Distinct roles of Fgf8, Foxi1, Dlx3b and Pax8/2 during otic vesicle induction and maintenance in medaka

The development of the vertebrate inner ear is a complex process that has been investigated in several model organisms. In this work, we examined genetic interactions regulating early development of otic structures in medaka. We demonstrate that misexpression of Fgf8, Dlx3b and Foxi1 during early gastrulation is sufficient to produce ectopic otic vesicles. Combined misexpression strongly increases the appearance of this phenotype. By using a heat-inducible promoter we were furthermore able to separate the regulatory interactions among Fgf8, Foxi1, Dlx3b, Pax8 and Pax2 genes, which are active during different stages of early otic development. In the preplacodal stage we suggest a central position of Foxi1 within a regulatory network of early patterning genes including Dlx3b and Pax8. Different pathways are active after the placodal stage with Dlx3b playing a central role. There Dlx3b regulates members of the Pax-Six-Eya-Dach network and also strongly affects the early dorsoventral marker genes Otx1 and Gbx2.     

Role of the hindbrain in patterning the otic vesicle: a study of the zebrafish vhnf1 mutant

The vertebrate inner ear develops from an ectodermal placode adjacent to rhombomeres 4 to 6 of the segmented hindbrain. The placode then transforms into a vesicle and becomes regionalised along its anteroposterior, dorsoventral and mediolateral axes. To investigate the role of hindbrain signals in instructing otic vesicle regionalisation, we analysed ear development in zebrafish mutants for vhnf1, a gene expressed in the caudal hindbrain during otic induction and regionalisation. We show that, in vhnf1 homozygous embryos, the patterning of the otic vesicle is affected along both the anteroposterior and dorsoventral axes. First, anterior gene expression domains are either expanded along the whole anteroposterior axis of the vesicle or duplicated in the posterior region. Second, the dorsal domain is severely reduced, and cell groups normally located ventrally are shifted dorsally, sometimes forming a single dorsal patch along the whole AP extent of the otic vesicle. Third, and probably as a consequence, the size and organization of the sensory and neurogenic epithelia are disturbed. These results demonstrate that, in zebrafish, signals from the hindbrain control the patterning of the otic vesicle, not only along the anteroposterior axis, but also, as in amniotes, along the dorsoventral axis. They suggest that, despite the evolution of inner ear structure and function, some of the mechanisms underlying the regionalisation of the otic vesicle in fish and amniotes have been conserved.     

Mouse FGF15 is the ortholog of human and chick FGF19, but is not uniquely required for otic induction

The inner ear develops from an ectodermal placode that is specified by inductive signals from the adjacent neurectoderm and underlying mesoderm. In chick, fibroblast growth factor (Fgf)-19 is expressed in mesoderm underlying the presumptive otic placode, and human FGF19 induces expression of otic markers in a tissue explant containing neural plate and surface ectoderm. We show here that mouse Fgf15 is the sequence homolog of chick and human Fgf19/FGF19. In addition, we show that FGF15, like FGF19, is sufficient to induce expression of otic markers in a chick explant assay, suggesting that these FGFs are orthologs. Mouse embryos lacking Fgf15, however, do not have otic abnormalities at E9.5-E10.5, suggesting that Fgf15 is not uniquely required for otic induction or early patterning of the otocyst. To compare FGF15 and FGF19 signaling components and assess where signals potentially redundant with FGF15 might function, we determined the expression patterns of Fgf15 and Fgf19. Unlike Fgf19, Fgf15 is not expressed in mesoderm underlying the presumptive otic placode, but is expressed in the adjacent neurectoderm. Fgfr4, which encodes the likely receptor for both FGF19 and FGF15, is expressed in the neurectoderm of both species, and is also expressed in the mesoderm only in chick. These results suggest the hypotheses that during otic induction, FGF19 signals in either an autocrine fashion to the mesoderm or a paracrine fashion to the neurectoderm, whereas FGF15 signals in an autocrine fashion to the neurectoderm. Thus, the FGFs that signal to the neurectoderm are the best potential candidates for redundancy with FGF15 during mouse otic development.     

Comparison of the chromosomes of Triticum timopheevi with related wheats using the techniques of C-banding and in situ hybridization

Summary The chromosomes of the tetraploid wheats Triticum timopheevi (Genome AAGG) and T. araraticum (Genome AAGG) were C-banded at mitosis. The identity of the banded and unbanded chromosomes was then established by firstly making comparisons with the hexaploid species T. zhukovskyi which has the genome formula AAAAGG. Secondly, the meiotic pairing in F₁ hybrids between T. timopheevi and diploid wheats was examined by means of C-banding. The results showed that the banded chromosomes belonged to the G genome, while the unbanded chromosomes belonged to the A genome. Only one of the two pairs of satellited chromosomes had strong heterochromatic bands. The relationship between the genomes of T. timopheevi and T. dicoccum (Genome AABB) was then assessed at meiosis in hybrids between these species, using the techniques of C-banding and in situ hybridisation of a cloned ribosomal RNA gene probe. It was concluded that there were differences both in the amount and distribution of heterochromatin and also translocation differences between the species.     

Identification and comparisons of the yolk polypeptides and the genes which code for them in D. melanogaster sibling species

Summary The yolk proteins stored in Drosophila, oocytes for utilisation during embryogenesis are an ideal system for studying the regulation of gene expression during development. The 3 major polypeptides found in yolk in D. melanogaster are synthesised in the fat body and ovarian follicle cells and selectively accumulated by the oocyte during vitellogenesis. In order to understand more about their regulation and the mechanism of uptake, studies on other species are necessary.Three yolk polypeptides have previously been identified in the D. melanogaster sibling species (D. melanogaster, D. simulans, D. mauritiana, D. erecta, D. teissieri, D. orena and D. yakuba). In D. melanogaster three genes located on the X chromosome are known to code for these yolk polypeptides. in this study genomic Southern transfers and in situ hybridisation experiments were carried out on the sibling species. Using the three cloned yolk protein genes from D. melanogaster, homologous sequences could be detected in the sibling species. It is suggested that three yolk protein genes occur in each of these species, all being located on the X chromosome, and that two of the genes are very closely linked in these same species. Yolk protein gene-homologous DNA sequences have also been identified in two more distantly related species D. funebris and D. virilis.     

Analysis and in situ detection of cholecalcin messenger RNA (9000 Mr CaBP) in the uterus of the pregnant rat

Summary The molecular cloning of a cDNA fragment synthesised from rat duodenal mRNA coding for cholecalcin (calbindin), a 9000 Mr vitamin D-induced calcium-binding protein (CaBP), has been previously described. DNA/RNA hybridisation assays have been used to examine CaBP mRNA production in the uterine horns and duodena of pregnant (21 day) rats using the cloned CaBP cDNA. Northern hybridisation studies showed that the ³²P cDNA sequence hybridised to a single 500–600 nucleotide species in both the uterus and the duodenum, thus demonstrating identical CaBP mRNA processing in both tissues. Dot blot hybridisation studies showed that the CaBP mRNA concentration was greatest in the duodenum while that of the uterine horns was about 10% of the duodenal level. The observed differences in CaBP mRNA levels correlate well with the in vivo CaBP concentrations. In situ hybridisation histochemistry using ³H cDNA revealed that CaBP mRNA visualised by silver grains was found in all the parts of the endometrium and the myometrium. However, CaBP mRNA was more concentrated in the outer and inner muscular fibres and in the luminal cells of the endometrium than in the stroma cells. These results demonstrate that the CaBP gene is expressed in specific cells of the rat uterus.     

Chromosomal analysis of Nicotiana asymmetric somatic hybrids by dot blotting and in situ hybridization

Summary A species-specific, dispersed repetitive DNA sequence was cloned from Nicotiana plumbaginifolia and used in dot blots and in situ hybridizations to analyze asymmetric somatic hybrids of N. tabacum(+)kanamycin-resistant N. plumbaginifolia. Dot blot hybridization data, using the cloned, species-specific repetitive DNA as a probe, showed that some of the hybrids contain only 1%–5% N. plumbaginifolia DNA, whereas others contain 15%–25%. In situ hybridization of the probe to chromosome spreads showed that the extremely asymmetric hybrids retain a single N. plumbaginifolia chromosome; the hybrids with higher dot blot values were found to have 8 to 12 N. plumbaginifolia chromosomes and chromosome fragments. In situ hybridization also revealed translocations between N. plumbaginifolia and N. tabacum chromosomes in 3 of 8 hybrids studied. RFLP analysis using a 5S gene probe showed the presence of N. plumbaginifolia-specific 5S banding patterns in most hybrids examined, including those that retain only a single N. plumbaginifolia chromosome.     

Detection of multiple hormones and their mRNAs in human neuroblastoma cell line NB-1 using in situ hybridization,immunocytochemistry and radioimmunoassay

The production and secretion of multiple peptide hormones and tyrosine hydroxylase by the human neuroblastoma cell line NB-1 and the effects of dibutyryl cAMP (Bt₂cAMP) and phorbol esters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on them were investigated. The presence of messenger RNA_s (mRNAs) of vasoactive intestinal peptide (VIP)/peptide histidine methionine (PHM), preprotachykinin, and tyrosine hydroxylase was detectable in the cytoplasm of cultured NB-1 cells by in situ hybridization. Treatment with Bt₂cAMP and TPA markedly increased the number of cells immunoreactive to VIP, PHM, neuropeptide Y, Met-enkephalin, substance P and tyrosine hydroxylase and also the contents of VIP and Met-enkephalin in the culture medium. Bt₂cAMP and TPA induced morphological changes characteristic of endocrine differentiation, such as an increase in neuroendocrine granules and the development of rough endoplasmic reticulum and Golgi apparatus. The results indicated that treatment with Bt²cAMP and TPA induces the expression of multiple genes of peptide hormone and tyrosine hydroxylase and increases hormone production and secretion through morphological changes into endocrine cells.     

Design of species-specific oligonucleotide probes for the detection of Bacteroides and Parabacteroides by fluorescence in situ hybridization and their application to the analysis of mouse caecal Bacteroides-Parabacteroides microbiota

Aims: To develop species‐specific monitoring techniques for rapid detection of Bacteroides and Parabacteroides inhabiting the mouse intestine by fluorescence in situ hybridization. Methods and Results: The specificity of oligonucleotide probes was evaluated by fluorescence whole‐cell hybridization. Oligonucleotide probes specific for each species hybridized only with the target bacteria. Using these probes, caecal Bacteroides–Parabacteroides microbiota of conventional mice and specific pathogen‐free (SPF) mice from three different breeders were analysed. It was shown that Bacteroides acidifaciens Group‐1, Group‐2 and Group‐3 were dominant in conventional mice and SPF mice from two out of three breeders. Bacteroides vulgatus and Parabacteroides distasonis were detected in one of these two SPF breeding colonies in addition to Bact. acidifaciens. SPF mice of the remaining breeder harboured characteristic Bacteroides–Parabacteroides microbiota, consisting of Bacteroides sp. ASF519 and Bacteroides caccae. Conclusions: Bacteroides acidifaciens is the dominant and most typical species in the mouse Bacteroides–Parabacteroides microbiota. The Group‐3 was identified as a novel group and revealed to occupy a major niche together with Bact. acidifaciens Group‐1 and Group‐2. Significance and Impact of the Study: The species‐specific probe set developed in this study was the efficient tool for rapid detection of target bacterial groups inhabiting the mouse intestine. The results of this study provide important new information on the mouse Bacteroides–Parabacteroides community.     

Use of dual section mRNA in situ hybridisation/immunohistochemistry to clarify gene expression patterns during the early stages of nephron development in the embryo and in the mature nephron of the adult mouse kidney

The kidney is the most complex organ within the urogenital system. The adult mouse kidney contains in excess of 8,000 mature nephrons, each of which can be subdivided into a renal corpuscle and 14 distinct tubular segments. The histological complexity of this organ can make the clarification of the site of gene expression by in situ hybridisation difficult. We have defined a panel of seven antibodies capable of identifying the six stages of early nephron development, the tubular nephron segments and the components of the renal corpuscle within the embryonic and adult mouse kidney. We have analysed in detail the protein expression of Wt1, Calb1 Aqp1, Aqp2 and Umod using these antibodies. We have then coupled immunohistochemistry with RNA in situ hybridisation in order to precisely identify the expression pattern of different genes, including Wnt4, Umod and Spp1. This technique will be invaluable for examining at high resolution, the structure of both the developing and mature nephron where standard in situ hybridisation and histological techniques are insufficient. The use of this technique will enhance the expression analyses of genes which may be involved in nephron formation and the function of the mature nephron in the mouse.