Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
PLoS One
2011 Jan 01;68:e23746. doi: 10.1371/journal.pone.0023746.
Show Gene links
Show Anatomy links
Acquisition of glial cells missing 2 enhancers contributes to a diversity of ionocytes in zebrafish.
Shono T
,
Kurokawa D
,
Miyake T
,
Okabe M
.
???displayArticle.abstract??? Glial cells missing 2 (gcm2) encoding a GCM-motif transcription factor is expressed in the parathyroid in amniotes. In contrast, gcm2 is expressed in pharyngeal pouches (a homologous site of the parathyroid), gills, and H(+)-ATPase-rich cells (HRCs), a subset of ionocytes on the skin surface of the teleost fish zebrafish. Ionocytes are specialized cells that are involved in osmotic homeostasis in aquatic vertebrates. Here, we showed that gcm2 is essential for the development of HRCs and Na(+)-Cl(-) co-transporter-rich cells (NCCCs), another subset of ionocytes in zebrafish. We also identified gcm2 enhancer regions that control gcm2 expression in ionocytes of zebrafish. Comparisons of the gcm2 locus with its neighboring regions revealed no conserved elements between zebrafish and tetrapods. Furthermore, We observed gcm2 expression patterns in embryos of the teleost fishes Medaka (Oryzias latipes) and fugu (Fugu niphobles), the extant primitive ray-finned fishes Polypterus (Polypterus senegalus) and sturgeon (a hybrid of Huso huso × Acipenser ruhenus), and the amphibian Xenopus (Xenopus laevis). Although gcm2-expressing cells were observed on the skin surface of Medaka and fugu, they were not found in Polypterus, sturgeon, or Xenopus. Our results suggest that an acquisition of enhancers for the expression of gcm2 contributes to a diversity of ionocytes in zebrafish during evolution.
???displayArticle.pubmedLink???
21858216
???displayArticle.pmcLink???PMC3157436 ???displayArticle.link???PLoS One
Figure 1. Expression of gcm2 in the gills and the cells on the skin surface of zebrafish.Whole-mount in situ hybridization of 5-day-old zebrafish with gcm2 probes. (A) gcm2 is expressed in the gills. (B) Magnification of a yolk sac area. Some cells in the yolk sac express gcm2 (arrowheads).
Figure 2. Scanning electron microscopy of the yolk sac membrane in 3-day-old zebrafish.(A, B) Magnification of the yolk sac membrane. (A) An embryo injected with a control morpholino (MO). (B) An embryo injected with a gcm2 MO. (C) Quantitative comparison of the ionocyte number per 0.04 mm2 between embryos injected with control MO and gcm2 MO. The ionocyte number on the yolk sac membrane area in gcm2 morphants is less than that in control MO. Arrowheads indicate ionocytes. Because it is likely that cells were still alive or had differentiated without expression of a marker gene by loss of function, we observed the external morphology of the skin surface in morphants using scanning electron microscopy.
Figure 3. gcm2 is essential for the development of HRCs and suppresses the development of NCCCs on the skin surface.(A, B) Whole-mount in situ hybridization of zebrafish 48 hours post-fertilization (hpf) with an atp6v1al probe as a HRC marker. (C, D) Whole-mount in situ hybridization with an atp1b1b probe as a NaRC marker. (E, F) Whole-mount in situ hybridization with a slc12a10.2 probe as a NCCC marker. (A, C, E) Zebrafish (48 hpf) injected with a control morpholino (MO). (B, D, F) Zebrafish (48 hpf) injected with a gcm2 MO. (G) Quantitative comparison of atp6v1al-positive cells, atp1b1b-positive cells, and slc12a10.2-positive cells in zebrafish injected with control MO and gcm2 MO.
Figure 4. Expression of gcm2 in Medaka, fugu, Polypterus, sturgeon and Xenopus.(A, B) Whole-mount in situ hybridization for gcm2 in 13-day-old Medaka. (C, D) Whole-mount in situ hybridization for gcm2 in 7-day-old fugu. (B, D) Magnification of the skin surface. gcm2 is expressed in the gills (arrowheads) and the cells on the skin surface of the teleostean fishes, Medaka and fugu. (E, F) Whole-mount in situ hybridization for gcm2 and atp1b1 probes in 48-hpf Polypterus. (E) gcm2 is expressed in the gills (arrowhead). (F) atp1b1 is expressed in the kidney and the cells on the skin surface. (G, H) Whole-mount in situ hybridization for gcm2 and atp1b1 in sturgeon at stage 32. (G) gcm2 is expressed in the gills (arrowhead). (H) atp1b1 is expressed in the cells on the skin surface. (I) Scanning electron microscopy on the yolk sac membrane of sturgeon at stage 32. Ionocytes (arrowheads) protrude between the epithelial cells. (J) Whole-mount in situ hybridization with the gcm2 probe in Xenopus at stage 37. gcm2 is expressed in the gills (arrowhead) but not on the skin surface of Polypterus, sturgeon, and Xenopus embryos.
Figure 5. Analysis of gcm2 enhancers specific for ionocytes on the skin surface of zebrafish.(A) Construction of the 160 kb BAC-Venus. The targeting construct was amplified by PCR from a plasmid containing Venus, polyA, and Kmr. Each primer contained 50 bp of the gcm2-derived sequences that served as homology arms for homologous recombination. The 160 kb BAC contained sequences that were 120 kb upstream and 40 kb downstream of the gcm2 locus. After homologous recombination, Venus was inserted into the translation site (160 kb BAC-Venus). (B) A 160 kb BAC-Venus transient transgenic zebrafish (72 hpf). Venus-expressing cells are observed on the skin surface but not in the gills. (C) Several cells on the yolk sac expressed Venus. (D) Ionocytes stained with MitoTracker. (E) Merge of (C) and (D), showing overlap in staining with a subset of ionocytes. (F) A 160 kb BAC-Venus transient transgenic 7-day-old fugu. Venus-expressing cells are observed on the skin surface.
Figure 6. Transient transgenic analysis of the gcm2 enhancer regions for HRCs specific to the skin surface.(A, B) The chart on the right shows the percentage of embryos with Venus-positive cells on the skin surface at 24 hpf. (C–F) In situ hybridization with an atp6v1al probe in combination with immunohistochemistry using anti-GFP on the yolk sac of transient transgenic 4-day-old zebrafish. (C, D) A zebrafish expressing pTolfV that contains the gcm2 enhancer region that is 8 kb upstream. (E, F) A zebrafish expressing pTolfV that contains the gcm2 enhancer region that is 41 kb downstream. The gcm2 loci at −8249 to −8004 bp upstream and at +41277 to +41497 bp downstream were required for Venus expression in HRCs.
Figure 7. Two gcm2 enhancers in zebrafish are not found in tetrapods.VISTA plots of zebrafish, Xenopus, chick, mouse, and human genomic sequences of the gcm2 locus with its neighboring genes, elovl2 and mak.
Akiyama,
The gcm-motif: a novel DNA-binding motif conserved in Drosophila and mammals.
1996, Pubmed
Akiyama,
The gcm-motif: a novel DNA-binding motif conserved in Drosophila and mammals.
1996,
Pubmed
Alfonso,
gcm2 promotes glial cell differentiation and is required with glial cells missing for macrophage development in Drosophila.
2002,
Pubmed
Altshuller,
Gcm1, a mammalian homolog of Drosophila glial cells missing.
1996,
Pubmed
Anson-Cartwright,
The glial cells missing-1 protein is essential for branching morphogenesis in the chorioallantoic placenta.
2000,
Pubmed
BALLARD,
NORMAL EMBRYONIC STAGES OF POLYODON SPATHULA (WALBAUM).
1964,
Pubmed
Basyuk,
Murine Gcm1 gene is expressed in a subset of placental trophoblast cells.
1999,
Pubmed
Bernardoni,
glide/gcm is expressed and required in the scavenger cell lineage.
1997,
Pubmed
Carlsson,
Forkhead transcription factors: key players in development and metabolism.
2002,
Pubmed
Carroll,
Evo-devo and an expanding evolutionary synthesis: a genetic theory of morphological evolution.
2008,
Pubmed
Chang,
The transcription factor, glial cell missing 2, is involved in differentiation and functional regulation of H+-ATPase-rich cells in zebrafish (Danio rerio).
2009,
Pubmed
Dubaissi,
Embryonic frog epidermis: a model for the study of cell-cell interactions in the development of mucociliary disease.
2011,
Pubmed
,
Xenbase
Esaki,
Mechanism of development of ionocytes rich in vacuolar-type H(+)-ATPase in the skin of zebrafish larvae.
2009,
Pubmed
Fisher,
Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish.
2006,
Pubmed
Gordon,
Gcm2 and Foxn1 mark early parathyroid- and thymus-specific domains in the developing third pharyngeal pouch.
2001,
Pubmed
Günther,
Genetic ablation of parathyroid glands reveals another source of parathyroid hormone.
2000,
Pubmed
Hanaoka,
Zebrafish gcmb is required for pharyngeal cartilage formation.
2004,
Pubmed
Hashemolhosseini,
Restricted expression of mouse GCMa/Gcm1 in kidney and thymus.
2002,
Pubmed
Hirose,
Molecular biology of major components of chloride cells.
2003,
Pubmed
Hoegg,
Phylogenetic timing of the fish-specific genome duplication correlates with the diversification of teleost fish.
2004,
Pubmed
Hogan,
Zebrafish gcm2 is required for gill filament budding from pharyngeal ectoderm.
2004,
Pubmed
Hosoya,
glial cells missing: a binary switch between neuronal and glial determination in Drosophila.
1995,
Pubmed
Hsiao,
A positive regulatory loop between foxi3a and foxi3b is essential for specification and differentiation of zebrafish epidermal ionocytes.
2007,
Pubmed
Hurley,
A new time-scale for ray-finned fish evolution.
2007,
Pubmed
Hwang,
Ion uptake and acid secretion in zebrafish (Danio rerio).
2009,
Pubmed
Inoue,
Basal actinopterygian relationships: a mitogenomic perspective on the phylogeny of the "ancient fish".
2003,
Pubmed
Jones,
glial cells missing: a genetic switch that controls glial versus neuronal fate.
1995,
Pubmed
Jänicke,
Foxi3 transcription factors and Notch signaling control the formation of skin ionocytes from epidermal precursors of the zebrafish embryo.
2007,
Pubmed
Kammerer,
GCMB, a second human homolog of the fly glide/gcm gene.
1999,
Pubmed
Kanemura,
Isolation and expression analysis of a novel human homologue of the Drosophila glial cells missing (gcm) gene.
1999,
Pubmed
Kawakami,
A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish.
2004,
Pubmed
Kikugawa,
Basal jawed vertebrate phylogeny inferred from multiple nuclear DNA-coded genes.
2004,
Pubmed
Kim,
Isolation and characterization of mammalian homologs of the Drosophila gene glial cells missing.
1998,
Pubmed
Lebestky,
Specification of Drosophila hematopoietic lineage by conserved transcription factors.
2000,
Pubmed
Lee,
A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA.
2001,
Pubmed
Lin,
Proton pump-rich cell secretes acid in skin of zebrafish larvae.
2006,
Pubmed
Liu,
Gcm2 is required for the differentiation and survival of parathyroid precursor cells in the parathyroid/thymus primordia.
2007,
Pubmed
Loots,
rVISTA 2.0: evolutionary analysis of transcription factor binding sites.
2004,
Pubmed
Meyer,
From 2R to 3R: evidence for a fish-specific genome duplication (FSGD).
2005,
Pubmed
Nagai,
A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.
2002,
Pubmed
Nait-Oumesmar,
Placental expression and chromosomal localization of the human Gcm 1 gene.
2000,
Pubmed
Okabe,
The origin of the parathyroid gland.
2004,
Pubmed
Philpott,
Tubular system membranes of teleost chloride cells: osmotic response and transport sites.
1980,
Pubmed
Quigley,
Specification of ion transport cells in the Xenopus larval skin.
2011,
Pubmed
,
Xenbase
Vincent,
Glide directs glial fate commitment and cell fate switch between neurones and glia.
1996,
Pubmed
Wagner,
Evolutionary novelties.
2010,
Pubmed
Wang,
Role of SLC12A10.2, a Na-Cl cotransporter-like protein, in a Cl uptake mechanism in zebrafish (Danio rerio).
2009,
Pubmed
Wray,
The evolutionary significance of cis-regulatory mutations.
2007,
Pubmed
