the CArGnear box disrupts the lacZ expression in the aorta; this mutation also drastically reduces its expression in visceral SMCs including stomach, uterus and bladder. Interestingly, mutating the distal CArG (CArGfar) box does not affect the lacZ expression in arterial, venous and visceral SMCs. Mutating both CArG boxes nearly abolishes lacZ expression in all SMCs. This study provides evidence supporting the generation of SM22 knockout mice by mutating the CArG boxes in the SM22 promoter using the CRISPR technology.

β-Catenin/Tcf signaling in murine oocytes identifies nonovulatory follicles

WNTS are secreted glycoprotein molecules that signal through one of three signaling pathways. The best-characterized pathway involves stabilization of the multifunctional protein β-catenin, which in concert with members of the T-cell factor (Tcf) family activates specific gene transcription. We have examined putative Wnt/β-catenin in the murine ovary using transgenic mice harboring a reporter construct that activates β-galactosidase (lacZ) expression in response to β-catenin/Tcf binding (TopGal mice). Primordial and primary follicles did not stain for lacZ, and the proportion of β-catenin/Tcf signaling oocytes was lower than that of nonsignaling oocytes throughout estrous cycle. β-Catenin/Tcf signaling oocytes were observed in follicles from the secondary stage of development and their proportion increased with follicular maturation (secondary follicles, 20%; early antral and antral follicles, 70%). In contrast, the majority (>90%) of ovulated oocytes did not stain for lacZ. As the oocyte possesses components for WNT signal transduction, our data suggest that β-catenin/Tcf signaling is involved in the development of follicular ovulatory capability and identifies nonovulatory follicles.

GATA6 reporter gene reveals myocardial phenotypic heterogeneity that is related to variations in gap junction coupling

This study examined transgenic mice whose expression of a β-galactosidase (lacZ) reporter is driven by a GATA6 gene enhancer. Previous investigations established that transcription of the transgene was associated with precardiac mesoderm and primary heart tube myocardium, which decreased progressively, so that its expression was no longer observed within ventricular myocardium by midgestation. Expression of this reporter in the adult was investigated for insights into myocyte homeostasis and cardiovascular biology. Morphometric analysis determined that <1% of myocytes, often found in small clusters, express this GATA6-associated reporter in the adult heart. LacZ expression was also found in the ascending aorta. Myocardial expression of the transgene was not associated with a proliferative phenotype or new myocyte formation, as lacZ-positive myocytes neither labeled with cell division markers nor following 5-bromodeoxyuridine pulse-chase experimentation. Despite exhibiting normal adherens junctions, these myocytes appeared to exhibit decreased connexin 43 gap junctions. Treatment with the gap junctional blocker heptanol both in vivo and in culture elevated myocardial β-galactosidase activity, suggesting that deficient gap junctional communication underlies expression of the transgenic reporter. LacZ expression within the myocardium was also enhanced in response to cryoinjury and isoproterenol-induced hypertrophy. These results reveal a previously uncharacterized phenotypic heterogeneity in the myocardium and suggest that decreased gap junctional coupling leads to induction of a signaling pathway that utilizes a unique GATA6 enhancer. Upregulation of lacZ reporter gene expression following cardiac injury indicates this transgenic mouse may serve as a model for examining the transition of the heart from healthy to pathological states.

Flavonoid-responsive nodY-lacZ expression in three phylogenetically different Bradyrhizobium groups

Previously, restriction fragment length polymorphism analysis using the nodD1YABC gene probe showed the genetic diversity of common nodD1ABC gene regions of Bradyrhizobium japonicum , Bradyrhizobium elkanii , and the Thai soybean Bradyrhizobium. The nodD1 sequences of representative strains of the 3 groups differed phylogenetically, suggesting that responses of NodD1 proteins of the 3 Bradyrhizobium groups to diverse flavonoids may differ. To confirm this hypothesis, 6 representative strains were chosen from the 3 Bradyrhizobium groups. Six reporter strains were constructed, all carrying the pZB32 plasmid, which contains a nod box and the nodY-lacZ fusion of B. japonicum USDA 110. Differences in nodY-lacZ expression among the strains in response to 37 flavonoid compounds at various concentrations were evaluated. Of those compounds, prunetin (4′,5-dihydroxy-7-methoxyisoflavone) and esculetin (6,7-dihydroxycoumarin) were identified as Bradyrhizobium group-specific nod gene inducers. Esculetin showed nod gene induction activities unique to Thai Bradyrhizobium strains. The levels of nodY-lacZ induction among B. japonicum and Thai Bradyrhizobium strains increased with increasing concentration of prunetin, whereas, those in B. elkanii strains did not.