16 research outputs found
Analysis of the Ush2a Gene in Medaka Fish (Oryzias latipes)
Patients suffering from Usher syndrome (USH) exhibit sensorineural hearing loss, retinitis pigmentosa (RP) and, in some cases, vestibular dysfunction. USH is the most common genetic disorder affecting hearing and vision and is included in a group of hereditary pathologies associated with defects in ciliary function known as ciliopathies. This syndrome is clinically classified into three types: USH1, USH2 and USH3. USH2 accounts for well over one-half of all Usher cases and mutations in the USH2A gene are responsible for the majority of USH2 cases, but also for atypical Usher syndrome and recessive non-syndromic RP. Because medaka fish (Oryzias latypes) is an attractive model organism for genetic-based studies in biomedical research, we investigated the expression and function of the USH2A ortholog in this teleost species. Ol-Ush2a encodes a protein of 5.445 aa codons, containing the same motif arrangement as the human USH2A. Ol-Ush2a is expressed during early stages of medaka fish development and persists into adulthood. Temporal Ol-Ush2a expression analysis using whole mount in situ hybridization (WMISH) on embryos at different embryonic stages showed restricted expression to otoliths and retina, suggesting that Ol-Ush2a might play a conserved role in the development and/or maintenance of retinal photoreceptors and cochlear hair cells. Knockdown of Ol-Ush2a in medaka fish caused embryonic developmental defects (small eyes and heads, otolith malformations and shortened bodies with curved tails) resulting in late embryo lethality. These embryonic defects, observed in our study and in other ciliary disorders, are associated with defective cell movement specifically implicated in left-right (LR) axis determination and planar cell polarity (PCP)
Cell fusion contributes to pericyte formation after stroke
6 páginas, 2 figuras.Recent reports have shown that bone marrow-derived cells (BMDCs) contribute to the formation of vasculature after stroke. However, the mechanism by which mural cells are formed from BMDC remains elusive. Here, we provide direct evidence that the cell fusion process contributes to the formation of pericytes after stroke. We generated mouse bone marrow chimeras using a cre/lox system that allows the detection of fusion events by X-gal staining. In these mice, we detected X-gal-positive cells that expressed vimentin and desmin, specific markers of mature murine pericytes. Electron microscopy confirmed that fused cells possessed basal lamina and characteristics of pericytes. Furthermore, induction of stroke increased significantly the presence of fused cells in the ischemic area. These cells expressed markers of developing pericytes such as NG2. We conclude that cell fusion participates actively in the generation of vascular tissue through pericyte formation under normal as well as pathologic conditions.This work was
supported by grants from Spanish Ministry of Health
(FIS 04/2744) and Regenerative Medicine Programme
from the CIPF. M.P-G and IZ were recipients
of PhD fellowships from CIPF and Generalitat
Valenciana, respectively.Peer reviewe
Enhanced Hematovascular Contribution of SCL 3′ Enhancer Expressing Fetal Liver Cells Uncovers Their Potential to Integrate in Extramedullary Adult Niches
13 páginas, 6 figuras, 1 tabla.-- et al.Fetal liver (FL) hematopoietic progenitors have superior blood engraftment competence compared with adult bone marrow (BM), however less is known about FL in vivo vascular capacity. Here we show in transplantation assays that FL cells possess enhanced vascular endothelial potential compared with adult bone marrow. We generated high-level hematopoietic chimeras using donor cells from mice transgenic for the stem cell leukaemia 3′ enhancer human placental alkaline phosphatase (SCL3′Enh-PLAP) reporter construct, active in vascular endothelium, and blood progenitor and stem cells. Long-term lineage tracing analysis revealed PLAP+ vascular-like patches in FL-derived chimeras, whereas adult BM-derived chimeras presented only rare and scattered PLAP+ cells. PLAP+ vascular-like patches were formed following transplantation into both newborn and adult recipient mice, although their frequency was reduced in adult recipients. Confocal analysis of multiple labeled tissues revealed that whereas most liver and heart PLAP+ vascular patch-associated cells were endothelial, PLAP+ vascular patches in the kidney contained endothelial, hematopoietic, and putative hemangioblastic cells. Moreover, fluorescence-activated cell sorting assays showed that only FL PLAPbright+ donor cells can generate PLAP+ vascular patches upon transplantation. Taken together, these data demonstrate superior vascular contribution potential of FL cells, and not only provide new insights into the developmental pathways controlling endothelial development but also may prove informative when addressing the mechanisms involved in vascular regeneration and hemangiogenic recovery in a clinical context.This work was supported
by the Spanish Ministry of Education and Science Grant
SAF64679, Junta de Andalucia Grants PAI-BIO-295 and
PAIDI-CTS1614, CONSOLIDER INGENIO 2010 Grant, the
Leukemia Research Fund, fellowship CONACYT-179065 to
A.M.G.-O., fellowship I3P-CSIC to C.Q., and fellowship UPO
to A.C. A.M.G.-O. and A.C. contributed equally to this work.Peer reviewe
Enhanced hemato-vascular contribution of SCL 3′ enhancer expressing fetal liver cells uncovers their potential to integrate in extramedullary adult niches
Fetal liver (FL) hematopoietic progenitors have superior blood engraftment competence compared with adult bone marrow (BM), however less is known about FL in vivo vascular capacity. Here we show in transplantation assays that FL cells possess enhanced vascular endothelial potential compared with adult bone marrow. We generated high‐level hematopoietic chimeras using donor cells from mice transgenic for the stem cell leukaemia 3′ enhancer human placental alkaline phosphatase (SCL3′Enh‐PLAP) reporter construct, active in vascular endothelium, and blood progenitor and stem cells. Long‐term lineage tracing analysis revealed PLAP+ vascular‐like patches in FL‐derived chimeras, whereas adult BM‐derived chimeras presented only rare and scattered PLAP+ cells. PLAP+ vascular‐like patches were formed following transplantation into both newborn and adult recipient mice, although their frequency was reduced in adult recipients. Confocal analysis of multiple labeled tissues revealed that whereas most liver and heart PLAP+ vascular patch‐associated cells were endothelial, PLAP+ vascular patches in the kidney contained endothelial, hematopoietic, and putative hemangioblastic cells. Moreover, fluorescence‐activated cell sorting assays showed that only FL PLAPbright+ donor cells can generate PLAP+ vascular patches upon transplantation. Taken together, these data demonstrate superior vascular contribution potential of FL cells, and not only provide new insights into the developmental pathways controlling endothelial development but also may prove informative when addressing the mechanisms involved in vascular regeneration and hemangiogenic recovery in a clinical context
Enhanced Hematovascular Contribution of SCL 3′ Enhancer Expressing Fetal Liver Cells Uncovers Their Potential to Integrate in Extramedullary Adult Niches
13 páginas, 6 figuras, 1 tabla.-- et al.Fetal liver (FL) hematopoietic progenitors have superior blood engraftment competence compared with adult bone marrow (BM), however less is known about FL in vivo vascular capacity. Here we show in transplantation assays that FL cells possess enhanced vascular endothelial potential compared with adult bone marrow. We generated high-level hematopoietic chimeras using donor cells from mice transgenic for the stem cell leukaemia 3′ enhancer human placental alkaline phosphatase (SCL3′Enh-PLAP) reporter construct, active in vascular endothelium, and blood progenitor and stem cells. Long-term lineage tracing analysis revealed PLAP+ vascular-like patches in FL-derived chimeras, whereas adult BM-derived chimeras presented only rare and scattered PLAP+ cells. PLAP+ vascular-like patches were formed following transplantation into both newborn and adult recipient mice, although their frequency was reduced in adult recipients. Confocal analysis of multiple labeled tissues revealed that whereas most liver and heart PLAP+ vascular patch-associated cells were endothelial, PLAP+ vascular patches in the kidney contained endothelial, hematopoietic, and putative hemangioblastic cells. Moreover, fluorescence-activated cell sorting assays showed that only FL PLAPbright+ donor cells can generate PLAP+ vascular patches upon transplantation. Taken together, these data demonstrate superior vascular contribution potential of FL cells, and not only provide new insights into the developmental pathways controlling endothelial development but also may prove informative when addressing the mechanisms involved in vascular regeneration and hemangiogenic recovery in a clinical context.This work was supported
by the Spanish Ministry of Education and Science Grant
SAF64679, Junta de Andalucia Grants PAI-BIO-295 and
PAIDI-CTS1614, CONSOLIDER INGENIO 2010 Grant, the
Leukemia Research Fund, fellowship CONACYT-179065 to
A.M.G.-O., fellowship I3P-CSIC to C.Q., and fellowship UPO
to A.C. A.M.G.-O. and A.C. contributed equally to this work.Peer reviewe
Retinal lamination observed in WT and 0.1 mM 5 dpf morphant embryos treated with PTU.
<p>Green: Alexa-fluor-488-phalloidin: actin. Blue: TOPRO: nuclei. Red: Zpr-1: double cone photoreceptors. hc: horizontal cells. os: outer segments. is: inner segments si.</p
Ultra structural architecture analysis of inner ear and stereocilia performed on MO1 morphant embryos using fluorescent staining.
<p>A. Phalloidin staining of WT larvae inner ear highlighting the three sensory areas containing stereocilia. B. Phalloidin staining of 0.1: A and B: 20 µm. C and D: 5 µm.</p
Evolutionary analysis of USH2A isoform_a.
<p>Alignment of the exons that correspond to the hypothetic short isoforms of <i>USH2A.</i> Only in Primates and most Eutherians the “KCV end” seems to be conserved.</p
Primers used to amplify and sequence the full-length open reading frame (ORF) of <i>Ol-Ush2a</i>.
<p>Primers used to amplify and sequence the full-length open reading frame (ORF) of <i>Ol-Ush2a</i>.</p
Spatial and temporal <i>Ol-Ush2a</i> expression pattern analysis performed by RT-PCR amplification.
<p><b>A</b>: RT-PCR amplification from adult organs. <b>B</b>: RT-PCR amplification from different embryonic developmental stages.</p