Marino Bortolussi

BMP signalling regulates anteroposterior endoderm patterning in zebrafish

In vertebrates, the embryonic dorsoventral asymmetry is regulated by the bone morphogenetic proteins (Bmp) activity gradient. In the present study, we have used dorsalized swirl (bmp2b) and ventralized chordino (chordin) zebrafish mutants to investigate the effects of dorsoventral signalling on endoderm patterning and on the differentiation and positioning of its derivatives. Alterations of dorsoventral Bmp signalling do not perturb the induction of endodermal precursors, as shown by normal amounts of cells expressing cas and sox17 in swirl and chordino gastrulae, but affect dramatically the expression pattern of her5, a regulator of endoderm anteroposterior patterning in zebrafish. In particular, increased levels of Bmp signalling in chordino gastrulae are associated with a markedly reduced her5 expression domain, that may be abolished by injecting bmp2b mRNA. Conversely, in swirl mutants, lacking Bmp2b, the her5 expression domain is expanded. Thus, a gradient of Bmp2b signalling defines the extension of the her5 expression domain at gastrulation and the allocation of anterior endodermal precursors. A balanced Bmp2b signalling is also required for the normal development of the pancreas, as shown by the sharp reduction of the pancreatic primordium in swirl embryos and its expansion in chordino mutants. In the latter, at 3 days post-fertilization, the increased Bmp signalling does not compromise the endocrine/exocrine pancreas compartmentalization, but the right/left positioning of the pancreas and liver is randomized. Our results suggest that by regulating the expression of her5, the Bmp2b/Chordin gradient directs the anteroposterior patterning of endoderm in zebrafish embryos.

Early appearance of pancreatic hormone-expressing cells in the zebrafish embryo.

Adult pancreatic islets comprise four cell types, alpha, beta, delta and PP, expressing glucagon, insulin, somatostatin and pancreatic-polypeptide, respectively, arising from cell lineages whose relationships during endocrine pancreas differentiation are still uncertain [Edlund, 1998. Diabetes 47, 1817-1823]. As zebrafish (Danio rerio) represents an attractive vertebrate model to study mutants affecting pancreatic organogenesis [Pack et al., 1996. Development 123, 321-328], we have investigated the expression patterns of islet hormones in zebrafish embryos, from the 16-somite (17 h) to 48-h stages, by whole-mount in situ hybridization and immunofluorescence. Results showed that in the zebrafish pancreatic primordium (a) insulin is the first hormone gene to be expressed, and (b) somatostatin colocalizes with insulin while glucagon-expressing cells, since their appearance, are distinct from insulin- or insulin/somatostatin-expressing cells. Notably, both somatostatin and glucagon, but not insulin, are first expressed in extrapancreatic regions.

Sertoli and Leydig cell numbers and gonadotropin receptors in rat testis from birth to puberty

SummaryIn testes of rats from 2 to 60 days of age, we examined the number of Sertoli cells (SC) and Leydig cells (LC) as well as the binding of radioiodinated gonadotropins to frozen sections and homogenates. The number of SC per testis increased only during the first 2 postnatal weeks, whereas that of LC was stable up to days 7–10 and increased thereafter. The uptake of 125I-labelled human follicle-stimulating hormone (125I-FSH) to frozen sections was confined to sex cords or seminiferous tubules, while that of 125I-labelled human choriogonadotropin (125I-hCG) matched the distribution of LC in the interstitium. High affinity receptors for FSH and hCG were found in homogenates at all stages studied. The number of FSH receptors per testis increased steadily, whereas that of hCG receptors was low until days 7–10 and rose afterwards. Thus, SC in rat testis appear to proliferate in the presence of fetal LC during the first 2 postnatal weeks and to differentiate concomitantly with the emergence of the adult LC generation after day 10. The complement of FSH receptors in SC remains constant as they proliferate and increases after day 21 as they differentiate. The hCG receptor number is relatively fixed in each LC generation, being higher in adult compared to fetal LC.

Prep1.1 has essential genetic functions in hindbrain development and cranial neural crest cell differentiation

In this study we analysed the function of the Meinox gene prep1.1 during zebrafish development. Meinox proteins form heterotrimeric complexes with Hox and Pbx members, increasing the DNA binding specificity of Hox proteins in vitro and in vivo. However, a role for a specific Meinox protein in the regulation of Hox activity in vivo has not been demonstrated. In situ hybridization showed that prep1.1 is expressed maternally and ubiquitously up to 24 hours post-fertilization (hpf), and restricted to the head from 48 hpf onwards. Morpholino-induced prep1.1 loss-of-function caused significant apoptosis in the CNS. Hindbrain segmentation and patterning was affected severely, as revealed by either loss or defective expression of several hindbrain markers (foxb1.2/mariposa, krox20, pax2.1 and pax6.1), including anteriorly expressed Hox genes (hoxb1a, hoxa2 and hoxb2), the impaired migration of facial nerve motor neurons, and the lack of reticulospinal neurons (RSNs) except Mauthner cells. Furthermore, the heads of prep1.1 morphants lacked all pharyngeal cartilages. This was not caused by the absence of neural crest cells or their impaired migration into the pharyngeal arches, as shown by expression of dlx2 and snail1, but by the inability of these cells to differentiate into chondroblasts. Our results indicate that prep1.1 has a unique genetic function in craniofacial chondrogenesis and, acting as a member of Meinox-Pbc-Hox trimers, it plays an essential role in hindbrain development.

Differential expression of two somatostatin genes during zebrafish embryonic development

We have identified the cDNAs of two new zebrafish preprosomatostatins, PPSS1 and PPSS3, in addition to the previously cloned PPSS2 (Argenton et al., 1999). PPSS1 is the orthologue of mammalian PPSSs, with a conserved C-terminal SS-14 sequence, PPSS2 is a divergent SS precursor and PPSS3 is a cortistatin-like prohormone. Using whole-mount in situ hybridisation, we have analysed the expression of PPSS1 and PPSS2 in zebrafish embryos up to 5 days post fertilisation. PPSS1 was expressed in the developing pancreas and central nervous system (CNS), whereas PPSS2 expression was exclusively pancreatic. In the CNS, PPSS1 was detected in several areas, in particular in the vagal motor nucleus and in cells that pioneer the tract of the postoptic commissure. PPSS1 was also expressed transiently in the telencephalon and spinal motor neurons. In all areas but the telencephalon PPSS1 was coexpressed with islet-1.

The binding of the RyR2 calcium channel to its gating protein FKBP12.6 is oppositely affected by ARVD2 and VTSIP mutations

Arrhythmogenic right ventricular dysplasia/cardiomyopathy type 2 (ARVD2, OMIM 600996) and stress-induced polymorphic ventricular tachycardia (VTSIP, OMIM 604772) are two cardiac diseases causing juvenile sudden death, both associated with mutations in the RyR2 calcium channel. By using a quantitative yeast two-hybrid system, we show that VTSIP- and ARVD2-associated point mutations influence positively and negatively, respectively, the binding of RyR2 to its gating protein FKBP12.6. These findings suggest that ARVD2 mutations increase RyR2-mediated calcium release to cytoplasm, while VTSIP mutations do not affect significantly cytosolic calcium levels, thereby explaining the clinical differences between the two diseases. The present two-hybrid system appears to be an efficient molecular tool to assay the binding of FKBP12s proteins to both cardiac RyR2 and skeletal muscle RyR1 isoforms, circumventing the full-length expression of this class of giant channels. We also provide evidence of the suitability of this system to test new drugs that target RyRs-FKBP12s interactions and do not affect yeast growth.

A histochemical study of the binding of 125I-HCG to the rat ovary throughout the estrous cycle.

SummaryChanges in the distribution of the in vitro uptake of 125I-HCG by the ovaries of adult rats were examined histochemically throughout the estrous cycle.Only in follicles wider than 500 μm, occurring mainly at diestrus and proestrus, could granulosa cells bind the labelled hormone. The labelling increased with follicular size and decreased in intensity from the peripheral granulosa cells inwards. No uptake occurred in the oocytes, in the cells of the cumulus oophorus nor in the granulosa cells of the atretic follicles.The binding capacity of the newly-formed corpora lutea of estrus was less than that of preovulatory follicles. The uptake of 125I-HCG by corpora lutea during the first cycle reached its maximum at diestrus but fell sharply by proestrus. The uptake was patchy in the corpora lutea of the second cycle and not significant in the older ones.The uptake of 125I-HCG by thecae increased with follicular size and was greater in the thecae of atretic follicles than in the thecae of growing follicles of like size. There was a greater uptake in the last formed interstitial tissue than there was in older tissue.At proestrus, the uptake of 125I-HCG was unaffected by the LH surge at 18.00h but had decreased slightly at 24.00 h.The implications of these data in relation to the regulation of receptor sites, is discussed.

Expression analysis of jagged genes in zebrafish embryos

The interaction of transmembrane Delta and Jagged/Serrate ligands with Notch receptors on neighboring cells is critically involved in cell specification during development. In zebrafish, the early expression of delta but not of jagged genes has been investigated in some detail. We have analyzed the sequence and embryonic expression pattern of the three zebrafish genes jagged1a, jagged1b, and jagged2. These genes, whose transcripts are detectable by in situ hybridization from early somitogenesis, are widely and dynamically expressed in embryos. Coexpression is limited, however, to the notochord and lens (jagged1a and jagged1b) and to the otic vesicle and pronephros (jagged1b and jagged2). Conversely, jagged1a and jagged2, both widely expressed in the central nervous system, are not coexpressed. jagged2 is also detected in the epidermis, newly formed somites, pharyngeal pouches, and pancreatic exocrine anlage and jagged1b in otic placodes and cell clusters close to the pancreatic islet. The similarities of the expression patterns of jagged and delta genes in zebrafish suggest that the Jagged and Delta ligands are functionally redundant or required in specific combinations in many differentiation processes. Developmental Dynamics 233:638–645, 2005.

Autoradiographic study of the distribution of LH(HCG) receptors in the ovary of untreated and gonadotrophin-primed immature rats.

SummaryThe LH(HCG) receptors in the ovaries of immature rats which were either untreated, or primed with PMSG and HCG, have been studied with a histochemical method which has proved to be as effective as when earlier used in the rat testis. This method, which consists of the topical application of 125I-HCG to picric acid-formaldehyde (PAF) fixed frozen sections followed by autoradiography, is also suitable for quantitative studies on the distribution of receptors.In the ovary of the immature 26 days old rat, the LH(HCG) receptors are localized exclusively in the interstitial and thecal tissues.After PMSG treatment many receptors appear in the granulosa of the large antral follicles. These receptors are most numerous in the outer layers of cells and least numerous in the inner. At the same time there are fewer receptors in the thecal and interstitial cells which have undergone the process of luteinization.After PMSG and HCG treatment the newly formed corpora lutea have few receptors, but these become progressively more numerous on subsequent days.It is suggested that, in the rat, the luteinization of the ovarian LH-target cells is associated with an initial decrease in the number of their LH(HCG) receptors.

Analysis of beta cell proliferation dynamics in zebrafish

Among the different mechanisms invoked to explain the beta cell mass expansion during postnatal stages and adulthood, self-replication is being considered the major cellular event occurring both under physiological conditions and in regenerating pancreas after partial pancreactomy. Neogenesis, i.e. differentiation from pancreatic progenitors, has been demonstrated to act concurrently with beta cell replication during pancreatic regeneration. Both phenomena have been largely elucidated in higher vertebrates (mouse, rat and guinea pig), but an extensive description of beta cell dynamics in other animal models is currently lacking. We, therefore, explored in zebrafish the cellular origins of new beta cells in both adult and larval stages. By integrating the results from in vivo time lapse analysis and immunostaining, we provide a detailed reconstruction of the major processes involved in fish beta cell genesis and maintenance. Moreover, by establishing the selective ablation of proliferating beta cells, through the ganciclovir-HSVTK system, we could show that in larval stages self-replication is the main mechanism of beta cells expansion. Since the same mechanism of proliferation has been observed to occur during early and late life stages, we suggest that zebrafish larvae can be used as an alternative tool for an in vivo exploration and screening of new potential mitogens specifically targeting beta cells.