Functional differentiation of bmp2a and bmp2b genes in zebrafish

Bone morphogenetic protein 2 plays an important role in the regulation of osteoblast proliferation and differentiation. Phylogenetic analysis showed that the bmp2 ortholog evolved from the same ancestral gene family in vertebrates and was duplicated in teleost, which were named bmp2a and bmp2b. The results of whole-mount in situ hybridization showed that the expression locations of bmp2a and bmp2b in zebrafish were different in different periods (24 hpf, 48 hpf, 72 hpf), which revealed potential functional differentiation between bmp2a and bmp2b. Phenotypic analysis showed that bmp2a mutations caused partial rib and vertebral deformities in zebrafish, while bmp2b^−/− embryos died massively after 12 hpf due to abnormal somite formation. We further explored the expression pattern changes of genes (bmp2a, bmp2b, smad1, fgf4, runx2b, alp) related to skeletal development at different developmental stages (20 dpf, 60 dpf, 90 dpf) in wild-type and bmp2a^−/− zebrafish. The results showed that the expression of runx2b in bmp2a^−/− was significantly downregulated at three stages and the expression of other genes were significantly downregulated at 90 dpf compared with wild-type zebrafish. The study revealed functional differentiation of bmp2a and bmp2b in zebrafish embryonic and skeletal development.     

Consequences of Lineage-Specific Gene Loss on Functional Evolution of Surviving Paralogs: ALDH1A and Retinoic Acid Signaling in Vertebrate Genomes

Genome duplications increase genetic diversity and may facilitate the evolution of gene subfunctions. Little attention, however, has focused on the evolutionary impact of lineage-specific gene loss. Here, we show that identifying lineage-specific gene loss after genome duplication is important for understanding the evolution of gene subfunctions in surviving paralogs and for improving functional connectivity among human and model organism genomes. We examine the general principles of gene loss following duplication, coupled with expression analysis of the retinaldehyde dehydrogenase Aldh1a gene family during retinoic acid signaling in eye development as a case study. Humans have three ALDH1A genes, but teleosts have just one or two. We used comparative genomics and conserved syntenies to identify loss of ohnologs (paralogs derived from genome duplication) and to clarify uncertain phylogenies. Analysis showed that Aldh1a1 and Aldh1a2 form a clade that is sister to Aldh1a3-related genes. Genome comparisons showed secondarily loss of aldh1a1 in teleosts, revealing that Aldh1a1 is not a tetrapod innovation and that aldh1a3 was recently lost in medaka, making it the first known vertebrate with a single aldh1a gene. Interestingly, results revealed asymmetric distribution of surviving ohnologs between co-orthologous teleost chromosome segments, suggesting that local genome architecture can influence ohnolog survival. We propose a model that reconstructs the chromosomal history of the Aldh1a family in the ancestral vertebrate genome, coupled with the evolution of gene functions in surviving Aldh1a ohnologs after R1, R2, and R3 genome duplications. Results provide evidence for early subfunctionalization and late subfunction-partitioning and suggest a mechanistic model based on altered regulation leading to heterochronic gene expression to explain the acquisition or modification of subfunctions by surviving ohnologs that preserve unaltered ancestral developmental programs in the face of gene loss.     

Control of cell migration in the development of the posterior lateral line: antagonistic interactions between the chemokine receptors CXCR4 and CXCR7/RDC1

Background  

The formation of the posterior lateral line of teleosts depends on the migration of a primordium that originates near the otic vesicle and moves to the tip of the tail. Groups of cells at the trailing edge of the primordium slow down at regular intervals and eventually settle to differentiate as sense organs. The migration of the primordium is driven by the chemokine SDF1 and by its receptor CXCR4, encoded respectively by the genes sdf1a and cxcr4b. cxcr4b is expressed in the migrating cells and is down-regulated in the trailing cells of the primordium. sdf1a is expressed along the path of migration. There is no evidence for a gradient of sdf1a expression, however, and the origin of the directionality of migration is not known.     

Distinct and Overlapping Functions of ptpn11 Genes in Zebrafish Development

The PTPN11 (protein-tyrosine phosphatase, non-receptor type 11) gene encodes SHP2, a cytoplasmic PTP that is essential for vertebrate development. Mutations in PTPN11 are associated with Noonan and LEOPARD syndrome. Human patients with these autosomal dominant disorders display various symptoms, including short stature, craniofacial defects and heart abnormalities. We have used the zebrafish as a model to investigate the role of Shp2 in embryonic development. The zebrafish genome encodes two ptpn11 genes, ptpn11a and ptpn11b. Here, we report that ptpn11a is expressed constitutively and ptpn11b expression is strongly upregulated during development. In addition, the products of both ptpn11 genes, Shp2a and Shp2b, are functional. Target-selected inactivation of ptpn11a and ptpn11b revealed that double homozygous mutants are embryonic lethal at 5–6 days post fertilization (dpf). Ptpn11a-/-ptpn11b-/- embryos showed pleiotropic defects from 4 dpf onwards, including reduced body axis extension and craniofacial defects, which was accompanied by low levels of phosphorylated Erk at 5 dpf. Interestingly, defects in homozygous ptpn11a-/- mutants overlapped with defects in the double mutants albeit they were milder, whereas ptpn11b-/- single mutants did not show detectable developmental defects and were viable and fertile. Ptpn11a-/-ptpn11b-/- mutants were rescued by expression of exogenous ptpn11a and ptpn11b alike, indicating functional redundance of Shp2a and Shp2b. The ptpn11 mutants provide a good basis for further unravelling of the function of Shp2 in vertebrate development.     

Molecular characterization and functional analysis of cyp11a and cyp11b in black rockfish (Sebastes schlegelii)

The cyp11 includes cyp11a and cyp11b in most mammals and teleosts, encoded cholesterol side chain lyase and 11β-hydroxylase, respectively. It is essential in steroid hormone synthesis. However, studies on the regulation of cyp11 are limited, especially in teleosts. In this study, the molecular characterization and function of cyp11a and cyp11b of black rockfish was investigated. Both of them showed high homology with other teleost counterparts by phylogenetic analysis. The expression of cyp11a and cyp11b exhibited a clear sexually dimorphic pattern, with a higher expression level in testis than that of in ovaries. During the different developmental stages (40 dpf, 80 dpf, 190 dpf, 360 dpf, 720 dpf), the expression of cyp11a was earlier than cyp11b. In situ hybridization results showed that cyp11a and cyp11b were mainly expressed in oogonia and oocytes of the ovary. They were located in spermatogonia and interstitial compartment in the 1.5-year-old gonads, and spermatocytesgonia and the peritubular myoid cell of the testis in the 2.5-year-old gonads. To explore the distinct roles of cyp11a and cyp11b in gonads, oestrogen and androgens were used to stimulate the primary testicular and ovarian cells. The expressions of cyp11a and cyp11b were tested under different dose of 17α-methyltestosterone (17α-MT) and 17β-estradiol (E2). The results showed cyp11a was significantly increased at 10⁻⁶ mol ml^–1 of 17α-MT and 10⁻⁸ mol ml^–1 of E2 in ovary and 10⁻¹⁰ mol ml^–1 of 17α-MT and E2 in testis, while cyp11b was significantly decreased after 17α-MT and E2 treatment. These results indicated that cyp11a and cyp11b were likely to have different functions, and also implied they might play an important roles in the differentiation of gonads and the synthesis of steroids in black rockfish.     

Duplication and distinct expression patterns of two thrombospondin-1 isoforms in teleost fishes

Two types of thrombospondin-1 (named TSP-1a and TSP-1b) were cloned from two species of teleosts, the Nile tilapia and medaka. Phylogenetic analysis of these TSP-1 sequences, together with those available from other vertebrates further demonstrated that two types of TSP-1 exist only in teleosts, extending the finding in fugu and tetraodon to two additional fish species. The expression of both genes was examined using tilapia at various developmental stages. Tilapia TSP-1a and TSP-1b were each expressed in a wide range of tissues examined. The early expression of TSP-1b in both XX and XY gonads from 5 dah (day after hatching) onwards suggested an important role in the formation of gonads, while the expression of TSP-1a only in ovaries during later stages of development (from 120 dah onwards) may suggest that TSP-1a is involved in oogenesis. During the 14-day spawning cycle, the expression of both types of TSP-1 exhibited distinct peaks at day 5 (peak of vitellogenesis) and day 12 (oocyte maturation). In situ hybridization analyses revealed differential expression, with TSP-1a occurring in granulosa cells and TSP-1b in theca cells. Furthermore, both TSP-1a and -1b were expressed in skeletal tissues but with clear temporal and spatial differences. In contrast, only TSP-1b was found in the myosepta. The positive signals of both TSP-1a and TSP-1b were also detected in the heart and spleen, and TSP-1a in brain and intestine by both RT-PCR and in situ hybridization.     

sept8a and sept8b mRNA expression in the developing and adult zebrafish

Septins are highly conserved GTP-binding proteins involved in numerous cellular processes. Despite a growing awareness of their roles in the cell biology, development and signal transmission in nervous systems, comparably little is known about precise septin expression. Here, we use the well-established model organism zebrafish (Danio rerio) to unravel the expression of sept8a and sept8b, with special focus on the CNS. We performed whole mount RNA in situ hybridization on zebrafish 1–4 dpf in combination with serial sectioning of epon-embedded samples as well as on brain sections of adult zebrafish to obtain precise histological mapping of gene expression. Our results show a common expression of both genes at embryonic stages, whereas sept8a is mainly restricted to the gill arches and sept8b to specific brain structures at later stages. Brains of adult zebrafish reveal a large spatial overlap of sept8a and sept8b expression with few regions uniquely expressing sept8a or sept8b. Our results indicate a neuronal expression of both genes, and additionally suggest expression of sept8b in glial cells. Altogether, this study provides a first detailed insight into the expression of sept8a and sept8b in zebrafish and contributes to a more comprehensive understanding of septin biology in vertebrate model systems.     

Zebrafish type XVII collagen: Gene structures,expression profiles,and morpholino “knock-down” phenotypes

The human COL17A1 gene encodes type XVII collagen (also known as the 180-kDa bullous pemphigoid antigen), an integral component of hemidesmosomes, attachment complexes providing integrity to the dermal–epidermal junction. Zebrafish, a useful model system to study skin development, displays fully developed hemidesmosomes at approximately 5 days post-fertilization (dpf). We have identified two COL17A1 orthologues in the zebrafish genome, col17a1a and col17a1b, which are expressed in the skin and the neural system, respectively. The proteins coded by these genes have structural module organizations homologous to the human type XVII collagen. “Knock-down” of the expression of col17a1a with a specific morpholino targeting the 5′ UTR of the gene resulted in a blistering phenotype and in perturbations in the basement membrane zone. “Knock-down” of col17a1b expression resulted in ablation or in marked reduction of neuromasts in the lateral line. Thus, zebrafish has two COL17A1 orthologues which may have evolved tissue-specific functions during vertebrate development. Collectively, zebrafish provides a model system to study the molecular aspects of skin development and offers insights into the corresponding human diseases.     

nr0b1 (DAX1) loss of function in zebrafish causes hypothalamic defects via abnormal progenitor proliferation and differentiation

The nuclear receptor DAX-1, encoded by the NR0B1 gene, is presented in the hypothalamic tissues in humans and other vertebrates. Human patients with NR0B1 mutations often have hypothalamic-pituitary defects, but the involvement of NR0B1 in hypothalamic development and function is not well understood. Here, we report the disruption of the nr0b1 gene in zebrafish causes abnormal expression of gonadotropins, a reduction in fertilization rate, and an increase in postfasting food intake, which are indicators of abnormal hypothalamic functions. We find that loss of nr0b1 increases the number of prodynorphin (pdyn)-expressing neurons but decreases the number of pro-opiomelanocortin (pomcb)-expressing neurons in the zebrafish hypothalamic arcuate region (ARC). Further examination reveals that the proliferation of progenitor cells is reduced in the hypothalamus of nr0b1 mutant embryos accompanying the decreased expression of genes in the Notch signaling pathway. Additionally, the inhibition of Notch signaling in wild-type embryos increases the number of pdyn neurons, mimicking the nr0b1 mutant phenotype. In contrast, ectopic activation of Notch signaling in nr0b1 mutant embryos decreases the number of pdyn neurons. Taken together, our results suggest that nr0b1 regulates neural progenitor proliferation and maintenance to ensure normal hypothalamic neuron development.     

Neuronal calcium sensor‐1 gene ncs‐1a is essential for semicircular canal formation in zebrafish inner ear

We have analyzed the functional role of neuronal calcium sensor‐1 (Ncs‐1) in zebrafish development. We identified two orthologs of the mammalian NCS‐1 gene. Full‐length cDNAs encoding zebrafish Ncs‐1a and Ncs‐1b polypeptides were cloned and characterized. Whole‐mount in situ hybridization revealed that ncs‐1a mRNA was expressed beginning at early somitogenesis. As development progressed, ncs‐1a mRNA was present throughout the embryo with expression detected in ventral hematopoietic mesoderm, pronephric tubules, CNS nuclei, and otic vesicle. By 4.5 days post fertilization (dpf), ncs‐1a expression was detected primarily in the brain. Expression of ncs‐1b mRNA was first detected at 36 hours post fertilization (hpf) and was restricted to the olfactory bulb. By 4.5 dpf, ncs‐1b was expressed at low levels throughout the brain. Knockdown of ncs‐1a mRNA translation with antisense morpholinos blocked formation of semicircular canals. These studies identify a novel function for ncs‐1a in inner ear development and suggest that this calcium sensor plays an important role in vestibular function. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Characterisation and differential expression during development of a duplicate Disabled-1 (Dab1) gene from zebrafish

We identified a new duplicated Dab1 gene (drDab1b) spanning around 25 kb of genomic DNA in zebrafish. Located in zebrafish chromosome 2, it is composed of 11 encoding exons and shows high sequence similarity to other Dab1 genes, including drDab1a, a zebrafish Dab1 gene previously characterised. drDab1b encodes by alternative splicing at least five different isoforms. Both drDab1a and drDab1b show differential gene expression levels in distinct adult tissues and during development. drDab1b is expressed in peripheral tissues (gills, heart, intestine, muscle), the immune system (blood, liver) and the central nervous system (CNS), whereas drDab1a is only expressed in gills, muscle and the CNS, suggesting a division of functions for two Dab1 genes in zebrafish adult tissues. RT-PCR analysis also reveals that both drDab1 genes show distinct developmental-specific expression patterns throughout development. drDab1b expression was higher than that of drDab1a, suggesting a major role of drDab1b in comparison with drDab1a during development and in different adult tissues. In addition, new putative Dab1 (a and/or b) from different teleost species were identified in silico and predicted protein products are compared with the previously characterised Dab1, demonstrating that the Dab1b group is more ancestral than their paralogue, the Dab1a group.     

Complete XY gonadal dysgenesis due to p.D293N homozygous mutation in theNR5A1 gene: a case study

TheSRY gene (sex-determining region on the Y chromosome; MIM *480000) is responsible for initiating male gonadal development. However, only 15–20% of the cases of XY gonadal dysgenesis are due to mutations in its sequence. Recently, heterozygous mutations in theNR5A1 gene (nuclear receptor subfamily 5, group A, member 1; MIM +184757) have been described in association with ovarian failure and disorders of testis development with or without adrenal failure. Here we describe a case of XY complete gonadal dysgenesis due to a p.D293N homozygous mutation in theNR5A1 gene, with normalSRY and no adrenal failure.