Transforming growth factor-β1 (TGF-

NHE3 is one of five plasma membrane Na+/H+ exchangers and is encoded by the mouse gene Slc9a3. It is expressed on apical membranes of renal proximal tubule and intestinal epithelial cells and is thought to play a major role in NaCl and HCO3- absorption. As the distribution of NHE3 overlaps with that of the NHE2 isoform in kidney and intestine, the function and relative importance of NHE3 in vivo is unclear. To analyse its physiological functions, we generated mice lacking NHE3 function. Homozygous mutant (Slc9a3-/-) mice survive, but they have slight diarrhoea and blood analysis revealed that they are mildly acidotic. HCO3- and fluid absorption are sharply reduced in proximal convoluted tubules, blood pressure is reduced and there is a severe absorptive defect in the intestine. Thus, compensatory mechanisms must limit gross perturbations of electrolyte and acid-base balance. Plasma aldosterone is increased in NHE3-deficient mice, and expression of both renin and the AE1 (Slc4a1) Cl-/HCO3- exchanger mRNAs are induced in kidney. In the colon, epithelial Na+ channel activity is increased and colonic H+,K+-ATPase mRNA is massively induced. These data show that NHE3 is the major absorptive Na+/H+ exchanger in kidney and intestine, and that lack of the exchanger impairs acid-base balance and Na+-fluid volume homeostasis.

Mice lacking TGF-β3 exhibit an incompletely penetrant failure of the palatal shelves to fuse leading to cleft palate. The defect appears to result from impaired adhesion of the apposing medial edge epithelia of the palatal shelves and subsequent elimination of the mid-line epithelial seam. No craniofacial abnormalities were observed. This result demonstrates that TGF-β3 affects palatal shelf fusion by an intrinsic, primary mechanism rather than by effects secondary to craniofacial defects.Members of the transforming growth factor-β (TGF-β) gene family have biological activities that control cell proliferation, migration and differentiation, regulation of extracellular matrix deposition and epithelial-mesenchymal transformation [1][2][3] . Mammals contain three highly conserved isoforms of TGF-β, termed TGF-β1, TGF-β2 and TGF-β3, which display distinctive, although at times overlapping, spatial and temporal expression patterns [4][5][6] . Previous studies suggested that TGF-β3 may play a crucial role during palatogenesis 7-9 , Meckel's cartilage formation 10 , cardiac morphogenesis 11 , mammary gland development 12 and wound healing 13 . Other tissues expressing TGF-β3 in significant levels are cartilage, bone, brain and lung [4][5][6]14 .In mammalian palatogenesis apposition of the palatal shelves, adhesion of the medial edge epithelia (MEE) and subsequent elimination of the epithelial seam lead to a seamless mesenchymal shelf separating the oral and nasal cavities 15 . In vitro organ culture studies indicate that TGF-β1 and TGF-β2 accelerate palatal shelf fusion 16,17 and that antisense oligodeoxynucleotides or neutralizing antibodies to TGF-β3, but not to TGF-β1 or TGF-β2, block the fusion process 9 . We have now created mice deficient in TGF-β3, and show that this factor has a role in palatal shelf fusion by means of an intrinsic, primary mechanism and not by effects secondary to craniofacial morphometrics. A comparison of this defect to the inflammatory disorder of TGF-β1-deficient mice [18][19][20][21] Mutation of TGF-β3 in ES cellsThe TGF-β3 gene was mutated in ES cells (Fig. 1a) by replacing exon 6, the first full exon encoding sequences of the active domain of the protein, with the neomycin-resistance gene from pMC1neo 22 . Diagnostic Southern blots of the clone I98 indicated that the locus was successfully targeted; the proper genomic regions flanking both sides of the target site remained intact (Fig. 1b). Probing with a neo-gene probe indicated that there was only one integration site (not shown). Consequently, only the TGF-β3 locus has been disrupted. RT-PCR analysis of whole 11.5- (Fig. 1c) and 15.5-day embryos (not shown) indicated no TGF-β3 expression in homozygous mutant embryos, and revealed no significant change in the expression of TGF-β1 or TGF-β2 in the absence of TGF-β3. Cleft palate in TGF-β3 null mutantsThe targeted ES cell clone I98 was used to produce chi-maeric mice, which were mated with CF-1, C57BL/6 or 129/Sv mice. Heterozygous offspring showed no apparent phenotype. Interc...

Phospholamban is the regulator of the Ca'+-ATPase in cardiac sarcoplasmic reticulum (SR), and it has been suggested to be an important determinant in the inotropic responses of the heart to 8-adrenergic stimulation. To determine the role of phospholamban in vivo, the gene coding for this protein was targeted in murine embryonic stem cells, and mice deficient in phospholamban were generated. The phospholamban-deficient mice showed no gross developmental abnormalities but exhibited enhanced myocardial performance without changes in heart rate. The time to peak pressure and the time to half-relaxation were significantly shorter in phospholamban-deficient mice compared with their wild-type homozygous littermates as assessed in work-performing mouse heart preparations under identical venous returns, afterloads, and heart rates. The first derivatives of intraventricular pressure (±dP/dt) were also significantly elevated, and this was associated with an increase in the affinity of the SR Ca +-ATPase for Ca`in the phospholamban-deficient hearts. Baseline levels of these parameters in the phospholamban-deficient hearts were equal to those observed in hearts of wild-type littermates maximally stimulated with the (-agonist isoproterenol. These findings indicate that phospholamban acts as a critical repressor of basal myocardial contractility and may be the key phosphoprotein in mediating the heart's contractile responses to f-adrenergic agonists. (Circ Res. 1994; 75:401-409.) Key Words * phospholamban * gene targeting . sarcoplasmic reticulum * cardiac contractility * l3-agonists C ardiac 8-adrenergic stimulation is associated with increases in the force of contraction and in the rates of rise and fall of force. These changes are mediated by increases in cAMP levels, which lead to phosphorylation of key regulatory proteins that may act as effectors of the adrenergic stimulation. One of these phosphoproteins is phospholamban, the regulator of the Ca`+-ATPase in cardiac sarcoplasmic reticulum (SR). Dephosphorylated phospholamban is an inhibitor of the Ca2`-ATPase activity, and phosphorylation relieves this inhibition.' The inhibition has been suggested to involve physical direct interaction between the two proteins,23 followed by conformational changes in the SR Ca2`-ATPase.

We show that fibroblast growth factor 2 (FGF2) and FGF receptors are transiently expressed by cells of the pseudostratified ventricular epithelium (PVE) during early neurogenesis. A single microinjection of FGF2 into cerebral ventricles of rat embryos at E15.5 increased the volume and total number of neurons in the adult cerebral cortex by 18% and 87%, respectively. Microinjection of FGF2 by the end of neurogenesis, at E20.5, selectively increased the number of glia. Mice lacking the FGF2 gene had fewer cortical neurons and glia at maturity. BrdU studies in FGF2-microinjected and FGF2-null animals suggested that FGF2 increases the proportion of dividing cells in the PVE without affecting the cell-cycle length. Thus, FGF2 increases the number of rounds of division of cortical progenitors.