L. Philip Sanford

Double-Outlet Right Ventricle and Overriding Tricuspid Valve Reflect Disturbances of Looping, Myocardialization, Endocardial Cushion Differentiation, and Apoptosis in TGF-β2–Knockout Mice

Background—Transforming growth factor-β2 (TGF-β2) is a member of a family of growth factors with the potential to modify multiple processes. Mice deficient in the TGF-β2 gene die around birth and show a variety of defects of different organs, including the heart. Methods and Results—We studied the hearts of TGF-β2–null mouse embryos from 11.5 to 18.5 days of gestation to analyze the types of defects and determine which processes of cardiac morphogenesis are affected by the absence of TGF-β2. Analysis of serial sections revealed malformations of the outflow tract (typically a double-outlet right ventricle) in 87.5%. There was 1 case of common arterial trunk. Abnormal thickening of the semilunar valves was seen in 4.2%. Associated malformations of the atrioventricular (AV) canal were found in 62.5% and were composed of perimembranous inlet ventricular septal defects (37.5%), AV valve thickening (33.3%), overriding tricuspid valve (25.0%), and complete AV septal defects (4.2%). Anomalies of the aorta and its...

Colonic anion secretory defects and metabolic acidosis in mice lacking the NBC1 Na+/HCO3- cotransporter

The NBC1 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Na}^{+}{/}\mathrm{HCO}_{3}^{-}\) \end{document} cotransporter is expressed in many tissues, including kidney and intestinal epithelia. NBC1 mutations cause proximal renal tubular acidosis in humans, consistent with its role in \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} absorption in the kidney. In intestinal and colonic epithelia, NBC1 localizes to basolateral membranes and is thought to function in anion secretion. To test the hypothesis that NBC1 plays a role in transepithelial \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} secretion in the intestinal tract, null mutant (NBC1-/-) mice were prepared by targeted disruption of its gene (Slc4a4). NBC1-/- mice exhibited severe metabolic acidosis, growth retardation, reduced plasma Na+, hyperal-dosteronism, splenomegaly, abnormal dentition, intestinal obstructions, and death before weaning. Intracellular pH (pHi) was not altered in cAMP-stimulated epithelial cells of NBC1-/- cecum, but pHi regulation during sodium removal and readdition was impaired. Bioelectric measurements of NBC1-/- colons revealed increased amiloride-sensitive Na+ absorption. In Ringer solution containing both Cl- and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document}, the magnitude of cAMP-stimulated anion secretion was normal in NBC1-/- distal colon but increased in proximal colon, with the increase largely supported by enhanced activity of the basolateral NKCC1 Na+-K+-2Cl- cotransporter. Anion substitution studies in which carbonic anhydrase was inhibited and transepithelial anion conductance was limited to \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} revealed a sharp decrease in both cAMP-stimulated \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} secretion and SITS-sensitive current in NBC1-/- proximal colon. These results are consistent with the known function of NBC1 in \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} absorption in the kidney and demonstrate that NBC1 activity is a component of the basolateral mechanisms for \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} uptake during cAMP-stimulated anion secretion in the proximal colon.

Mice with a Targeted Disruption of the AE2 Exchanger Are Achlorhydric

The AE2 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Cl}^{-}{/}\mathrm{HCO}_{3}^{-}\) \end{document} exchanger is expressed in numerous cell types, including epithelial cells of the kidney, respiratory tract, and alimentary tract. In gastric epithelia, AE2 is particularly abundant in parietal cells, where it may be the predominant mechanism for \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{HCO}_{3}^{-}\) \end{document} efflux and Cl- influx across the basolateral membrane that is needed for acid secretion. To investigate the hypothesis that AE2 is critical for parietal cell function and to assess its importance in other tissues, homozygous null mutant (AE2-/-) mice were prepared by targeted disruption of the AE2 (Slc4a2) gene. AE2-/- mice were emaciated, edentulous (toothless), and exhibited severe growth retardation, and most of them died around the time of weaning. AE2-/- mice exhibited achlorhydria, and histological studies revealed abnormalities of the gastric epithelium, including moderate dilation of the gastric gland lumens and a reduction in the number of parietal cells. There was little evidence, however, that parietal cell viability was impaired. Ultrastructural analysis of AE2-/- gastric mucosa revealed abnormal parietal cell structure, with severely impaired development of secretory canaliculi and few tubulovesicles but normal apical microvilli. These results demonstrate that AE2 is essential for gastric acid secretion and for normal development of secretory canalicular and tubulovesicular membranes in mouse parietal cells.

Uroguanylin knockout mice have increased blood pressure and impaired natriuretic response to enteral NaCl load

Guanylin and uroguanylin, peptides synthesized in the intestine and kidney, have been postulated to have both paracrine and endocrine functions, forming a potential enteric-renal link to coordinate salt ingestion with natriuresis. To explore the in vivo role of uroguanylin in the regulation of sodium excretion, we created gene-targeted mice in which uroguanylin gene expression had been ablated. Northern and Western analysis confirmed the absence of uroguanylin message and protein in knockout mice, and cGMP levels were decreased in the mucosa of the small intestine. Ussing chamber analysis of jejunum revealed that Na+/H+ exchanger-mediated Na+ absorption and tissue conductance was not altered in the knockout animals, but short-circuit current, an index of electrogenic anion secretion, was reduced. Renal clearance measurements showed that uroguanylin deficiency results in impaired ability to excrete an enteral load of NaCl, primarily due to an inappropriate increase in renal Na+ reabsorption. Finally, telemetric recordings of blood pressure demonstrated increased mean arterial pressure in uroguanylin knockout animals that was independent of the level of dietary salt intake. Together, these findings establish a role for uroguanylin in an enteric-renal communication axis as well as a fundamental principle of this axis in the maintenance of salt homeostasis in vivo.

Loss of the Atp2c1 Secretory Pathway Ca2+-ATPase (SPCA1) in Mice Causes Golgi Stress, Apoptosis, and Midgestational Death in Homozygous Embryos and Squamous Cell Tumors in Adult Heterozygotes

Loss of one copy of the human ATP2C1 gene, encoding SPCA1 (secretory pathway Ca2+-ATPase isoform 1), causes Hailey-Hailey disease, a skin disorder. We performed targeted mutagenesis of the Atp2c1 gene in mice to analyze the functions of this Golgi membrane Ca2+ pump. Breeding of heterozygous mutants yielded a normal Mendelian ratio among embryos on gestation day 9.5; however, null mutant (Spca1-/-) embryos exhibited growth retardation and did not survive beyond gestation day 10.5. Spca1-/- embryos had an open rostral neural tube, but hematopoiesis and cardiovascular development were ostensibly normal. Golgi membranes of Spca1-/- embryos were dilated, had fewer stacked leaflets, and were expanded in amount, consistent with increased Golgi biogenesis. The number of Golgi-associated vesicles was also increased, and rough endoplasmic reticulum had fewer ribosomes. Coated pits, junctional complexes, desmosomes, and basement membranes appeared normal in mutant embryos, indicating that processing and trafficking of proteins in the secretory pathway was not massively impaired. However, apoptosis was increased, possibly the result of secretory pathway stress, and a large increase in cytoplasmic lipid was observed in mutant embryos, consistent with impaired handling of lipid by the Golgi. Adult heterozygous mice appeared normal and exhibited no evidence of Hailey-Hailey disease; however, aged heterozygotes had an increased incidence of squamous cell tumors of keratinized epithelial cells of the skin and esophagus. These data show that loss of the Golgi Ca2+ pump causes Golgi stress, expansion of the Golgi, increased apoptosis, and embryonic lethality and demonstrates that SPCA1 haploinsufficiency causes a genetic predisposition to cancer.

Ligand-specific function of transforming growth factor beta in epithelial-mesenchymal transition in heart development.

The ligand specificity of transforming growth factor beta (TGFβ) in vivo in mouse cardiac cushion epithelial‐to‐mesenchymal transition (EMT) is poorly understood. To elucidate the function of TGFβ in cushion EMT, we analyzed Tgfb1−/−, Tgfb2−/−, and Tgfb3−/− mice between embryonic day (E) 9.5 and E14.5 using both in vitro and in vivo approaches. Atrioventricular (AV) canal collagen gel assays at E9.5 indicated normal EMT in both Tgfb1−/− and Tgfb3−/− mice. However, analysis of Tgfb2−/− AV explants at E9.5 and E10.5 indicated that EMT, but not cushion cell proliferation, was initially delayed but later remained persistent. This was concordant with the observation that Tgfb2−/− embryos, and not Tgfb1−/− or Tgfb3−/− embryos, develop enlarged cushions at E14.5 with elevated levels of well‐validated indicators of EMT. Collectively, these data indicate that TGFβ2, and not TGFβ1 or TGFβ3, mediates cardiac cushion EMT by promoting both the initiation and cessation of EMT. Developmental Dynamics 238:431–442, 2009.

Impaired gastric acid secretion in mice with a targeted disruption of the NHE4 Na+/H+ exchanger

The NHE4 Na+/H+ exchanger is abundantly expressed on the basolateral membrane of gastric parietal cells. To test the hypothesis that it is required for normal acid secretion, NHE4-null mutant (NHE4–/–) mice were prepared by targeted disruption of the NHE4 (Slc9a4) gene. NHE4–/– mice survived and appeared outwardly normal. Analysis of stomach contents revealed that NHE4–/– mice were hypochlorhydric. The reduction in acid secretion was similar in 18-day-old, 9-week-old, and 6-month-old mice, indicating that the hypochlorhydria phenotype did not progress over time, as was observed in mice lacking the NHE2 Na+/H+ exchanger. Histological abnormalities were observed in the gastric mucosa of 9-week-old NHE4–/– mice, including sharply reduced numbers of parietal cells, a loss of mature chief cells, increased numbers of mucous and undifferentiated cells, and an increase in the number of necrotic and apoptotic cells. NHE4–/– parietal cells exhibited limited development of canalicular membranes and a virtual absence of tubulovesicles, and some of the microvilli had centrally bundled actin. We conclude that NHE4, which may normally be coupled with the AE2 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Cl}^{-}{/}\mathrm{HCO}_{3}^{-}\) \end{document} exchanger, is important for normal levels of gastric acid secretion, gastric epithelial cell differentiation, and development of secretory canalicular and tubulovesicular membranes.

Developmental expression of the TGFβs in the mouse cochlea

Abstract Mice with targeted disruption of the TGFβ2 gene display defects in epithelial-mesenchymal tissue interactions in several tissues including the developing cochlea. Specifically, the region of the spiral limbus and the overlying interdental cells, structures putatively involved in endolymphatic fluid homeostasis, display morphogenetic abnormalities. These findings prompted us to explore the pre-natal and post-natal expression of all three mammalian TGFβ genes in the developing mouse inner ear. TGFβ2 mRNA expression was identified throughout the cochlear epithelium at all of the developmental stages examined. TGFβ3 mRNA expression was identified in the mesenchymal tissues of the cochlea surrounding the otic epithelium. We found no evidence for compensation by the other two TGFβ isoforms in the cochleas of the TGFβ2 mutants.

Gene Targeted Ablation of High Molecular Weight Fibroblast Growth Factor-2

Fibroblast growth factor‐2 (FGF2) is produced as high molecular weight isoforms (HMW) and a low molecular weight isoform (LMW) by means of alternative usage of translation start sites in a single Fgf2 mRNA. Although the physiological function of FGF2 and FGF2 LMW has been investigated in myocardial capillarogenesis during normal cardiac growth, the role of FGF2 HMW has not been determined. Here, we report the generation of FGF2 HMW‐deficient mice in which FGF2 HMW isoforms are ablated by the Tag‐and‐Exchange gene targeting technique. These mice are normal and fertile with normal fecundity, and have a normal life span. Histological, immunohistochemical, and morphometric analyses indicate normal myocardial architecture, blood vessel, and cardiac capillary density in young adult FGF2 HMW‐deficient mice. These mice along with the FGF2‐ and FGF2 LMW‐deficient mice that we have generated previously will be very useful for elucidating the differential functions of FGF2 isoforms in pathophysiology of cardiovascular diseases. Developmental Dynamics 238:351–357, 2009.

Altered Renin Synthesis and Secretion in the Kidneys of Heterozygous Mice with a Null Mutation in the TGF-β2 Gene

Transforming growth factors β (TGF-βs) are peptides involved in autocrine and paracrine control of cell growth and differentiation. In the kidneys, TGF-β2 has been shown to localize specifically in renin-producing cells in various conditions stimulating the renin response. To test in vivo the functional role of TGF-β2, the renin response was investigated in mice heterozygous for a null mutation of the TGF-β2 gene, which had a twofold reduction in the amount of TGF-β2 mRNA. Although the increase in plasma renin concentration triggered by dehydration was not different from wild-type mice, renal renin mRNA and protein levels were higher in mutant mice under hydrated or dehydrated conditions. These data suggest that TGF-β2 exerts an inhibitory effect on renin synthesis and release from the juxtaglomerular apparatuses.