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Conserved Noncoding Sequences Regulate lhx5 Expression in the Zebrafish Forebrain.

Sun L, Chen F, Peng G - PLoS ONE (2015)

Bottom Line: We found a proximately located enhancer gave rise to robust reporter EGFP expression in the forebrain regions.We also identify an enhancer located approximately 40 kb upstream of the lhx5 coding region that is required for expression in the prethalamus (ventral thalamus).Together our results suggest discrete enhancer elements control lhx5 expression in different regions of the forebrain.

View Article: PubMed Central - PubMed

Affiliation: Institutes of Brain Science, State Key Laboratory of Medical Neurobiology and Collaborative Innovation Center for Brain Science, Fudan University, Shanghai, China.

ABSTRACT
The LIM homeobox family protein Lhx5 plays important roles in forebrain development in the vertebrates. The lhx5 gene exhibits complex temporal and spatial expression patterns during early development but its transcriptional regulation mechanisms are not well understood. Here, we have used transgenesis in zebrafish in order to define regulatory elements that drive lhx5 expression in the forebrain. Through comparative genomic analysis we identified 10 non-coding sequences conserved in five teleost species. We next examined the enhancer activities of these conserved non-coding sequences with Tol2 transposon mediated transgenesis. We found a proximately located enhancer gave rise to robust reporter EGFP expression in the forebrain regions. In addition, we identified an enhancer located at approximately 50 kb upstream of lhx5 coding region that is responsible for reporter gene expression in the hypothalamus. We also identify an enhancer located approximately 40 kb upstream of the lhx5 coding region that is required for expression in the prethalamus (ventral thalamus). Together our results suggest discrete enhancer elements control lhx5 expression in different regions of the forebrain.

No MeSH data available.


Region specific enhancer activity of the identified CNSs.(A-B) CNS8 and CNS9, located in the vicinity of the lhx5 promoter region give rise to broad reporter EGFP expression in the forebrain regions. (C) CNS2 located approximately 50 kb upstream of the lhx5 coding region gives rise to restricted EGFP signal in the anterior ventral forebrain. (D) CNS4 located 40 kb upstream of the lhx5 coding region, gives rise to restricted EGFP expression in the diencephalic region. (E) Vector construct gives rise to basal non-tissue specific EGFP expression in transient expression assay. Lateral view of the forebrain regions of embryos at 24 hpf, anterior to the left. Scale bar: 50μm.
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pone.0132525.g002: Region specific enhancer activity of the identified CNSs.(A-B) CNS8 and CNS9, located in the vicinity of the lhx5 promoter region give rise to broad reporter EGFP expression in the forebrain regions. (C) CNS2 located approximately 50 kb upstream of the lhx5 coding region gives rise to restricted EGFP signal in the anterior ventral forebrain. (D) CNS4 located 40 kb upstream of the lhx5 coding region, gives rise to restricted EGFP expression in the diencephalic region. (E) Vector construct gives rise to basal non-tissue specific EGFP expression in transient expression assay. Lateral view of the forebrain regions of embryos at 24 hpf, anterior to the left. Scale bar: 50μm.

Mentions: We then injected each construct into one-cell stage embryos obtained from a UAS:EGFP report line fish [25]. Three independent microinjections were performed for each of the 10 CNS constructs, and the fluorescent signals were examined at 24, 48 and 72 hours post fertilization (hpf). These transient reporter EGFP assays indicated CNS2, CNS4, CNS8, and CNS9 had tissue specific enhancer activities. While the CNS8 and CNS9 constructs gave rise to broad reporter EGFP expression in the forebrain regions, the CNS2 and CNS4 constructs produced more restricted EGFP expression in the forebrain (data not shown). In contrast, the construct carrying the basal promoter produced little or weak non-tissue specific fluorescent signals (Fig 2E). It should be noted that the CNS8 construct likely also carried basal promoter sequences from the endogenous lhx5 gene due to proximity of the CNS8 element and the lhx5 transcription start site. Thus it was unclear whether the CNS8 element contained region specific enhancer activity by itself.


Conserved Noncoding Sequences Regulate lhx5 Expression in the Zebrafish Forebrain.

Sun L, Chen F, Peng G - PLoS ONE (2015)

Region specific enhancer activity of the identified CNSs.(A-B) CNS8 and CNS9, located in the vicinity of the lhx5 promoter region give rise to broad reporter EGFP expression in the forebrain regions. (C) CNS2 located approximately 50 kb upstream of the lhx5 coding region gives rise to restricted EGFP signal in the anterior ventral forebrain. (D) CNS4 located 40 kb upstream of the lhx5 coding region, gives rise to restricted EGFP expression in the diencephalic region. (E) Vector construct gives rise to basal non-tissue specific EGFP expression in transient expression assay. Lateral view of the forebrain regions of embryos at 24 hpf, anterior to the left. Scale bar: 50μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4492605&req=5

pone.0132525.g002: Region specific enhancer activity of the identified CNSs.(A-B) CNS8 and CNS9, located in the vicinity of the lhx5 promoter region give rise to broad reporter EGFP expression in the forebrain regions. (C) CNS2 located approximately 50 kb upstream of the lhx5 coding region gives rise to restricted EGFP signal in the anterior ventral forebrain. (D) CNS4 located 40 kb upstream of the lhx5 coding region, gives rise to restricted EGFP expression in the diencephalic region. (E) Vector construct gives rise to basal non-tissue specific EGFP expression in transient expression assay. Lateral view of the forebrain regions of embryos at 24 hpf, anterior to the left. Scale bar: 50μm.
Mentions: We then injected each construct into one-cell stage embryos obtained from a UAS:EGFP report line fish [25]. Three independent microinjections were performed for each of the 10 CNS constructs, and the fluorescent signals were examined at 24, 48 and 72 hours post fertilization (hpf). These transient reporter EGFP assays indicated CNS2, CNS4, CNS8, and CNS9 had tissue specific enhancer activities. While the CNS8 and CNS9 constructs gave rise to broad reporter EGFP expression in the forebrain regions, the CNS2 and CNS4 constructs produced more restricted EGFP expression in the forebrain (data not shown). In contrast, the construct carrying the basal promoter produced little or weak non-tissue specific fluorescent signals (Fig 2E). It should be noted that the CNS8 construct likely also carried basal promoter sequences from the endogenous lhx5 gene due to proximity of the CNS8 element and the lhx5 transcription start site. Thus it was unclear whether the CNS8 element contained region specific enhancer activity by itself.

Bottom Line: We found a proximately located enhancer gave rise to robust reporter EGFP expression in the forebrain regions.We also identify an enhancer located approximately 40 kb upstream of the lhx5 coding region that is required for expression in the prethalamus (ventral thalamus).Together our results suggest discrete enhancer elements control lhx5 expression in different regions of the forebrain.

View Article: PubMed Central - PubMed

Affiliation: Institutes of Brain Science, State Key Laboratory of Medical Neurobiology and Collaborative Innovation Center for Brain Science, Fudan University, Shanghai, China.

ABSTRACT
The LIM homeobox family protein Lhx5 plays important roles in forebrain development in the vertebrates. The lhx5 gene exhibits complex temporal and spatial expression patterns during early development but its transcriptional regulation mechanisms are not well understood. Here, we have used transgenesis in zebrafish in order to define regulatory elements that drive lhx5 expression in the forebrain. Through comparative genomic analysis we identified 10 non-coding sequences conserved in five teleost species. We next examined the enhancer activities of these conserved non-coding sequences with Tol2 transposon mediated transgenesis. We found a proximately located enhancer gave rise to robust reporter EGFP expression in the forebrain regions. In addition, we identified an enhancer located at approximately 50 kb upstream of lhx5 coding region that is responsible for reporter gene expression in the hypothalamus. We also identify an enhancer located approximately 40 kb upstream of the lhx5 coding region that is required for expression in the prethalamus (ventral thalamus). Together our results suggest discrete enhancer elements control lhx5 expression in different regions of the forebrain.

No MeSH data available.