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Ptena and ptenb genes play distinct roles in zebrafish embryogenesis.

ABSTRACT: PTEN is a tumor suppressor gene associated with multiple tumor types. PTEN function is essential for early embryonic development and is involved in the regulation of cell size, number, and survival. By dephosphorylating PIP(3), PTEN normally acts to inhibit the PI3-Kinase/AKT pathway. Here we have identified two zebrafish orthologs, ptena and ptenb, of the single mammalian PTEN gene and analyzed the role of these genes in zebrafish development. Ptena transcripts were expressed throughout the embryo at early somitogenesis. By 24 hpf, expression was predominant in the central nervous system, axial vasculature, retina, branchial arches, ear, lateral line primordium, and pectoral fin bud. Ptenb was also ubiquitously expressed early in somitogenesis, but transcripts became more restricted to the somites and central nervous system as development progressed. By 48 hpf, ptena and ptenb were expressed predominantly in the central nervous system, branchial arches, pectoral fins, and eye. Antisense morpholinos were used to knock down translation of ptena and ptenb mRNA in zebrafish embryos. Knockdown of either pten gene caused increased levels of phosphorylated Akt in morphant embryos, indicating that Ptena and Ptenb each possess PIP(3) lipid phosphatase activity. Ptena morphants had irregularities in notochord shape (73%), vasculogenesis (83%), head shape (72%), and inner ear development (59%). The most noticeable defects in ptenb morphants were upward hooked tails (73%), domed heads (83%), and reduced yolk extensions (90%). These results indicate that ptena and ptenb encode functional enzymes and that each pten gene plays a distinct role during zebrafish embryogenesis.

SUBMITTER: Croushore JA

PROVIDER: S-EPMC2611959 | biostudies-literature | 2005 Dec

REPOSITORIES: biostudies-literature

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Ptena and ptenb genes play distinct roles in zebrafish embryogenesis.

Croushore Jessica A JA Blasiole Brian B Riddle Ryan C RC Thisse Christine C Thisse Bernard B Canfield Victor A VA Robertson Gavin P GP Cheng Keith C KC Levenson Robert R

Developmental dynamics : an official publication of the American Association of Anatomists 20051201 4

PTEN is a tumor suppressor gene associated with multiple tumor types. PTEN function is essential for early embryonic development and is involved in the regulation of cell size, number, and survival. By dephosphorylating PIP(3), PTEN normally acts to inhibit the PI3-Kinase/AKT pathway. Here we have identified two zebrafish orthologs, ptena and ptenb, of the single mammalian PTEN gene and analyzed the role of these genes in zebrafish development. Ptena transcripts were expressed throughout the emb ...[more]

PMID: 16193492

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Project description:Despite the well-described discrepancy between ATG (macroautophagy/autophagy-related) genes in the regulation of hematopoiesis, varying essentiality of core ATG proteins in vertebrate definitive hematopoiesis remains largely unclear. Here, we employed zebrafish (Danio rerio) to compare the functions of six core atg genes, including atg13, becn1 (beclin1), atg9a, atg2a, atg5, and atg3, in vertebrate definitive hematopoiesis via clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 ribonucleoprotein and morpholino targeting. Zebrafish with various atg mutations showed autophagic deficiency and presented partially consistent hematopoietic abnormalities during early development. All six atg mutations led to a declined number of spi1b+ (Spi-1 proto-oncogene b) myeloid progenitor cells. However, only becn1 mutation resulted in the expansion of myb+ (v-myb avian myeloblastosis viral oncogene homolog) hematopoietic stem and progenitor cells (HSPCs) and transiently increased coro1a+ (coronin, actin binding protein, 1A) leukocytes, whereas atg3 mutation decreased the number of HSPCs and leukocytes. Proteomic analysis of caudal hematopoietic tissue identified sin3aa (SIN3 transcription regulator family member Aa) as a potential modulator of atg13- and becn1-regulated definitive hematopoiesis. Disruption of sin3aa rescued the expansion of HSPCs and leukocytes in becn1 mutants and exacerbated the decrease of HSPCs in atg13 mutants. Double mutations were also performed to examine alternative functions of various atg genes in definitive hematopoiesis. Notably, becn1 mutation failed to induce HSPCs expansion with one of the other five atg mutations. These findings demonstrated the distinct roles of atg genes and their interplays in zebrafish definitive hematopoiesis, thereby suggesting that the vertebrate definitive hematopoiesis is regulated in an atg gene-dependent manner.Abbreviations: AGM: aorta-gonad-mesonephros; AO: acridine orange; atg: autophagy related; becn1: beclin 1, autophagy related; CHT: caudal hematopoietic tissue; CKO: conditional knockout; coro1a: coronin, actin binding protein, 1A; CQ: chloroquine; CRISPR: clustered regularly interspaced short palindromic repeats; dpf: days post fertilization; FACS: fluorescence-activated cell sorting; hbae1.1: hemoglobin, alpha embryonic 1.1; HSCs: hematopoietic stem cells; HSPCs: hematopoietic stem and progenitor cells; KD: knockdown; KO: knockout; map1lc3/lc3: microtubule-associated protein 1 light chain 3; MO: morpholino; mpeg1.1: macrophage expressed 1, tandem duplicate 1; mpx: myeloid-specific peroxidase; myb: v-myb avian myeloblastosis viral oncogene homolog; PE: phosphatidylethanolamine; p-H3: phospho-H3 histone; PtdIns3K: class 3 phosphatidylinositol 3-kinase; rag1: recombination activating 1; rb1cc1/fip200: RB1-inducible coiled-coil 1; RFLP: restriction fragment length polymorphism; RNP: ribonucleoprotein; sin3aa: SIN3 transcription regulator family member Aa; spi1b: Spi-1 proto-oncogene b; ulk: unc-51 like autophagy activating kinase; vtg1: vitellogenin 1; WISH: whole-mount in situ hybridization.

Project description:The HCN4 channel is essential for heart rate regulation in vertebrates by generating pacemaker potentials in the sinoatrial node. HCN4 channel abnormality may cause bradycardia and sick sinus syndrome, making it an important target for clinical research and drug discovery. The zebrafish is a popular animal model for cardiovascular research. They are potentially suitable for studying inherited heart diseases, including cardiac arrhythmia. However, it has not been determined how similar the ion channels that underlie cardiac automaticity are in zebrafish and humans. In the case of HCN4, humans have one gene, whereas zebrafish have two ortholog genes (DrHCN4 and DrHCN4L; 'Dr' referring to Danio rerio). However, it is not known whether the two HCN4 channels have different physiological functions and roles in heart rate regulation. In this study, we characterized the biophysical properties of the two zebrafish HCN4 channels in Xenopus oocytes and compared them to those of the human HCN4 channel. We found that they showed different gating properties: DrHCN4L currents showed faster activation kinetics and a more positively shifted G-V curve than did DrHCN4 and human HCN4 currents. We made chimeric channels of DrHCN4 and DrHCN4L and found that cytoplasmic domains were determinants for the faster activation and the positively shifted G-V relationship in DrHCN4L. The use of a dominant-negative HCN4 mutant confirmed that DrHCN4 and DrHCN4L can form a heteromultimeric channel in Xenopus oocytes. Next, we confirmed that both are sensitive to common HCN channel inhibitors/blockers including Cs+, ivabradine, and ZD7288. These HCN inhibitors successfully lowered zebrafish heart rate during early embryonic stages. Finally, we knocked down the HCN4 genes using antisense morpholino and found that knocking down either or both of the HCN4 channels caused a temporal decrease in heart rate and tended to cause pericardial edema. These findings suggest that both DrHCN4 and DrHCN4L play a significant role in zebrafish heart rate regulation.

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Ptena and ptenb genes play distinct roles in zebrafish embryogenesis.

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Ptena and ptenb genes play distinct roles in zebrafish embryogenesis.

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