T-Box Genes and Developmental Gene Regulatory Networks in Ascidians.

Ascidians are invertebrate chordates with a biphasic life cycle characterized by a dual body plan that displays simplified versions of chordate structures, such as a premetamorphic 40-cell notochord topped by a dorsal nerve cord and postmetamorphic pharyngeal slits. These relatively simple chordates are characterized by rapid development, compact genomes and ease of transgenesis, and thus provide the opportunity to rapidly characterize the genomic organization, developmental function, and transcriptional regulation of evolutionarily conserved gene families. This review summarizes the current knowledge on members of the T-box family of transcription factors in Ciona and other ascidians. In both chordate and nonchordate animals, these genes control a variety of morphogenetic processes, and their mutations are responsible for malformations and developmental defects in organisms ranging from flies to humans. In ascidians, T-box transcription factors are required for the formation and specialization of essential structures, including notochord, muscle, heart, and differentiated neurons. In recent years, the experimental advantages offered by ascidian embryos have allowed the rapid accumulation of a wealth of information on the molecular mechanisms that regulate the expression of T-box genes. These studies have also elucidated the strategies employed by these transcription factors to orchestrate the appropriate spatial and temporal deployment of the numerous target genes that they control.

Ultrasonographic caput epididymis diameter is reduced in non-obstructive azoospermia compared with normozoospermia but is not predictive for successful sperm retrieval after TESE.

STUDY QUESTION: Is the ultrasonographic determination of the caput epididymis diameter predictive for sperm retrieval after testicular sperm extraction (TESE) in non-obstructive azoospermia (NOA)? SUMMARY ANSWER: Ultrasonographic determination of the caput epididymis diameter did not give any relevant clinical information in NOA and was not predictive for positive sperm retrieval after TESE. WHAT IS KNOWN ALREADY: The diameter of the caput epididymis in ultrasonography (US) has a diagnostic relevance in azoospermic men to correctly identify obstructive azoospermia; however, its clinical value in NOA is not yet determined. STUDY DESIGN, SIZE, DURATION: We performed a retrospective study of 100 azoospermic and 160 normozoospermic men attending a university infertility clinic. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were submitted to scrotal US to determine the mean value of bilateral testicular volumes (ml), the bilateral longitudinal caput diameter (mm) and the antero-posterior diameter of the corpus (mm) epididymis. The number of spermatozoa retrieved after TESE and the testicular histology of azoospermic men was obtained and the percentage of seminiferous tubules with elongated spermatids (%T) was used to classify cases with normal spermatogenesis (obstructive azoospermia) (OA) (n = 20; %T ≥ 80) or with NOA (n = 80; %T < 70). MAIN RESULTS AND THE ROLE OF CHANCE: The US testes volumes and caput diameters were reduced (P < 0.05) in NOA compared with OA and with normozoospermia, but the corpus values were not different. The caput diameter in the side submitted to biopsy was significantly reduced when germinal epithelium was absent (Sertoli cell only) (P < 0.05) and the lowest value of caput diameter was observed when the seminiferous epithelium and tubule lumen were absent (testicular hyalinosis). On the contrary, a total arrest of spermatogenesis at the first meiosis level, or a defect of spermiogenesis resulting in scattered elongated spermatids in each tubule, did not show a reduced diameter of caput epididymis compared with normozoospermia. The caput diameter did not show any difference between NOA patients with or without successful sperm retrieval at TESE. On the contrary testicular volume was significantly reduced in NOA patients with no sperm retrieval (P = 0.0037). The caput diameter was not correlated with the number of retrieved sperm, the serum level of follicle stimulating hormone, or with the percentage of tubules with elongated spermatids at histological analysis. LIMITATIONS, REASONS FOR CAUTION: The aetiology of NOA was not included in the statistical analysis due to the low rate of cases with a specific aetiology for a testicular failure. Larger studies should exclude the possibility that besides testicular histology, aetiology of NOA might influence the diameter of caput epididymis. Moreover, whether a reduced diameter of caput epididymis is only a result of a testicular pathologic phenotype or whether it may underscore a primitive dysfunction influencing the number of ejaculated spermatozoa is not yet determined. WIDER IMPLICATIONS OF THE FINDINGS: We reported that US diameter of the caput epididymis is reduced in cases of NOA but, in contrast with the testicular volume, it is independent of the completion of spermatogenesis and subsequent presence of spermatozoa in the epididymis. Therefore ultrasonographic determination of caput epididymis diameter is not predictive for positive sperm retrieval after TESE in cases of a primitive testicular failure. Our novel findings may help to define which reproducible parameters of scrotal US should be assessed in the work-up of male infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Ministero dell'Università e Ricerca (I) PRIN 2009. The authors declare no competing interest.

The role of laparoscopic myomectomy in women of reproductive age.

Laparoscopic surgery has gained wide popularity for the treatment of uterine fibroids in women of reproductive age. The aim of this study is to evaluate the safety of the present surgical technique in order to preserve fertility and to achieve a satisfactory uterine repair so as to obtain an uncomplicated, full-term pregnancy. Between March 1988 and April 2001, 1170 uterine myomata were laparoscopically removed in 635 patients. The number of myomata removed from each patient varied from one to nine. The main steps of the surgical technique are described. No serious complication occurred. All the myomata proved to be benign. A second look was performed in 121 patients, and in two cases adhesions were found. A total of 105 patients achieved pregnancy (one triplet and three twin) and 91 delivered. No uterine rupture or scar dehiscence was observed. Out of 148 patients who were infertile with one or more myomata larger than 30 mm, 74 achieved pregnancy, 63 spontaneously and 11 after IVF.

The regulation of forkhead/HNF-3beta expression in the Ciona embryo.

The Ciona forkhead/HNF-3beta gene (Ci-fkh) is expressed in the primary axial tissues of the developing tadpole, including the notochord, endoderm, and rudimentary floor plate of the CNS. In an effort to determine the basis for this complex pattern of expression we have conducted a detailed analysis of the Ci-fkh 5'-regulatory region. Different 5' sequences were attached to a lacZ reporter gene and analyzed in electroporated Ciona embryos. A short regulatory sequence (AS) located approximately 1.7 kb upstream of the transcribed region is shown to be essential for expression in all three axial tissues. The proximal 20 bp of the AS contains overlapping Snail repressor elements and a T-box motif. Deleting these sequences causes the loss of reporter gene expression in the endoderm, as well as expanded expression in the neural tube. These results suggest that a T-box gene such as Ci-VegTR activates Ci-fkh expression in the endoderm, while the Ci-Sna repressor excludes expression from the lateral ependymal cells and restricts the Ci-fkh pattern to the rudimentary floor plate in ventral regions of the neural tube. We also present evidence for Ci-fkh positive autofeedback, whereby the Ci-Fkh protein binds to critical activator sites within the Ci-fkh 5'-regulatory region and helps maintain high levels of expression. We discuss these results with respect to forkhead/HNF-3beta regulation in vertebrates.

Identification and developmental expression of three Distal-less homeobox containing genes in the ascidian Ciona intestinalis.

Several homeobox-containing genes related to Drosophila Distal-less (Dll) have been isolated from a wide variety of organisms and have been shown to function as developmental regulators. While in Drosophila only one Dll gene has been described so far, in Vertebrates many components of the Dlx multigenic family have been characterized. This suggests that, during the evolution of the Chordate phylum, the Dlx genes arose from an ancestral Dll/Dlx gene via gene duplication. We have previously reported the isolation of two Dll-related homeoboxes from the protochordate Ciona intestinalis, and described their clustered arrangement (Gene 156 (1995) 253). Here we present the detailed genomic organization and spatial-temporal expression of these two genes, Ci-Dll-A and Ci-Dll-B, and describe the isolation and characterization of another member of the ascidian family of Dll-related genes, which we tentatively named Ci-Dll-C.

Regulation of Ci-tropomyosin-like, a Brachyury target gene in the ascidian, Ciona intestinalis.

Brachyury is a sequence-specific transcriptional activator that is essential for notochord differentiation in a variety of chordates. In vertebrates, Brachyury is expressed throughout the presumptive mesoderm, but becomes restricted to the notochord at later stages of development. In ascidians, such as Ciona intestinalis, Brachyury is expressed exclusively in the notochord and does not exhibit an early pan-mesodermal pattern. Subtractive hybridization screens were recently used to identify potential Ciona Brachyury (Ci-Bra) target genes (Takahashi, H., Hotta, K., Erives, A., Di Gregorio, A., Zeller, R. W., Levine, M. and Satoh, N. (1999). Genes Dev. 13, 1519-1523). Of the genes that were identified in this screen, one corresponds to a new member of the tropomyosin superfamily, Ciona tropomyosin (Ci-trop). Here we show that Ci-trop is specifically expressed in the developing notochord beginning at gastrulation, and expression persists in the notochord during tailbud and tadpole stages. A 3 kb region of the Ci-trop 5'-flanking sequence was characterized via electroporation of lacZ fusion genes into fertilized Ciona eggs. A minimal, 114 bp enhancer was identified that is sufficient to direct the expression of a heterologous promoter in the notochord. DNA binding assays indicate that this enhancer contains two sets of low-affinity Brachyury half-sites, which are bound in vitro by a GST/Ci-Bra fusion protein. Deletion of the distal sites inactivates the notochord-specific staining pattern mediated by an otherwise normal Ci-trop/lacZ transgene. These results suggest that Ci-trop is a direct target gene of Ci-Bra and that Brachyury plays an immediate role in the cellular morphogenesis of the notochord.

Identification and developmental expression of Ci-msxb: a novel homologue of Drosophila msh gene in Ciona intestinalis.

We report the cloning and expression pattern of Ci-msxb the second Ciona intestinalis homeobox gene homologue to the Drosophila muscle segment homeobox (msh) gene. Northern blot analysis showed that transcripts appeared at gastrula stage, peaked in the early tailbud and decreased during the tailed stages. Whole mount in situ hybridization showed that the Ci-msxb expression first is detected at 110 cell-stage in the blastomeres that are precursors of different tissue (muscle, spinal cord, endodermal strand, brain, mesenchyme, pigmented cells and primordial pharynx). Transcript level declined in mesoderm cells after the completion of gastrulation, but mRNAs were still present in the folding neural plate during neurulation and in the pigmented cells. Later, at larval stage, transcripts were present around the otolith and ocellus, in a restricted part of the nervous system and in the primordial pharynx; the gene expression was conserved after metamorphosis in the juvenile.

Four-week nebulized beclomethasone dipropionate in stable COPD patients with exertional dyspnoea.

Twenty male outpatients with severe-but-stable chronic obstructive pulmonary disease handicapped by exertional dyspnoea (aged 69.7 +/- 5.68 yrs; forced expiratory volume in one second (FEV1) 1.02 +/- 0.18 L or 34.6 +/- 6.5% of the predicted value; forced vital capacity (FVC) 2.51 +/- 0.34 L; arterial oxygen tension (Pa,O2) 9.11 +/- 0.32 kPa (68.5 +/- 2.4 mmHg); arterial carbon dioxide tension (Pa,CO2) 5.20 +/- 0.23 kPa (39.1 +/- 1.7 mmHg)) completed a randomized double-blind crossover study to evaluate the effects of a 4-week regular treatment with inhaled beclomethasone dipropionate via nebulizers at a dosage of 2 mg twice daily. After active and placebo treatment, no peak expiratory flow rate variation in FEV1, FVC, rescue use of beta 2-agonists, exercise tolerance and dyspnoea was observed. In conclusion, a regular short-term treatment with nebulized beclomethasone dipropionate does not give any improvement in lung function or exercise capacity in severe-but-stable chronic obstructive pulmonary disease patients handicapped by exertional dyspnoea.

Brachyury downstream notochord differentiation in the ascidian embryo.

The ascidian tadpole represents the most simplified chordate body plan. It contains a notochord composed of just 40 cells, but as in vertebrates Brachyury is essential for notochord differentiation. Here, we show that the misexpression of the Brachyury gene (Ci-Bra) of Ciona intestinalis is sufficient to transform endoderm into notochord. Subtractive hybridization screens were conducted to identify potential Brachyury target genes that are induced upon Ci-Bra misexpression. Of 501 independent cDNA clones that were surveyed, 38 were specifically expressed in notochord cells. These potential Ci-Bra downstream genes appear to encode a broad spectrum of divergent proteins associated with notochord formation.

Suppressor of hairless activates brachyury expression in the Ciona embryo.

The Ciona Brachyury gene (Ci-Bra) is regulated, in part, by a 434-bp enhancer that mediates restricted expression in the notochord. Here we present evidence that a Ciona Suppressor of Hairless ¿Ci-Su(H)¿ protein functions as an activator of this enhancer. Point mutations that reduce the binding of a GST/Ci-Su(H) fusion protein in vitro diminish the expression of mutagenized Ci-Bra/lacZ transgenes in electroporated embryos. Overexpression of a Ci-Su(H) fusion protein containing the Drosophila Hairy repression domain interferes with notochord differentiation, producing mutant tadpoles with shortened tails. Expression of a constitutively activated Xotch receptor in the notochord, endoderm, and CNS also alters tail morphogenesis. These results suggest that a Notch-Su(H) pathway might participate in notochord differentiation in Ciona.

Acute respiratory failure due to gentamicin aerosolization.

We report a paradoxical episode of near-fatal bronchoconstriction which occurred in an adult bronchiectatic subject, with chronic Pseudomonas aeruginosa airways colonization, immediately after his first inhalation of a gentamicin solution. This adverse reaction may be due to the gentamicin itself, the physical properties of the solution, or preservatives in the (commercially available) gentamicin solution.

Ascidian embryogenesis and the origins of the chordate body plan.

For more than a century, ascidians have been a widely used system for classic embryological studies. Ascidians possess simple, well-defined cell-lineages, compact genomes, rapid development and world-wide distribution. Transgenic DNA can be introduced into developing embryos using simple electroporation methods. The ascidian larva represents the most simplified chordate body plan and provides a useful model for studying the molecular pathways underlying the morphogenesis and differentiation of the notochord and neural tube.

Developmental regulation and tissue-specific localization of calmodulin mRNA in the protochordate Ciona intestinalis.

A full-length cDNA encoding a highly conserved calmodulin was isolated from a cDNA library prepared from hatched larvae of the ascidian Ciona intestinalis. Sequence analysis has identified a 447 b.p. open reading frame, encoding a putative protein of 149 amino acid residues, with a predicted molecular weight of 16.8 kDa, showing 85-98% identity to known calmodulins. Northern blot analysis revealed a single transcript of about 0.8 kb in length, which was maternally expressed and progressively increased during development, until late tail-bud stage. Whole-mount in situ hybridizations, carried out on embryos at different stages of development, showed that starting from the neurula stage, the C. intestinalis calmodulin (Ci-CaM) expression became restricted to the neuroectoderm and that in larvae it was specifically detected in the nervous system.

Cihox5, a new Ciona intestinalis Hox-related gene, is involved in regionalization of the spinal cord.

In this paper we report the cloning, sequence and expression analysis of a new Ciona intestinalis hox gene. On the basis of sequence comparison with mammalian and Amphioxus homologues, we called this gene Cihox5. Northern blot analysis reveals a single transcript of about 1.3 kb in length, that is present from neurula until larva stage. Whole-mount in situ hybridization shows restricted expression of this gene in putative blood cells precursors and in a regional domain of the spinal chord. Expression in the spinal cord is attributed to ependymal cells. This result implies a role for this gene in primitive regionalization of spinal cord along the anteroposterior axis in the absence of neuronal bodies.

Dorsoventral patterning of the vertebrate neural tube is conserved in a protochordate.

The notochord and dorsal ectoderm induce dorsoventral compartmentalization of the vertebrate neural tube through the differential regulation of genes such as HNF-3beta, Pax3, Pax6 and snail. Here we analyze the expression of HNF-3beta and snail homologues in the ascidian, Ciona intestinalis, a member of the subphylum Urochordata, the earliest branch in the chordate phylum. A combination of in situ hybridization and promoter fusion analyses was used to demonstrate that the Ciona HNF-3beta homologue is expressed in the ventralmost ependymal cells of the neural tube, while the Ciona snail homologue is expressed at the junction between the invaginating neuroepithelium and dorsal ectoderm, similar to the patterns seen in vertebrates. These findings provide evidence that dorsoventral compartmentalization of the chordate neural tube is not an innovation of the vertebrates. We propose that precursors of the floor plate and neural crest were present in a common ancestor of both vertebrates and ascidians.

Ovarian pregnancy and operative laparoscopy: report of eight cases.

We report a series of eight cases of ovarian pregnancy treated by operative laparoscopy during the last 12 years. This rare ectopic pregnancy (2.6% of all extra-uterine pregnancies in our experience) is difficult to diagnose prior to surgery. Earlier diagnosis is now possible, owing to the availability of highly specific radioimmunoassay for human chorionic gonadotrophin and the development of transvaginal ultrasonography. Primary ovarian pregnancy is distinguished by some authors from distal tubal pregnancy, in which a secondary ovarian implantation is possible. The therapy is surgical and currently more conservative than in the past, because of improvement in operative laparoscopy. Laparoscopy allows a short hospital stay, less physical stress and a favourable cost-benefit ratio. Moreover, its low risk of adhesion formation is important with regard to reproductive prognosis: in the light of this, since the patients are generally young and desire future childbearing, laparoscopy may be the treatment of choice.

Cloning of ascidian homeobox genes provides evidence for a primordial chordate cluster.

In order to isolate genes important in controlling embryonic development in Tunicates, a genomic library from the ascidian Ciona intestinalis was screened with a degenerate oligodeoxyribonucleotide encoding the third helix of Antennapedia-type homeoboxes. Fourteen C. intestinalis homeobox genes, corresponding to several classes of homeodomains, have been identified. Five of the isolated homeoboxes show their highest homology to members of the Vertebrate HOX clusters. mRNAs for two of the isolated homeoboxes are present in unfertilized C. intestinalis eggs.

Cloning and characterization of a developmentally regulated sea urchin cDNA encoding glutamine synthetase.

A 2935-bp cDNA clone encoding glutamine synthetase (GS) was isolated from a cDNA library prepared from four-blastomere Paracentrotus lividus sea urchin embryos. The sequence consists of a 75-bp 5' untranslated region (5'-UTR) followed by a 1095-bp coding region corresponding to a 365-amino-acid (aa) protein, a 1747-bp 3'-UTR and a terminal 18-bp poly(A) tail. The encoded protein shows about 66% identical residues, as compared with human and lobster class-II GS. The sequence contains the Mn(2+)-binding aa and the highly conserved aa regions observed in other GS. Northern blot analyses show that the GS mRNA is present in the sea urchin egg and is developmentally regulated in the embryo.