Ontogeny of the GnRH systems in zebrafish brain: in situ hybridization and promoter-reporter expression analyses in intact animals

The ontogeny of two gonadotropin-releasing-hormone (GnRH) systems, salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II), was investigated in zebrafish (Danio rerio). In situ hybridization (ISH) first detected sGnRH mRNA-expressing cells at 1 day post-fertilization (pf) anterior to the developing olfactory organs. Subsequently, cells were seen along the ventral olfactory organs and the olfactory bulbs, reaching the terminal nerve (TN) ganglion at 5–6 days pf. Some cells were detected passing posteriorly through the ventral telencephalon (10–25 days pf), and by 25–30 days pf, sGnRH cells were found in the hypothalamic/preoptic area. Continuous documentation in live zebrafish was achieved by a promoter-reporter expression system. The expression of enhanced green fluorescent protein (EGFP) driven by the sGnRH promoter allowed the earlier detection of cells and projections and the migration of sGnRH neurons. This expression system revealed that long leading processes, presumably axons, preceded the migration of the sGnRH neuron somata. cGnRH-II mRNA expressing cells were initially detected (1 day pf) by ISH analysis at lateral aspects of the midbrain and later on (starting at 5 days pf) at the midline of the midbrain tegmentum. Detection of red fluorescent protein (DsRed) driven by the cGnRH-II promoter confirmed the midbrain expression domain and identified specific hindbrain and forebrain cGnRH-II-cells that were not identified by ISH. The forebrain DsRed-expressing cells seemed to emerge from the same site as the sGnRH-EGFP-expressing cells, as revealed by co-injection of both constructs. These studies indicate that zebrafish TN and hypothalamic sGnRH cell populations share a common embryonic origin and migratory path, and that midbrain cGnRH-II cells originate within the midbrain. This study was supported by the US-Israel Bi-national Agricultural Research and Development (BARD) Foundation (grant 3428-03).     

XHas2 activity is required during somitogenesis and precursor cell migration in Xenopus development

In vertebrates, hyaluronan biosynthesis is regulated by three transmembrane catalytic enzymes denoted Has1, Has2 and Has3. We have previously cloned the Xenopus orthologues of the corresponding genes and defined their spatiotemporal distribution during development. During mammalian embryogenesis, Has2 activity is known to be crucial, as its abrogation in mice leads to early embryonic lethality. Here, we show that, in Xenopus, morpholino-mediated loss-of-function of XHas2 alters somitogenesis by causing a disruption of the metameric somitic pattern and leads to a defective myogenesis. In the absence of XHas2, early myoblasts underwent apoptosis, failing to complete their muscle differentiation programme. XHas2 activity is also required for migration of hypaxial muscle cells and trunk neural crest cells (NCC). To approach the mechanism whereby loss of HA, following XHas2 knockdown, could influence somitogenesis and precursor cell migration, we cloned the orthologue of the primary HA signalling receptor CD44 and addressed its function through an analogous knockdown approach. Loss of XCD44 did not disturb somitogenesis, but strongly impaired hypaxial muscle precursor cell migration and the subsequent formation of the ventral body wall musculature. In contrast to XHas2, loss of function of XCD44 did not seem to be essential for trunk NCC migration, suggesting that the HA dependence of NCC movement was rather associated with an altered macromolecular composition of the ECM structuring the cells' migratory pathways. The presented results, extend our knowledge on Has2 function and, for the first time, demonstrate a developmental role for CD44 in vertebrates. On the whole, these data underlie and confirm the emerging importance of cell-ECM interactions and modulation during embryonic development.     

A genetic map of melon highly enriched with fruit quality QTLs and EST markers, including sugar and carotenoid metabolism genes

A genetic map of melon enriched for fruit traits was constructed, using a recombinant inbred (RI) population developed from a cross between representatives of the two subspecies of Cucumis melo L.: PI 414723 (subspecies agrestis) and ‘Dulce’ (subspecies melo). Phenotyping of 99 RI lines was conducted over three seasons in two locations in Israel and the US. The map includes 668 DNA markers (386 SSRs, 76 SNPs, six INDELs and 200 AFLPs), of which 160 were newly developed from fruit ESTs. These ESTs include candidate genes encoding for enzymes of sugar and carotenoid metabolic pathways that were cloned from melon cDNA or identified through mining of the International Cucurbit Genomics Initiative database (http://www.icugi.org/). The map covers 1,222 cM with an average of 2.672 cM between markers. In addition, a skeleton physical map was initiated and 29 melon BACs harboring fruit ESTs were localized to the 12 linkage groups of the map. Altogether, 44 fruit QTLs were identified: 25 confirming QTLs described using other populations and 19 newly described QTLs. The map includes QTLs for fruit sugar content, particularly sucrose, the major sugar affecting sweetness in melon fruit. Six QTLs interacting in an additive manner account for nearly all the difference in sugar content between the two genotypes. Three QTLs for fruit flesh color and carotenoid content were identified. Interestingly, no clear colocalization of QTLs for either sugar or carotenoid content was observed with over 40 genes encoding for enzymes involved in their metabolism. The RI population described here provides a useful resource for further genomics and metabolomics studies in melon, as well as useful markers for breeding for fruit quality.     

Combined association,segregation and aggregation analysis on case-control family data

Recently genetic epidemiologists have begun using case-control family study designs to investigate the role of genetic and environmental risk factors in disease etiology. The objective of these studies is to assess the association of environmental factors with the disease trait; to characterize the disease genes using segregation analysis; and to quantify the residual familial aggregation after controlling for environmental and genetic factors. Typically these objectives are achieved by conducting separate studies and analysis. This paper describes an estimating equation based approach for a combined association, segregation and aggregation analysis on data from case-control family studies. Simulations indicate that the method performs well in a variety of settings. The method is illustrated using simulated family history data made available to participants in a recent Genetic Analysis Workshop.     

Antiproliferative activity of CCN3: involvement of the C-terminal module and post-translational regulation

Previous work had suggested that recombinant CCN3 was partially inhibiting cell proliferation. Here we show that native CCN3 protein secreted into the conditioned medium of glioma transfected cells indeed induces a reduction in cell proliferation. Large amounts of CCN3 are shown to accumulate both cytoplasmically and extracellularly as cells reach high density, therefore highlighting new aspects on how cell growth may be regulated by CCN proteins. Evidence is presented establishing that the amount of CCN3 secreted into cell culture medium is regulated by post-translational proteolysis. As a consequence, the production of CCN3 varies throughout the cell cycle and CCN3 accumulates at the G2/M transition of the cycle. We also show that CCN3-induced inhibition of cell growth can be partially reversed by specific antibodies raised against a C-terminal peptide of CCN3. The use of several clones expressing various portions of CCN3 established that the CT module of CCN3 is sufficient to induce cell growth inhibition.     

Chromosome aberration and environmental physical activity: Down syndrome and solar and cosmic ray activity, Israel, 1990–2000

The possibility that environmental effects are associated with chromosome aberrations and various congenital pathologies has been discussed previously. Recent advances in the collection and computerization of data make studying these potential associations more feasible. The aim of this study was to investigate a possible link between the number of Down syndrome (DS) cases detected prenatally or at birth yearly in Israel over a 10-year period compared with the levels of solar and cosmic ray activity 1 year before the detection or birth of each affected child. Information about 1,108,449 births was collected for the years 1990–2000, excluding 1991, when data were unavailable. A total of 1,310 cases of DS were detected prenatally or at birth—138 in the non-Jewish community and 1,172 in the Jewish population. Solar activity indices—sunspot number and solar radio flux 2,800 MHz at 10.7 cm wavelength for 1989–1999—were compared with the number of DS cases detected. Pearson correlation coefficients (r) and their probabilities (P) were established for the percentage of DS cases in the whole population. There was a significant inverse correlation between the indices of solar activity and the number of cases of DS detected—r=–0.78, P=0.008 for sunspot number and r=–0.76, P=0.01 for solar flux. The possibility that cosmophysical factors inversely related to solar activity play a role in the pathogenesis of chromosome aberrations should be considered. We have confirmed a strong trend towards an association between the cosmic ray activity level and the incidence of DS.