Laurent Fasano

The gene teashirt is required for the development of Drosophila embryonic trunk segments and encodes a protein with widely spaced zinc finger motifs

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.

Chaetognath phylogenomics: a protostome with deuterostome-like development

Traditional textbook phylogeny splits bilaterians into protostomes and deuterostomes according to whether their mouth derives from the blastopore or not. This scheme has been largely confirmed by small subunit ribosomal RNA (SSU) molecular phylogeny. However, some phyla, such as the lophphorate phyla Phoronida and Brachiopoda as well as the Chaetognatha exhibit classical deuterostome embryological features such as formation of the mesoderm from the gut (enterocoely) and secondary opening of the mouth.

Three putative murine Teashirt orthologues specify trunk structures in Drosophila in the same way as the Drosophila teashirt gene.

Drosophila teashirt (tsh) functions as a region-specific homeotic gene that specifies trunk identity during embryogenesis. Based on sequence homology, three tsh-like (Tsh) genes have been identified in the mouse. Their expression patterns in specific regions of the trunk, limbs and gut raise the possibility that they may play similar roles to tsh in flies. By expressing the putative mouse Tsh genes in flies, we provide evidence that they behave in a very similar way to the fly tsh gene. First, ectopic expression of any of the three mouse Tsh genes, like that of tsh, induces head to trunk homeotic transformation. Second, mouse Tsh proteins can rescue both the homeotic and the segment polarity phenotypes of a tsh null mutant. Third, following ectopic expression, the three mouse Tsh genes affect the expression of the same target genes as tsh in the Drosophila embryo. Fourth, mouse Tsh genes, like tsh, are able to induce ectopic eyes in adult flies. Finally, all Tsh proteins contain a motif that recruits the C-terminal binding protein and contributes to their repression function. As no other vertebrate or fly protein has been shown to induce such effects upon ectopic expression, these results are consistent with the idea that the three mouse Tsh genes are functionally equivalent to the Drosophila tsh gene when expressed in developing Drosophila embryos.

Characterization of the two zebrafish orthologues of the KAL-1 gene underlying X chromosome-linked Kallmann syndrome

The gene underlying X chromosome-linked Kallmann syndrome, KAL-1, has been identified for several years, yet its role in development is still poorly understood. In order to take advantage of the zebrafish as a model in developmental genetics, we isolated the two KAL-1 orthologues, kal1.1 and kal1.2, in this species. Comparison of deduced protein sequences with the human one shows 75.5 and 66.5% overall homology, respectively. The most conserved domains are the whey acidic protein-like domain and the first of four fibronectin-like type III repeats. However, kal1.2 putative protein lacks the basic C-terminal domain (20 residues) found in kal1.1 and KAL-1. The expressions of kal1.1 and kal1.2 were studied in the embryo between 6 and 96 hours post fertilization using whole-mount in situ hybridization. Although a few structures express both genes, kal1.1 and kal1.2 expression patterns are largely non-overlapping. Taken together, these patterns match fairly well those previously reported for human KAL-1 and chicken kal1. As regards the olfactory system, kal1.1 is expressed, from 37 h.p.f. onward, in the presumptive olfactory bulbs, whereas kal1.2 transcript is only detected, from 48 h.p.f., in the epithelium of the nasal cavity. The relevance of the zebrafish as an animal model for studying both the function of KAL-1 in normal development and the developmental failure leading to the olfactory defect in Kallmann syndrome, is discussed.

Teashirt 3 Regulates Development of Neurons Involved in Both Respiratory Rhythm and Airflow Control

Neonatal breathing in mammals involves multiple neuronal circuits, but its genetic basis remains unclear. Mice deficient for the zinc finger protein Teashirt 3 (TSHZ3) fail to breathe and die at birth. Tshz3 is expressed in multiple areas of the brainstem involved in respiration, including the pre-Bötzinger complex (preBötC), the embryonic parafacial respiratory group (e-pF), and cranial motoneurons that control the upper airways. Tshz3 inactivation led to pronounced cell death of motoneurons in the nucleus ambiguus and induced strong alterations of rhythmogenesis in the e-pF oscillator. In contrast, the preBötC oscillator appeared to be unaffected. These deficits result in impaired upper airway function, abnormal central respiratory rhythm generation, and altered responses to pH changes. Thus, a single gene, Tshz3, controls the development of diverse components of the circuitry required for breathing.

Hox gene survey in the chaetognath Spadella cephaloptera: evolutionary implications

We present the isolation of six Hox genes in the chaetognath Spadella cephaloptera. We identified one member of the paralogy group 3, four median genes and a mosaic gene that shares features of both median and posterior classes (SceMedPost). Several hypotheses may account for the presence of a mosaic Hox gene in this animal. Here we propose that SceMedPost may represent an ancestral gene, which has not diverged totally into a posterior or a median one. This hypothesis has interesting implications for the reconstruction of the evolutionary history of Hox genes and suggests that Chaetognatha lineage divergence could predate the deuterostome/protostome split. Such a phylogenetic position is considered in the light of their embryological and morphological characters.

Ureter Myogenesis: Putting Teashirt into Context

After the basic shape of the mammalian ureter is established, its epithelia mature and a coat of smooth muscle cells differentiate around nascent urothelia. The ureter actively propels tubular fluid from the renal pelvis to the bladder, and this peristalsis, which starts in the fetal period, requires coordinated smooth muscle contraction. Teashirt-3 (Tshz3) is expressed in smooth muscle cell precursors that form the wall of the forming mammalian ureter. The Teashirt gene family was first identified in Drosophila where Teashirt (Tsh) protein acts as a transcription factor directing embryonic anterior-posterior patterning and leg and eye development. In fly embryonic renal tubules, Tsh is expressed in mesodermally derived stellate cells intercalating between principal cells, and a paralogue, tiptop, is expressed in forming tubules. Teashirt is a component of several gene networks in flies and it is notable that similar networks control mammalian renal tract development. Null mutation of Tshz3 in mice leads to failure of functional muscularization in the top of the ureter and this is followed by congenital hydronephrosis. A signaling pathway can be envisaged, starting with sonic hedgehog secreted by the nascent ureteric urothelium and ending with ureteric smooth muscle cell differentiation, with Tshz3 downstream of bone morphogenetic protein 4 and upstream of myocardin and smooth muscle cell contractile protein synthesis. The phenotype of Tshz3 mutant mice resembles that of human congenital pelviureteric junction obstruction, and we suggest these individuals may have mutations of genes encoding molecules in the differentiation pathway mediated by Tshz3.

Teashirt-3, a Novel Regulator of Muscle Differentiation, Associates with BRG1-associated Factor 57 (BAF57) to Inhibit Myogenin Gene Expression

In adult muscles and under normal physiological conditions, satellite cells are found in a quiescent state but can be induced to enter the cell cycle by signals resulting from exercise, injury-induced muscle regeneration, or specific disease states. Once activated, satellite cells proliferate, self-renew, and differentiate to form myofibers. In the present study, we found that the zinc finger-containing factor Teashirt-3 (TSHZ3) was expressed in quiescent satellite cells of adult mouse skeletal muscles. We showed that following treatment with cardiotoxin TSHZ3 was strongly expressed in satellite cells of regenerating muscles. Moreover, immunohistochemical analysis indicated that TSHZ3 was expressed in both quiescent and activated satellite cells on intact myofibers in culture. TSHZ3 expression was maintained in myoblasts but disappeared with myotube formation. In C2C12 myoblasts, we showed that overexpression of Tshz3 impaired myogenic differentiation and promoted the down-regulation of myogenin (Myog) and up-regulation of paired-box factor 7 (Pax7). Moreover, knockdown experiments revealed a selective effect of Tshz3 on Myog regulation, and transcriptional reporter experiments indicated that TSHZ3 repressed Myog promoter. We identified the BRG1-associated factor 57 (BAF57), a subunit of the SWI/SNF complex, as a partner of TSHZ3. We showed that TSHZ3 cooperated with BAF57 to repress MYOD-dependent Myog expression. These results suggest a novel mechanism for transcriptional repression by TSHZ3 in which TSHZ3 and BAF57 cooperate to modulate MyoD activity on the Myog promoter to regulate skeletal muscle differentiation.

Analysis of TSHZ2 and TSHZ3 genes in congenital pelvi-ureteric junction obstruction

BACKGROUND Congenital pelvi-ureteric junction obstruction (PUJO) affects 0.3% of human births. It may result from aberrant smooth muscle development in the renal pelvis, resulting in hydronephrosis. Mice that are null mutant for the Teashirt3 (Tshz3) gene exhibit congenital PUJO with defective smooth muscle differentiation and absent peristalsis in the proximal ureter. METHODS Given the phenotype of Tshz3 mutant mice, we considered that Teashirt genes, which code for a family of transcription factors, might represent candidate genes for human PUJO. To evaluate this possibility, we used in situ hydridization to analyse the three mammalian Tshz genes in mouse embryonic ureters and determined whether TSHZ3 was expressed in the human embryonic ureter. TSHZ2 and TSHZ3 were sequenced in index cases with non-syndromic PUJO. RESULTS Tshz2 and Tshz3 genes were detected in mouse ureters and TSHZ3 was expressed in the human embryonic renal pelvis. Direct sequencing of TSHZ2 and TSHZ3 did not identify any mutations in an initial cohort of 48 PUJO index cases, excluding these genes as a major cause of this condition. A polymorphic missense change (E469G) in TSHZ3 was identified at a residue highly conserved throughout evolution in all Teashirt proteins, although subsequently no significant difference between the E469G allele frequency in Albanian and Macedonian PUJO index cases (3.2%) versus 633 control individuals (1.7%) was found (P = 0.18). CONCLUSIONS Mutations in TSHZ2 and TSHZ3 are not a major cause of PUJO, at least in Albanian and Macedonian populations. Expression of these genes in the human fetal ureter emphasizes the importance of analysing these genes in other groups of patients with renal tract malformations.

Expression of a reporter gene resembles that of its neighbour: an insertion in the hairy gene of Drosophila

SummaryRandom insertions of a promotor fused to a reporter gene, such as Lac-Z, reveal regulatory sequences that confer temporal and spatial patterns of gene expression in eukaryotes. These patterns may reflect the activity of a neighbouring gene and thus lead to the isolation of new genes essential for normal development. Here, we demonstrate that this hypothesis is true for an insertion into the well characterized segmentation gene, hairy, in Drosophila. The insertion is homozygous lethal and fails to complement other hairy alleles, giving the phenotype described for hairy mutations. The insertion is located at 66D on the polytene chromosome map, is within 300–600 bp 5′ to the first hairy exon, and is orientated in the same sense (5′-3′) as the hairy transcription unit. Expression of β-galactosidase (β-gal), deriving from the insertion, follows closely the spatio-temporal patterns of expression of hairy gene product during embryogenesis. In addition, other sites of β-galactosidase expression are shown in the third larval instar stage and in the adult ovary. The results show that some insertions, giving restricted patterns of reporter gene expression, will reflect the temporo-spatial activity of a nearby gene.