Pod-1, a mesoderm-specific basic-helix-loop-helix protein expressed in mesenchymal and glomerular epithelial cells in the developing kidney.

Basic-helix-loop-helix (bHLH) proteins are transcriptional regulatory proteins that govern cell fate determination and differentiation in a variety of tissues. We have identified a novel bHLH protein, named Pod-1, that belongs to a recently-described subfamily of bHLH proteins that have essential roles in the embryonic development of mesodermal tissues. In the adult human and mouse, Pod-1 was most highly expressed in the kidney, lung and heart. In developing mouse embryos, Pod-1 was selectively expressed in mesenchymal cells at sites of epithelial-mesenchymal interaction in the kidney, lung, intestine and pancreas. Pod-1 was also expressed in visceral glomerular epithelial cells (podocytes) in the kidney, and its expression coincided with the onset of podocyte differentiation. The expression of Pod-1 in embryonic kidney explants was inhibited using antisense oligonucleotides. Inhibition of Pod-1 expression resulted in decreased mesenchymal cell condensation around the ureteric bud and a 40% decrease in ureteric branching. Pod-1 is the first tissue-restricted basic-helix-loop-helix protein that has been identified in the developing kidney where it may play a role in the regulation of morphogenetic events.

A conserved Hox axis in the mouse and human female reproductive system: late establishment and persistent adult expression of the Hoxa cluster genes.

The mammalian female reproductive system arises from the uniform paramesonephric duct. The molecular mechanisms that establish differential development along this axis are unknown. We determined the pattern and timing of genes of the Hoxa axis in the development of the Müllerian tract. Hoxa-9, Hoxa-10, Hoxa-11, and Hoxa-13 are all expressed along the length of the paramesonephric duct in the embryonic mouse. After birth, a spatial Hox axis is established, corresponding to the postnatal differentiation of this organ system in the mouse. Hoxa-9 is expressed in the fallopian tubes, Hoxa-10 in the uterus, Hoxa-11 in the uterus and uterine cervix, and Hoxa-13 in the upper vagina. This expression pattern follows the paradigm of spatial colinearity but is a novel exception to temporal colinearity that has been considered typical of Hox genes. These genes remain expressed in the adult mouse and are expressed in the same pattern in the human. The female reproductive system undergoes dramatic structural and functional changes during the estrous cycle and in pregnancy, retaining a high degree of developmental plasticity. The late establishment of a Hox axis and persistent expression of Hox genes in the adult may play an important role in preserving this plasticity.

A unique variant of a homeobox gene related to Drosophila cut is expressed in mouse testis.

Mammalian homologues of the Drosophila cut homeobox gene encode transcriptional repressors that are involved in tissue-specific and developmental gene regulation. We examined the expression of a murine cut homologue (Cux-1) in the adult mouse. In many somatic tissues, Cux-1 was expressed as a 13-kb transcript. However, the highest expression of Cux-1 was in the testis, where a unique 2.4-kb splice variant was identified. Less abundant transcripts of 5 kb, 6.5 kb, and 8.5 kb were also detected only in the testis. The nucleotide sequence of the 2.4-kb Cux-1 transcript was identical to the 13-kb transcript in the region of overlap, but the testis-specific transcript encoded a truncated protein that contained only one Cut repeat in addition to the Cut-related homeodomain. Studies of mice homozygous for the atrichosis (at/at) mutation suggested that the 2.4-kb transcript was expressed in germ cells in the testis. In situ hybridization verified that Cux-1 was transiently expressed in post-meiotic germ cells at the round spermatid stage. Immunoblot analysis of nuclear extracts showed that the testis-specific Cux-1 transcripts encoded a 55-kDa protein. These results demonstrate that multiple products of a cut-related homeobox gene are expressed in the testis. The highly restricted pattern of expression of Cux-1 in the testis suggests that it may be involved in regulation of postmeiotic gene expression.

Expression of a cut-related homeobox gene in developing and polycystic mouse kidney.

Cut is a diverged homeobox gene that is essential for normal development of the Malpighian tubules in Drosophila melanogaster. Homologues of Drosophila cut that encode transcriptional repressors have been identified in several mammalian species and cell lineages. We examined the expression of a murine cut homologue (named Cux-1) in the developing mouse using Northern blot analysis and in situ hybridization. At 12.5 d.p.c. and 13.5 d.p.c., Cux-1 was highly expressed in a subset of embryonic tissues, including the developing metanephros. Within the metanephros, Cux-1 was expressed in the nephrogenic zone including both mesenchymal cells (uninduced and condensed mesenchyme) and epithelial cells (ureteric buds, renal vesicles, S-shaped bodies). During later stages of nephrogenesis, Cux-1 was down-regulated such that there was minimal expression in mature glomeruli and tubules. In addition, Cux-1 was detected in the mesonephros, mesonephric duct, and bladder. Expression of Cux-1 was also examined in polycystic kidneys from C57BL/6J-cpk/ cpk mice. At 21 days of age, Cux-1 was highly expressed in cyst epithelium of polycystic kidneys but was minimally expressed in kidneys from phenotypically normal littermates. These results demonstrate that a cut-related homeobox gene is expressed in the developing kidney and urinary tract of the mouse. Expression of Cux-1 in the kidney is inversely related to degree of cellular differentiation. Cux-1 may encode a transcriptional repressor that inhibits terminally differentiated gene expression during early stages of nephrogenesis.

Carboxy terminal sequence and synthesis of rat kidney laminin gamma 1 chain.

We used antibodies against mouse Englebreth-Holm-Swarm (EHS) tumor laminin to screen a newborn rat kidney lambda gt11 expression library and isolated three overlapping cDNA clones, termed 2b-11 (401 bp), 10-b7 (779 bp), and 2a (2,095 bp). DNA sequence analysis identified these cDNAs as encoding much of the carboxy terminal domain I/II of laminin gamma 1 chain (formerly referred to as B2e), and 1436 bp of the 3' untranslated region. In situ hybridization of fetal (E15) rat sections localized laminin gamma 1 chain mRNA primarily to meninges of the brain, auditory and peripheral nerve fibers, gastrointestinal system, and developing lung airway epithelium. Intense hybridization was also found in early nephric structures and glomeruli of fetal kidneys. In kidneys of three-day-old rats, hybridization persisted over early nephric figures, developing glomeruli, and collecting ducts, but considerably less hybridization was seen over tubules. On Northern blots of neonatal kidney RNA, the three cDNA clones hybridized to two species of 7.5 and 5.5 kb, suggesting that developing rat kidney laminin gamma 1 mRNAs are processed using two different polyadenylation signals.

Cloning, embryonic expression, and alternative splicing of a murine kidney-specific Na-K-Cl cotransporter.

A full-length cDNA encoding the murine renal Na-K-Cl cotransporter (NKCC2) was cloned using library screening and anchored polymerase chain reaction. The deduced protein sequence contained 1,095 amino acids and was 93.5% identical to rabbit NKCC2 and 97.6% identical to rat BSC1. Two potential sites of phosphorylation by adenosine 3',5'-cyclic monophosphate-dependent protein kinase and seven potential sites of phosphorylation by protein kinase C, which were previously identified in the rabbit and rat sequences, were phylogenetically conserved in the mouse. The expression of NKCC2 in the mouse was examined with Northern blot analysis and in situ hybridization. Expression of NKCC2 was kidney specific in both adult and embryonic mice. In the developing metanephros, NKCC2 was induced at 14.5 days post coitus and was expressed in distal limbs of immature loops of Henle but was absent from the ureteric bud, S-shaped bodies, and earlier nephrogenic structures. Similar to the rabbit, isoforms of NKCC2 that differed in the sequence of a 96-bp segment were identified in the mouse. In situ hybridization revealed that the isoforms exhibited different patterns of expression in the mature thick ascending limb of the loop of Henle as follows: isoform F was most highly expressed in the inner stripe of outer medulla, isoform A was most highly expressed in the outer stripe of the outer medulla, and isoform B was most highly expressed in the cortical thick ascending limb. To verify that the isoforms were generated by alternative splicing of mutually exclusive cassette exons, genomic clones encoding murine NKCC2 were characterized. Cassette exons were identified that corresponded to each of the three isoforms and were flanked by consensus splice donor and acceptor sequences.

Quantitation and localization of laminin A, B1, and B2 chain RNA transcripts in developing kidney.

To quantitate expression of laminin A, B1, and B2 chains during kidney development, we prepared riboprobes of similar size (318-365 nucleotides) and carried out scanning densitometry of Northern blots from newborn mouse and rat kidneys. Much higher expression of B1 than A or B2 mRNAs was observed with an A-to-B1-to-B2 ratio of approximately 1:8:4 in mice and approximately 1:12:1 in rats. In addition to the 9.5-kb laminin A chain transcript, we also observed specific hybridization of the A chain probe to approximately 6-kb band in both newborn mice and rats. To localize these mRNAs, newborn rat kidneys were sieved to obtain separate glomerular and tubule fractions. Northern blots from these samples showed that most of the B1, B2, and 9.5-kb laminin A chain mRNAs were in glomerular fractions, whereas tubules contained more of the 6-kb A chain band. Analyses of poly(A)+ mRNA from newborn and adult kidneys showed severalfold decreases in adult RNA encoding the three laminin chains and the alpha 1-chain type IV collagen. However, only a 20% decline from newborn levels was seen for heparan sulfate proteoglycan (HSPG) core protein message. These quantitative results provide additional evidence that different laminin isoforms are present in separate kidney basement membranes and that HSPG core protein synthesis remains at relatively high levels in adult kidneys.