Seleit A, Krämer I, Ambrosio E, Dross N, Engel U,Centanin L. (2017) Sequential Organogenesis Sets Two Parallel Sensory Lines in Medaka. Development doi: 10.1242/dev.142752
Agaallaei N, Gruhl F, Schaefer CG, Wernet T, Weinhardt V, Centanin L, Loosli F, Baumbach T, Wittbrodt J. (2016) Identification, Visualization and Clonal Analysis of Intestinal Stem Cells in Fish. Development Vol.143: 3470-3480
Lust K, Sinn R, Pérez Saturnino A, Centanin L#, Wittbrodt J#.(2016) De novo Neurogenesis by Targeted Expression of Atoh7 to Müller Glia Cells. Development Vol.143: 1874-1883. #co-corresponding authors
Reinhardt, R.*, Centanin, L.*#, Tavhelidse, T., Inoue, D., Wittbrodt, B., Concordet, J.-P., Martinez-Morales, J.-R., and Wittbrodt, J.# (2015). Sox2, Tlx, Gli3, and Her9 converge on Rx2 to define retinal stem cells in vivo. EMBO J 34, 1572–1588. *Equal contribution, #Co-corresponding.
Centanin, L.#, Ander, J.-J., Hoeckendorf, B., Lust, K., Kellner, T., Kraemer, I., Urbany, C., Hasel, E., Harris, W. A., Simons, B. D., Wittbrodt, J.#(2014). Exclusive multipotency and preferential asymmetric divisions in post-embryonic neural stem cells of the fish retina. Development 141, 3472–3482. #Co-corresponding.
Centanin, L.#, and Wittbrodt, J.# (2014). Retinal neurogenesis. Development 141, 241–244.#Co-corresponding.
Schaafhausen, M. K., Yang, W.-J., Centanin, L., Wittbrodt, J., Bosserhoff, A., Fischer, A., Schartl, M., and Meierjohann, S. (2013). Tumor angiogenesis is caused by single melanoma cells in a manner dependent on reactive oxygen species and NF-κB. J Cell Sci 126, 3862–3872.
Centanin L.#, Hoeckendorf B, Wittbrodt J.# (2011). Fate restriction and multipotency in retinal stem cells. Cell Stem Cell. 9(6):553-62.#Co-corresponding.
Acevedo JM*, Centanin L*
, Dekanty A, Wappner P. (2010
). Oxygen sensing in Drosophila: multiple isoforms of the prolyl hydroxylase fatiga have different capacity to regulate HIFalpha/Sima. PLoS One.
BACKGROUND: The Hypoxia Inducible Factor (HIF) mediates cellular adaptations to low oxygen. Prolyl-4-hydroxylases are oxygen sensors that hydroxylate the HIF alpha-subunit, promoting its proteasomal degradation in normoxia. Three HIF-prolyl hydroxylases, encoded by independent genes, PHD1, PHD2, and PHD3, occur in mammals. PHD2, the longest PHD isoform includes a MYND domain, whose biochemical function is unclear. PHD2 and PHD3 genes are induced in hypoxia to shut down HIF dependent transcription upon reoxygenation, while expression of PHD1 is oxygen-independent. The physiologic significance of the diversity of the PHD oxygen sensors is intriguing. METHODOLOGY AND PRINCIPAL FINDINGS: We have analyzed the Drosophila PHD locus, fatiga, which encodes 3 isoforms, FgaA, FgaB and FgaC that are originated through a combination of alternative initiation of transcription and alternative splicing. FgaA includes a MYND domain and is homologous to PHD2, while FgaB and FgaC are shorter isoforms most similar to PHD3. Through a combination of genetic experiments in vivo and molecular analyses in cell culture, we show that fgaB but not fgaA is induced in hypoxia, in a Sima-dependent manner, through a HIF-Responsive Element localized in the first intron of fgaA. The regulatory capacity of FgaB is stronger than that of FgaA, as complete reversion of fga loss-of-function phenotypes is observed upon transgenic expression of the former, and only partial rescue occurs after expression of the latter. CONCLUSIONS AND SIGNIFICANCE: Diversity of PHD isoforms is a conserved feature in evolution. As in mammals, there are hypoxia-inducible and non-inducible Drosophila PHDs, and a fly isoform including a MYND domain co-exists with isoforms lacking this domain. Our results suggest that the isoform devoid of a MYND domain has stronger regulatory capacity than that including this domain.
Irisarri M, Lavista-Llanos S, Romero NM, Centanin L
, Dekanty A, Wappner P. (2009
). Central role of the oxygen-dependent degradation domain of Drosophila HIFalpha/Sima in oxygen-dependent nuclear export. Mol Biol Cell.
The Drosophila HIFalpha homologue, Sima, is localized mainly in the cytoplasm in normoxia and accumulates in the nucleus upon hypoxic exposure. We have characterized the mechanism governing Sima oxygen-dependent subcellular localization and found that Sima shuttles continuously between the nucleus and the cytoplasm. We have previously shown that nuclear import depends on an atypical bipartite nuclear localization signal mapping next to the C-terminus of the protein. We show here that nuclear export is mediated in part by a CRM1-dependent nuclear export signal localized in the oxygen-dependent degradation domain (ODDD). CRM1-dependent nuclear export requires both oxygen-dependent hydroxylation of a specific prolyl residue (Pro850) in the ODDD, and the activity of the von Hippel Lindau tumor suppressor factor. At high oxygen tension rapid nuclear export of Sima occurs, whereas in hypoxia, Sima nuclear export is largely inhibited. HIFalpha/Sima nucleo-cytoplasmic localization is the result of a dynamic equilibrium between nuclear import and nuclear export, and nuclear export is modulated by oxygen tension.
, Gorr TA, Wappner P. (2009
). Tracheal remodelling in response to hypoxia. J Insect Physiol.
The insect tracheal system is a continuous tubular network that ramifies into progressively thinner branches to provide air directly to every organ and tissue throughout the body. During embryogenesis the basic architecture of the tracheal system develops in a stereotypical and genetically controlled manner. Later, in larval stages, the tracheal system becomes plastic, and adapts to particular oxygen needs of the different tissues of the body. Oxygen sensing is mediated by specific prolyl-4-hydroxylases that regulate protein stability of the alpha subunit of oxygen-responsive transcription factors from the HIF family. Tracheal cells are exquisitely sensitive to oxygen levels, modulating the expression of hypoxia-inducible proteins that mediate sprouting of tracheal branches in direction to oxygen-deprived tissues.
, Dekanty A, Romero N, Irisarri M, Gorr TA, Wappner P. (2008
). Cell autonomy of HIF effects in Drosophila: tracheal cells sense hypoxia and induce terminal branch sprouting. Dev Cell.
Drosophila tracheal terminal branches are plastic and have the capacity to sprout out projections toward oxygen-starved areas, in a process analogous to mammalian angiogenesis. This response involves the upregulation of FGF/Branchless in hypoxic tissues, which binds its receptor Breathless on tracheal cells. Here, we show that extra sprouting depends on the Hypoxia-Inducible Factor (HIF)-alpha homolog Sima and on the HIF-prolyl hydroxylase Fatiga that operates as an oxygen sensor. In mild hypoxia, Sima accumulates in tracheal cells, where it induces breathless, and this induction is sufficient to provoke tracheal extra sprouting. In nontracheal cells, Sima contributes to branchless induction, whereas overexpression of Sima fails to attract terminal branch outgrowth, suggesting that HIF-independent components are also required for full induction of the ligand. We propose that the autonomous response to hypoxia that occurs in tracheal cells enhances tracheal sensitivity to increasing Branchless levels, and that this mechanism is a cardinal step in hypoxia-dependent tracheal sprouting.
Rojas DA, Perez-Munizaga DA, Centanin L
, Antonelli M, Wappner P, Allende ML, Reyes AE. (2006
). Cloning of hif-1alpha and hif-2alpha and mRNA expression pattern during development in zebrafish. Gene Expr Patterns.
Hypoxia-inducible factors (HIFs) regulate gene expression in response to hypoxia and in vertebrates they are known to participate in several developmental processes, including angiogenesis, vasculogenesis, heart and central nervous system development. Over the last decade, major progress in unraveling the molecular mechanisms that mediate regulation of HIF proteins by oxygen tension has been reported, but our knowledge on their developmental regulation during embryogenesis in model organisms is limited. Expression of hif-1alpha and hif-2alpha genes has been characterized during normal mouse development and they were found to be expressed from stages E7.5, later in E9.5 and E15.5 in several different tissues such as the brain, heart and blood vessels. However, there is no detailed temporal information on their expression at other embryonic stages, even though orthologous genes have been described in several different vertebrate species. In this study, we describe the cloning and detailed expression pattern of zebrafish hif-1alpha and hif-2alpha genes. Sequence analysis revealed that zebrafish Hif proteins are highly homologous to other vertebrate orthologues. Zebrafish hif-1alpha and hif-2alpha are both expressed throughout development in discrete territories in a dynamic pattern. Interestingly, in the notochord the expression of hif-1alpha is switched off, while hif-2alpha transcription is turned on, signifying that the two genes might have partially overlapping, although non-redundant functions in development. This is the first time that a detailed comparison of the expression of hif-1alpha and hif-2alpha is directly assessed in a vertebrate model system throughout development.
, Ratcliffe PJ, Wappner P. (2005
). Reversion of lethality and growth defects in Fatiga oxygen-sensor mutant flies by loss of hypoxia-inducible factor-alpha/Sima. EMBO Rep.
Hypoxia-Inducible Factor (HIF) prolyl hydroxylase domains (PHDs) have been proposed to act as sensors that have an important role in oxygen homeostasis. In the presence of oxygen, they hydroxylate two specific prolyl residues in HIF-alpha polypeptides, thereby promoting their proteasomal degradation. So far, however, the developmental consequences of the inactivation of PHDs in higher metazoans have not been reported. Here, we describe novel loss-of-function mutants of fatiga, the gene encoding the Drosophila PHD oxygen sensor, which manifest growth defects and lethality. We also report a null mutation in dHIF-alpha/sima, which is unable to adapt to hypoxia but is fully viable in normoxic conditions. Strikingly, loss-of-function mutations of sima rescued the developmental defects observed in fatiga mutants and enabled survival to adulthood. These results indicate that the main functions of Fatiga in development, including control of cell size, involve the regulation of dHIF/Sima.
Lavista-Llanos S, Centanin L
, Irisarri M, Russo DM, Gleadle JM, Bocca SN, Muzzopappa M, Ratcliffe PJ, Wappner P. (2002
). Control of the hypoxic response in Drosophila melanogaster by the basic helix-loop-helix PAS protein similar. Mol Cell Biol.
In mammalian systems, the heterodimeric basic helix-loop-helix (bHLH)-PAS transcription hypoxia-inducible factor (HIF) has emerged as the key regulator of responses to hypoxia. Here we define a homologous system in Drosophila melanogaster, and we characterize its activity in vivo during development. By using transcriptional reporters in developing transgenic flies, we show that hypoxia-inducible activity rises to a peak in late embryogenesis and is most pronounced in tracheal cells. We show that the bHLH-PAS proteins Similar (Sima) and Tango (Tgo) function as HIF-alpha and HIF-beta homologues, respectively, and demonstrate a conserved mode of regulation for Sima by oxygen. Sima protein, but not its mRNA, was upregulated in hypoxia. Time course experiments following pulsed ectopic expression demonstrated that Sima is stabilized in hypoxia and that degradation relies on a central domain encompassing amino acids 692 to 863. Continuous ectopic expression overrode Sima degradation, which remained cytoplasmic in normoxia, and translocated to the nucleus only in hypoxia, revealing a second oxygen-regulated activation step. Abrogation of the Drosophila Egl-9 prolyl hydroxylase homologue, CG1114, caused both stabilization and nuclear localization of Sima, indicating a central involvement in both processes. Tight conservation of the HIF/prolyl hydroxylase system in Drosophila provides a new focus for understanding oxygen homeostasis in intact multicellular organisms.