Yoko Tabata

Specification of the Retinal Fate of Mouse Embryonic Stem Cells by Ectopic Expression of Rx/rax, a Homeobox Gene

ABSTRACT With the goal of generating retinal cells from mouse embryonic stem (ES) cells by exogenous gene transfer, we introduced the Rx/rax transcription factor, which is expressed in immature retinal cells, into feeder-free mouse ES cells (CCE). CCE cells expressing Rx/rax as well as enhanced green fluorescent protein (CCE-RX/E cells) proliferated and remained in the undifferentiated state in the presence of leukemia inhibitory factor, as did parental ES cells. We made use of mouse embryo retinal explant cultures to address the differentiation ability of grafted ES cells. Dissociated embryoid bodies were treated with retinoic acid for use as donor cells and cocultured with retina explants for 2 weeks. In contrast to the parental CCE cells, which could not migrate into host retinal cultures, CCE-RX/E cells migrated into the host retina and extended their process-like structures between the host retinal cells. Most of the grafted CCE-RX/E cells became located in the ganglion cell and inner plexiform layers and expressed ganglion and horizontal cell markers. Furthermore, these grafted cells had the electrophysiological properties expected of ganglion cells. Our data thus suggest that subpopulations of retinal neurons can be generated in retinal explant cultures from grafted mouse ES cells ectopically expressing the transcription factor Rx/rax.

Negative regulation of retinal-neurite extension by beta-catenin signaling pathway.

Although there have been many studies on the regulation of neurite extension in mouse brain, such a mechanism in neural retina has remained to be clarified. To delineate the role of Wnt signaling in retinal development, we used a retrovirus-vector-mediated expression system to express various mutants forms of Wnt signaling members in E17.5 mouse retinal explant cultures, which are an excellent system to examine retinal development in vitro. Expression of constitutively active β-catenin or Lef-1 in the retinal cells resulted in failure of neurite extension, suggesting that β-catenin negatively regulates neurite extension in the retina through Lef-1 transcriptional activity. However, proliferation and differentiation of retinal cells into mature retinal cells such as rod-photoreceptor cells and Muller glia cells were not affected by perturbation of the Wnt-Lef-1 pathway. As in retinal cells, activation of β-catenin-Lef-1 signaling inhibited NGF-induced neurite extension in PC12 cells without affecting their proliferation. Interestingly, the Wnt-Lef-1 signaling pathway suppressed neurite extension without affecting Mek-1 signal activity, which is known to promote neurite extension. We found that MAPK was activated in retinal explant cultures, but that perturbation of MAPK signals did not affect neurite extension. Taken together, our data suggest that the Wnt pathway functions in proper neurite extension by opposing positive signals for promotion of neurite extension that are distinct from those of the MAPK pathway.

CD73, a Novel Cell Surface Antigen That Characterizes Retinal Photoreceptor Precursor Cells

PURPOSE The authors sought to identify cell surface markers of photoreceptor and its precursor cells. METHODS The expression of surface CD antigens that label both temporally and spatially distinct populations of mouse retinal cells were examined. Of the antibodies that showed positive signals in retinal cells, CD73 was focused on for more detailed analyses. RESULTS Mouse retinal subpopulations that expressed CD73 first appeared around birth and subsequently increased dramatically in number, eventually representing more than 90% of the retinal cells in the adult. CD73(+) cells were postmitotic and mostly rhodopsin-negative at postnatal day 1. However, in the adult retina, most of these cells expressed rhodopsin but not s-opsin. In reaggregation cultures, CD73(+) cells differentiated into rhodopsin-positive cells more rapidly than CD73(-) cells, which supports the idea that CD73 is an early photoreceptor lineage marker. The effects of ectopic expression in retinal cells of Nrl and Crx, both of which are transcription factors known to be expressed in photoreceptor lineage, suggest that CD73 is genetically downstream of Crx in the rod cell differentiation lineage. Adult retina of the common marmoset monkey also showed correlation of the expression pattern of rhodopsin and CD73. CONCLUSIONS CD73 is a cell surface marker of cone/rod common precursors and mature rod cells in mice and is genetically localized between Nrl and Crx. The expression of CD73 was conserved in primate rod cells, and CD73 provides an useful tool to purify photoreceptor cells for transplantation aimed at the regeneration of photoreceptors.

Forkhead transcription factor foxe1 regulates chondrogenesis in zebrafish

Forkhead transcription factor (Fox) e1 is a causative gene for Bamforth-Lazarus syndrome, which is characterized by hypothyroidism and cleft palate. Applying degenerate polymerase chain reaction using primers specific for the conserved forkhead domain, we identified zebrafish foxe1 (foxe1). Foxe1 is expressed in the thyroid, pharynx, and pharyngeal skeleton during development; strongly expressed in the gill and weakly expressed in the brain, eye, and heart in adult zebrafish. A loss of function of foxe1 by morpholino antisense oligo (MO) exhibited abnormal craniofacial development, shortening of Meckels cartilage and the ceratohyals, and suppressed chondrycytic proliferation. However, at 27 hr post fertilization, the foxe1 MO-injected embryos showed normal dlx2, hoxa2, and hoxb2 expression, suggesting that the initial steps of pharyngeal skeletal development, including neural crest migration and specification of the pharyngeal arch occurred normally. In contrast, at 2 dpf, a severe reduction in the expression of sox9a, colIIaI, and runx2b, which play roles in chondrocytic proliferation and differentiation, was observed. Interestingly, fgfr2 was strongly upregulated in the branchial arches of the foxe1 MO-injected embryos. Unlike Foxe1-null mice, normal thyroid development in terms of morphology and thyroid-specific marker expression was observed in foxe1 MO-injected zebrafish embryos. Taken together, our results indicate that Foxe1 plays an important role in chondrogenesis during development of the pharyngeal skeleton in zebrafish, probably through regulation of fgfr2 expression. Furthermore, the roles reported for FOXE1 in mammalian thyroid development may have been acquired during evolution.

Melk-Like Kinase Plays a Role in Hematopoiesis in the Zebra Fish

ABSTRACT A serine/threonine kinase, Melk, was initially cloned in mouse oocytes as a maternal gene, but whose function was unknown. In adult mice, Melk was strongly expressed in the thymus and bone marrow, suggesting a role for Melk in hematopoiesis. We cloned a Melk-like gene from zebra fish (zMelk). zMelk-like gene was expressed in the brain and lateral mesoderm at 12 hours postfertilization (hpf) and in several tissues of adult fish, including the kidney and spleen, both of which are known to be hematopoietic tissues in zebra fish. Abrogation of zMelk-like gene function by zMelk-like gene-specific Morpholino (MO) resulted in abnormal swelling around the tectum region. In addition, the start of blood circulation was severely delayed but, in contrast, the vessel formation seemed normal. Expression of scl, gata-1, and lmo-2 was down regulated at 12 to 14 hpf in the zMelk-like gene MO-injected embryos, and the coexpression of gata-1 rescued the anemic phenotype induced by zMelk-like gene MO. Expression of the zMelk-like gene in embryos enhanced gata-1 promoter-dependent enhanced green fluorescent protein expression, suggesting that the zMelk-like gene affects gata-1 expression at the transcriptional level. Taken together, our data suggest that the zMelk-like gene may play a role in primitive hematopoiesis by affecting the expression of genes critical for hematopoiesis.

Zebrafish Numb homologue : Phylogenetic evolution and involvement in regulation of left-right asymmetry

Numb and its homologue, Numb-like (Numbl), play important roles in mammalian development, but their role in embryonic development of lower vertebrates remains unknown. We cloned a zebrafish numb homologue (znumb) by searching database. znumb shows approximately 60% identity with mammalian Numb orthologs. Interstingly, znumb lacks two specific sequence motifs unique to Numbl orthologs. However, chromosomal localization of znumb gene revealed colinearity with genes located around mouse and human Numbl genes. Furthermore, multi-species comparisons of conserved phosphotyrosine-binding (PTB) domain sequences in Numb and Numbl proteins suggest that znumb is more closely related to Numbl than Numb. znumb mRNA was expressed in a wide variety of zebrafish adult tissues. Overexpression of znumb in embryos resulted in an absence, or reversal, of the normal leftward shift of the developing heart tube. Furthermore, no or bi-lateral transcripts of lefty2 were observed in znumb-expressing embryos, suggesting that the Notch signaling was essential for left-right field formation and maintenance in zebrafish, and that znumb perturbed this process through down-regulation of endogenous Notch signaling.

Transcriptional Repressor foxl1 Regulates Central Nervous System Development by Suppressing shh Expression in Zebra Fish

ABSTRACT We identified zebra fish forkhead transcription factor l1 (zfoxl1) as a gene strongly expressed in neural tissues such as midbrain, hindbrain, and the otic vesicle at the early embryonic stage. Loss of the function of zfoxl1 effected by morpholino antisense oligonucleotide resulted in defects in midbrain and eye development, and in that of formation of the pectoral fins. Interestingly, ectopic expression of shh in the midbrain and elevated pax2a expression in the optic stalk were observed in foxl1 MO-injected embryos. In contrast, expression of pax6a, which is negatively regulated by shh, was suppressed in the thalamus and pretectum regions, supporting the idea of augmentation of the shh signaling pathway by suppression of foxl1. Expression of zfoxl1-EnR (repressing) rather than zfoxl1-VP16 (activating) resulted in a phenotype similar to that induced by foxl1-mRNA, suggesting that foxl1 may act as a transcriptional repressor of shh in zebra fish embryos. Supporting this notion, foxl1 suppressed isolated 2.7-kb shh promoter activity in PC12 cells, and the minimal region of foxl1 required for its transcriptional repressor activity showed strong homology with the groucho binding motif, which is found in genes encoding various homeodomain proteins. In view of all of our data taken together, we propose zfoxl1 to be a novel regulator of neural development that acts by suppressing shh expression.

CD138/syndecan-1 and SSEA-1 mark distinct populations of developing ciliary epithelium that are regulated differentially by Wnt signal.

Ciliary epithelium (CE), which consists of nonpigmented and pigmented layers, develops from the optic vesicle. However, the molecular mechanisms underlying CE development have not been closely examined, in part because cell‐surface markers suitable for specific labeling of subregions of the retina were unknown. Here, we identified CD138/syndecan‐1 and stage specific embryonic antigen‐1 (SSEA‐1) CD15 as cell‐surface antigens marking nonpigmented and pigmented CE, respectively. During retinal development, both CD138 and SSEA‐1 were expressed in the early stage, and segregation of these markers in the tissue began at around embryonic day (E) 10. As a result, CD138‐positive (CD138+) cells were found at the most distal tip of the retina, and SSEA‐1+ cells were found in the periphery adjacent to the area of CD138 expression. In vitro characterization of isolated CD138+ or SSEA‐1+ cell subpopulations revealed that CD138+ cells lose their retinal progenitor characteristics between E13 and E16, suggesting that they commit to becoming nonpigmented CE cells within this period. By in vivo mouse models, we found that stabilized β‐catenin expanded the area of CD138+ nonpigmented CE and that elimination of β‐catenin inhibited development of nonpigmented CE cells. These findings are the first to use cell‐surface markers to ascertain the spatial and temporal transitions that occur in developing CE.

Critical roles of DNase1l3l in lens nuclear degeneration in zebrafish.

The vertebrate lens undergoes organelle and nuclear degradation during lens development, allowing the lens to become transparent. DNase2b is an enzyme responsible for nuclear degradation in the mouse lens; however, dnase2b expression in zebrafish showed a distribution pattern that differed from that in mice. No zebrafish dnase2b was detected by reverse-transcription polymerase chain reaction until around 120 h postfertilization (hpf), suggesting that dnase2b is not expressed in the critical period for lens nuclear degradation, which corresponds to 56-74 hpf. However, public database searches have indicated that dnase1l3l is strongly and specifically expressed in embryonic zebrafish lens. Whole mount in situ hybridization showed that dnase1l3l expression began around 36 hpf and was found exclusively in the lens until the adult stage. Morpholino (MO)-dependent downregulation of dnase1l3l expression during early development in zebrafish led to the failure of nuclear degradation in the lens. Immunostaining of lens sections showed that expression of Pax6, Prox1 and β-catenin was comparable to the control in the early stage of development in dnase1l3l-MO injected embryos. However, downregulation of expression of these genes in lens was not observed in dnase1l3l-MO-treated zebrafish at 72 hpf, suggesting that the lens development was halted. Taken together, we showed that dnase1l3l plays major roles in nuclear degradation in zebrafish lens development. No homologous gene was found in other species in public databases, suggesting that dnase1l3l developed and acquired its function specifically in zebrafish.