Cytokeratins 8 and 19 in the mouse placental development.

To investigate the expression and biological roles of cytokeratin 19 (K19) in development and in adult tissues, we inactivated the mouse K19 gene (Krt1-19) by inserting a bacterial beta-galactosidase gene (lacZ) by homologous recombination in embryonic stem cells, and established germ line mutant mice. Both heterozygous and homozygous mutant mice were viable, fertile, and appeared normal. By 7.5-8.0 days post coitum (dpc), heterozygous mutant embryos expressed lacZ in the notochordal plate and hindgut diverticulum, reflecting the fact that the notochord and the gut endoderm are derived from the axial mesoderm-originated cells. In the adult mutant, lacZ was expressed mainly in epithelial tissues. To investigate the possible functional cooperation and synergy between K19 and K8, we then constructed compound homozygous mutants, whose embryos died approximately 10 dpc. The lethality resulted from defects in the placenta where both K19 and K8 are normally expressed. As early as 9. 5 dpc, the compound mutant placenta had an excessive number of giant trophoblasts, but lacked proper labyrinthine trophoblast or spongiotrophoblast development, which apparently caused flooding of the maternal blood into the embryonic placenta. These results indicate that K19 and K8 cooperate in ensuring the normal development of placental tissues.

Disruption of ClC-3, a chloride channel expressed on synaptic vesicles, leads to a loss of the hippocampus.

Several plasma membrane chloride channels are well characterized, but much less is known about the molecular identity and function of intracellular Cl- channels. ClC-3 is thought to mediate swelling-activated plasma membrane currents, but we now show that this broadly expressed chloride channel is present in endosomal compartments and synaptic vesicles of neurons. While swelling-activated currents are unchanged in mice with disrupted ClC-3, acidification of synaptic vesicles is impaired and there is severe postnatal degeneration of the retina and the hippocampus. Electrophysiological analysis of juvenile hippocampal slices revealed no major functional abnormalities despite slightly increased amplitudes of miniature excitatory postsynaptic currents. Mice almost lacking the hippocampus survive and show several behavioral abnormalities but are still able to acquire motor skills.

Idiopathic weight reduction in mice deficient in the high-mobility-group transcription factor Sox8.

Sox8, Sox9, and Sox10 constitute subgroup E within the Sox family of transcription factors. Many Sox proteins are essential regulators of development. Sox9, for instance, is required for chondrogenesis and male sex determination; Sox10 plays key roles in neural crest development and peripheral gliogenesis. The function of Sox8 has not been studied so far. Here, we generated mice deficient in this third member of subgroup E. In analogy to the case for the related Sox9 and Sox10, we expected severe developmental defects in these mice. Despite strong expression of Sox8 in many tissues, including neural crest, nervous system, muscle, cartilage, adrenal gland, kidney, and testis, homozygous mice developed normally in utero, were born at Mendelian frequencies, and were viable. A substantial reduction in weight was observed in these mice; however, this reduction was not attributable to significant structural deficits in any of the Sox8-expressing tissues. Because of frequent coexpression with either Sox9 or Sox10, the mild phenotype of Sox8-deficient mice might at least in part be due to functional redundancy between group E Sox proteins.

Placental failure in mice lacking the mammalian homolog of glial cells missing, GCMa.

The GCM family of transcription factors consists of Drosophila melanogaster GCM, an important regulator of gliogenesis in the fly, and its two mammalian homologs, GCMa and GCMb. To clarify the function of these mammalian homologs, we deleted GCMa in mice. Genetic ablation of murine GCMa (mGCMa) is embryonic lethal, with mice dying between 9.5 and 10 days postcoitum. At the time of death, no abnormalities were apparent in the embryo proper. Nervous system development, in particular, was not impaired, as might have been expected in analogy to Drosophila GCM. Instead, placental failure was the cause of death. In agreement with the selective expression of mGCMa in labyrinthine trophoblasts, mutant placentas did not develop a functional labyrinth layer, which is necessary for nutrient and gas exchange between maternal and fetal blood. Only a few fetal blood vessels entered the placenta, and these failed to thrive and branch normally. Labyrinthine trophoblasts did not differentiate. All other layers of the placenta, including spongiotrophoblast and giant cell layer, formed normally. Our results indicate that mGCMa plays a critical role in trophoblast differentiation and the signal transduction processes required for normal vascularization of the placenta.

Complete genomic structure of the CLCN6 and CLCN7 putative chloride channel genes(1).

The CLC family of voltage-gated chloride channels comprises nine members in mammals. CLCN6 and CLCN7 belong to a novel, poorly characterized subbranch of this family. We investigated the genomic organization of the human CLCN6 gene, as well as the murine CLCN6 and CLCN7 genes. The human and murine CLCN6 genes both consist of 23 exons and share a nearly identical genomic structure. The coding region of mouse CLCN7 is composed of 25 exons. Comparison of the genomic organization of CLCN6 and CLCN7 genes shows that just eight introns are located at corresponding cDNA positions. Moreover, no significant gene structure homology to other members of the CLC family could be detected indicating a great structural diversity of mammalian CLC genes.

Cloning, structural analysis and mapping of the mouse selenocysteine tRNA([Ser]Sec) gene (Trsp).

The tRNA([Ser]Sec) molecule mediates the synthesis of selenoproteins by incorporating selenocysteine into specific UGA codons upon translation of mRNAs that encode selenocysteine-containing proteins. The mouse gene encoding tRNA([Ser]Sec) (Trsp) was isolated from a genomic library and sequenced. The mouse sequence is colinear with its tRNA product, and contains a C to T transition relative to the homologous genes in other vertebrates except rat. Transcriptional control motifs found 5' to the tRNA coding region included a TATA element, a PSE element and an SPH motif which is associated with an octamer motif. A Northern hybridization analysis showed highest expression in the testis, followed by thymus, spleen, kidney, ovary, brain, liver, heart and skeletal muscle. Surprisingly, the expression level was lowest in embryonic stem cells. These results suggest a tissue-specific transcriptional control. Using restriction fragment length variants (RFLVs) in interspecific backcross mice between Mus musculus (C3H strain) and Mus spretus, the Trsp gene was mapped to the proximal region of mouse Chr 7, cosegregating with octamer-binding transcription factor-2 (Otf2).

Early embryonic lethality caused by targeted disruption of the mouse selenocysteine tRNA gene (Trsp).

Selenoprotein biosynthesis is mediated by tRNASec, which inserts selenocysteine at UGA codons in a complex, context-specific manner. This opal suppressor serves in the conversion of serine to selenocysteine as well. The mouse tRNASec gene (Trsp) maps to a proximal segment of chromosome 7. We constructed mice carrying a targeted deletion of the Trsp gene. The heterozygous mutants were viable, fertile, and appeared normal. Although the level of tRNASec was reduced to about 50%-80% of the wild type in most organs, one of the selenoproteins, glutathione peroxidase, remained unaffected in the levels of its mRNA, protein, and enzyme activity, indicating that the haploid amount of tRNASec is not limiting in its biosynthesis. In contrast, the homozygous mutants died shortly after implantation, and the embryos were resorbed before 6.5 days post coitum. When the preimplantation embryos were placed in culture, however, the trophoectoderm cells showed outgrowths and the inner cell mass cells of the homozygous embryos were able to proliferate. These results indicate that Trsp expression is essential for early development of the embryo, and its lack causes peri-implantation lethality. However, the lethality does not appear to be due to a cell-autonomous function of tRNASec.

ClC-5 Cl- -channel disruption impairs endocytosis in a mouse model for Dent's disease.

Dent's disease is an X-linked disorder associated with the urinary loss of low-molecular-weight proteins, phosphate and calcium, which often leads to kidney stones. It is caused by mutations in ClC-5, a renal chloride channel that is expressed in endosomes of the proximal tubule. Here we show that disruption of the mouse clcn5 gene causes proteinuria by strongly reducing apical proximal tubular endocytosis. Both receptor-mediated and fluid-phase endocytosis are affected, and the internalization of the apical transporters NaPi-2 and NHE3 is slowed. At steady state, however, both proteins are redistributed from the plasma membrane to intracellular vesicles. This may be caused by an increased stimulation of luminal parathyroid hormone (PTH) receptors owing to the observed decreased tubular endocytosis of PTH. The rise in luminal PTH concentration should also stimulate the hydroxylation of 25(OH) vitamin D3 to the active hormone. However, this is counteracted by a urinary loss of the precursor 25(OH) vitamin D3. The balance between these opposing effects, both of which are secondary to the defect in proximal tubular endocytosis, probably determines whether there will be hypercalciuria and kidney stones.

Male germ cells and photoreceptors, both dependent on close cell-cell interactions, degenerate upon ClC-2 Cl(-) channel disruption.

The functions of some CLC Cl(-) channels are evident from human diseases that result from their mutations, but the role of the broadly expressed ClC-2 Cl(-) channel is less clear. Several important functions have been attributed to ClC-2, but contrary to these expectations ClC-2-deficient mice lacked overt abnormalities except for a severe degeneration of the retina and the testes, which led to selective male infertility. Seminiferous tubules did not develop lumina and germ cells failed to complete meiosis. Beginning around puberty there was a massive death of primary spermatocytes and later also of spermatogonia. Tubules were filled with abnormal Sertoli cells, which normally express ClC-2 in patches adjacent to germ cells. In the retina, photoreceptors lacked normal outer segments and degenerated between days P10 and P30. The current across the retinal pigment epithelium was severely reduced at P36. Thus, ClC-2 disruption entails the death of two cell types which depend on supporting cells that form the blood-testes and blood-retina barriers. We propose that ClC-2 is crucial for controlling the ionic environment of these cells.

Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man.

Chloride channels play important roles in the plasma membrane and in intracellular organelles. Mice deficient for the ubiquitously expressed ClC-7 Cl(-) channel show severe osteopetrosis and retinal degeneration. Although osteoclasts are present in normal numbers, they fail to resorb bone because they cannot acidify the extracellular resorption lacuna. ClC-7 resides in late endosomal and lysosomal compartments. In osteoclasts, it is highly expressed in the ruffled membrane, formed by the fusion of H(+)-ATPase-containing vesicles, that secretes protons into the lacuna. We also identified CLCN7 mutations in a patient with human infantile malignant osteopetrosis. We conclude that ClC-7 provides the chloride conductance required for an efficient proton pumping by the H(+)-ATPase of the osteoclast ruffled membrane.