HpEts, an ets-related transcription factor implicated in primary mesenchyme cell differentiation in the sea urchin embryo

Kurokawa, Daisuke; Kitajima, Takashi; Mitsunaga‐Nakatsubo, Keiko; Amemiya, Shonan; Shimada, Hiraku; Akasaka, Koji

doi:10.1016/s0925-4773(98)00192-0

Cited by 108 publications

(127 citation statements)

References 46 publications

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“…To explore the possibility of inserting a reporter cassette into a genomic site of interest in the sea urchin, H. pulcherrimus, we selected the HpEts1L and HpEts1R ZFNs, whose target sites (5′-GGGGTTGACG-3′ and 5′-GATGATGACT-3′, respectively) are located upstream of the stop codon of the HpEts1 gene responsible for PMC differentiation (16), by bacterial one-hybrid (B1H) and single-strand annealing (SSA) screenings (8) (Fig. 1A and Fig.…”

Section: Resultsmentioning

confidence: 99%

“…2 D-I and Table 1). In some embryos, GFP fluorescence was observed in the nuclei of PMCs, in which HpEts1 is expressed (16,17), and was never detected in other cell types. Consistent with the genomic PCR analysis, GFP-expressing embryos were more frequently observed after injection of Ets-HRD+T, HpEts1 ZFNs, and DNlig4 mRNAs than after injections with other combinations.…”

Section: Resultsmentioning

confidence: 99%

“…To explore this possibility, we examined the fluorescence intensity of GFP-expressing PMCs and the ingression of PMCs in embryos injected with HpEts1 ZFN mRNAs, DN-lig4 mRNA, Ets-HRD+T, and a moderate dose of an mRNA for a dominant-negative form of HpEts1 (ΔHpEts) (ref. 16; Fig. 4).…”

Section: Quantitative Imaging Of Endogenous Gene Expression In Livingmentioning

confidence: 94%

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Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos

Ochiai

Sakamoto

Fujita

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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To understand complex biological systems, such as the development of multicellular organisms, it is important to characterize the gene expression dynamics. However, there is currently no universal technique for targeted insertion of reporter genes and quantitative imaging in multicellular model systems. Recently, genome editing using zinc-finger nucleases (ZFNs) has been reported in several models. ZFNs consist of a zinc-finger DNA-binding array with the nuclease domain of the restriction enzyme FokI and facilitate targeted transgene insertion. In this study, we successfully inserted a GFP reporter cassette into the HpEts1 gene locus of the sea urchin, Hemicentrotus pulcherrimus. We achieved this insertion by injecting eggs with a pair of ZFNs for HpEts1 with a targeting donor construct that contained ∼1-kb homology arms and a 2A-histone H2B-GFP cassette. We increased the efficiency of the ZFNmediated targeted transgene insertion by in situ linearization of the targeting donor construct and cointroduction of an mRNA for a dominant-negative form of HpLig4, which encodes the H. pulcherrimus homolog of DNA ligase IV required for error-prone nonhomologous end joining. We measured the fluorescence intensity of GFP at the single-cell level in living embryos during development and found that there was variation in HpEts1 expression among the primary mesenchyme cells. These findings demonstrate the feasibility of ZFN-mediated targeted transgene insertion to enable quantification of the expression levels of endogenous genes during development in living sea urchin embryos.live imaging | quantitative biology

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos

Ochiai

Sakamoto

Fujita

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…Recent evidence suggests that a member of the ets gene family is involved in PMC differentiation (Kurokawa et al 1999).…”

Section: Discussionmentioning

confidence: 99%

Hbox1 and Hbox7 are involved in pattern formation in sea urchin embryos

et al. 1999

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In spite of their potential importance in evolution, there is little information about Hox genes in animal groups that are related to ancestors of deuterostome. It has been reported that only two Hox genes (Hbox1 and Hbox7) are expressed significantly in sea urchin embryos. Expression of Hbox1 protein is restricted to the aboral ectoderm, and Hbox7 expression is restricted to oral ectoderm, endoderm and secondary mesenchyme cells in sea urchin embryos after the gastrula stage. With the aim of gaining insight into the role of Hbox1 and Hbox7 in sea urchin development, Hbox1 and Hbox7 overexpression experiments were performed. Overexpression of Hbox1 repressed the development of oral ectoderm, endoderm and mesenchyme cells. On the contrary, overexpression of Hbox7 repressed the development of aboral ectoderm and primary mesenchyme cells. The data suggest that Hbox1 and Hbox7 are expressed in distinct non‐overlapping territories, and overexpression of either one inhibits territory‐specific gene expression in the domain of the other. It is proposed that an important function of both Hbox1 and Hbox7 genes is to maintain specific territorial gene expression by each one, in its domain of expression, while repressing the expression of the other in this same domain.

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“…2 A. They are four essential regulators of downstream micromere lineage function: alx1 (29), ets1 (9,33), tbr (34,35), and tel (9), plus SoxC, which in this lineage plays no role as it turns itself off shortly after activation.…”

Section: The Genomic Program For Initial Specification Of the Micromerementioning

confidence: 99%

Global regulatory logic for specification of an embryonic cell lineage

Oliveri

Davidson

2008

Proc. Natl. Acad. Sci. U.S.A.

358

532

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HpEts, an ets-related transcription factor implicated in primary mesenchyme cell differentiation in the sea urchin embryo

Cited by 108 publications

References 46 publications

Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos

Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos

Hbox1 and Hbox7 are involved in pattern formation in sea urchin embryos

Global regulatory logic for specification of an embryonic cell lineage

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