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Smooth muscle cells are of key importance for the proper functioning of different visceral organs including those of the urogenital system. In the mouse ureter, the two transcriptional regulators TSHZ3 and SOX9 are independently required for initiation of smooth muscle differentiation from uncommitted mesenchymal precursor cells. However, it has remained unclear whether TSHZ3 and SOX9 act independently or as part of a larger regulatory network. Here, we set out to characterize the molecular function of TSHZ3 in the differentiation of the ureteric mesenchyme. Using a yeast-two-hybrid screen, we identified SOX9 as an interacting protein. We show that TSHZ3 also binds to the master regulator of the smooth muscle program, MYOCD, and displaces it from the coregulator SRF, thereby disrupting the activation of smooth muscle specific genes. We found that the initiation of the expression of smooth muscle specific genes in MYOCD-positive ureteric mesenchyme coincides with the down regulation of Sox9 expression, identifying SOX9 as a possible negative regulator of smooth muscle cell differentiation. To test this hypothesis, we prolonged the expression of Sox9 in the ureteric mesenchyme in vivo. We found that Sox9 does not affect Myocd expression but significantly reduces the expression of MYOCD/SRF-dependent smooth muscle genes, suggesting that down-regulation of Sox9 is a prerequisite for MYOCD activity. We propose that the dynamic expression of Sox9 and the interaction between TSHZ3, SOX9 and MYOCD provide a mechanism that regulates the pace of progression of the myogenic program in the ureter.

Ureteric contractions propel foetal urine from the kidney to the urinary bladder. Here, we show that mouse ureteric smooth muscle cell (SMC) precursors express the transcription factor teashirt 3 (TSHZ3), and that Tshz3-null mutant mice have congenital hydronephrosis without anatomical obstruction. Ex vivo, the spontaneous contractions that occurred in proximal segments of wild-type embryonic ureter explants were absent in Tshz3 mutant ureters. In vivo, prior to the onset of hydronephrosis, mutant proximal ureters failed to express contractile SMC markers, whereas these molecules were detected in controls. Mutant embryonic ureters expressed Shh and Bmp4 transcripts as normal, with appropriate expression of Ptch1 and pSMAD1/5/8 in target SM precursors, whereas myocardin, a key regulator for SMC differentiation, was not expressed in Tshz3-null ureters. In wild-type embryonic renal tract explants, exogenous BMP4 upregulated Tshz3 and myocardin expression. More interestingly, in Tshz3 mutant renal tract explants, exogenous BMP4 did not improve the Tshz3 phenotype. Thus, Tshz3 is required for proximal ureteric SMC differentiation downstream of SHH and BMP4. Furthermore, the Tshz3 mutant mouse model of ;functional' urinary obstruction resembles congenital pelvi-ureteric junction obstruction, a common human malformation, suggesting that TSHZ, or related, gene variants may contribute to this disorder.

teashirt (tsh) encodes a zinc-finger protein that is thought to be part of a network that contributes to regionalization of the Drosophila embryo and establishes the domains of Hox protein function. tsh and the Hox gene Sex combs reduced (Scr) are essential to establish the identity of the first thoracic segment. We used the development of the first thoracic segment as a paradigm for Scr dependent regional morphological distinctions. In this specific context, we asked whether Tsh protein could have a direct influence on Scr activity. Here we present evidence that Tsh interacts directly with Scr and this interaction depends in part on the presence of a short domain located in the N-terminal half of Teashirt called "acidic domain". In vivo, expression of full length Tsh can rescue the tsh null phenotype throughout the trunk whereas Tsh lacking the Scr interacting domain rescues all the trunk defects except in the prothorax. We suggest this provides insights into the mechanism by which Tsh, in concert with Scr, specifies the prothoracic identity.

The teashirt gene encodes a protein with three widely spaced zinc finger motifs that is crucial for specifying trunk identity in Drosophila embryos. Here, we describe a gene called tiptop, which encodes a protein highly similar to Teashirt. We have analyzed the expression patterns and functions of these two genes in the trunk of the embryo. Initially, teashirt and tiptop expressions are detected in distinct domains; teashirt in the trunk and tiptop in parts of the head and tail. In different mutant situations, we show that, in the trunk and head, they repress each other's expression. Unlike teashirt, we found that deletion of tiptop is homozygous viable and fertile. However, embryos lacking both gene activities display a more severe trunk phenotype than teashirt mutant embryos alone. Ectopic expression of either gene produces an almost identical phenotype, indicating that Teashirt and Tiptop have, on the whole, common activities. We conclude that Teashirt and Tiptop repress each other's expression and that Teashirt has a crucial role for trunk patterning that is in part masked by ectopic expression of Tiptop.

We have carried out a genetic screen designed to isolate regulators of teashirt expression. One of these regulators is the Grunge gene, which encodes a protein with motifs found in human arginine-glutamic acid dipeptide repeat, Metastasis-associated-like and Atrophin-1 proteins. Grunge is the only Atrophin-like protein in Drosophila, whereas several exist in humans. We provide evidence that Grunge is required for the proper regulation of teashirt but also has multiple activities in fly development. First, Grunge is crucial for correct segmentation during embryogenesis via a failure in the repression of at least four segmentation genes known to regulate teashirt. Second, Grunge acts positively to regulate teashirt expression in proximoventral parts of the leg. Grunge has other regulatory functions in the leg, including the patterning of ventral parts along the entire proximodistal axis and the proper spacing of bristles in all regions.

InDrosophila imaginal discs, pattern formation requires the activity of three positional information systems, antero-posterior (A/P), dorso-ventral (D/V) and proximo-distal (P/D). Three genes,Decapentaplegic, Distal-less androtund (rn), involved in pattern formation along the P/D axis have been characterized. Thern gene is required in a sub-distal region, localized at a similar position along the P/D axis in all appendages; it encodes two major transcripts, m1.7 and m5.3, both expressed in the central region of all the major imaginal discs. The present study of these transcripts in severalrn mutant favours m5.3 as encodingrn morphogenetic function in the imaginal discs. The fine characterization of its distribution partitions all major imaginal discs in domains along the P/D axis. The ventral and dorsal discs appear to be similarly but not identically organized: two P/D domains are evident in the wing and haltere discs whilst the leg and antenna discs appear to be composed of at least three. We also show that m5.3 is sex-regulated in the genital disc and thatrn function is required for proper development of a sub-distal structure of the female genitalia. This suggests that the primordia of the female genitalia may be organized in a similar way to the other imaginal discs, and strongly supports the hypothesis thatrn function is specific to pattern formation along the P/D axis and that it may be involved in the establishment or maintenance of this pattern.

A chimeric Xdh gene was constructed in vitro, by recombining DNA sequences from the Dipterans Drosophila melanogaster and Calliphora vicina. The ry506 strain, an eye-colour mutant of Drosophila that is deficient for Xdh, was genetically transformed with the recombinant gene. Transformed flies with ry+ eye phenotype and increased resistance to purine were obtained, showing that the chimeric XDH is physiologically active in Drosophila. XDH activity was detected in crude extracts from transformed flies, yet at lower levels than in wild-type controls. The amounts of Xdh transcripts in the transformants were found to be 8 to 16% of the amount of wild-type ry mRNA, suggesting that Calliphora Xdh sequences may be relatively inefficient for mRNA production in Drosophila, or may produce unstable mRNA.

We have discovered a reporter gene insertion that is expressed in the trunk region of Drosophila embryos. Genetic and molecular details of a new regulatory gene neighboring the reporter gene insertion, which we call teashirt (tsh), are described. In situ hybridization of a tsh probe to embryos shows that this gene is expressed in a way similar to the reporter gene. Mutations of tsh show that the gene is required for normal development of the ventral trunk region of embryos, which correlates with the spatial expression of the gene in the anteroposterior axis but not in the dorsoventral axis. Sequencing of a tsh cDNA shows that the putative protein possesses three distantly spaced CX2CX12HX5H zinc finger motifs.

We used a molecular approach to search for maternally expressed genes in Drosophila melanogaster. The relative merits of differential and competition screens were analyzed in a series of reconstruction experiments using either purified phage plaques or derivative DNA sequences. In the course of this study, we isolated 5 clones whose RNA level varies during early embryogenesis. Three gastrula differential clones, b4, b8 and d3, are present in numerous copies in the genome; clone b4 hybridizes with the copia-like B104 repetitive sequence described by Scherer et al. We also isolated 2 maternally-expressed genes, not previously identified in either classical genetic or similarly molecular-based screens. These clones, b11 and d6, map at cytogenetic positions 98F and 4F respectively, on the polytene chromosome map.

In this article we describe some in vivo properties of a coldsensitive ribosomal mutant from Escherichia coli. The mutation affects the rplV gene which is the structural gene of ribosomal protein L22. Our work shows that at 22 degrees C, the biosynthesis of both ribosomal subunits and the maturation processing of 15S and 23S ribosomal RNA are impaired. Integration of our results in a general model of in vivo ribosomal assembly in E. coli is presented.