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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.


CNS2 contains hypothalamic enhancer activity and responses to FGF signaling.(A-B) Double in situ hybridization results indicate CNS2 contains hypothalamic enhancer activity. The hypothalamic marker nkx2.1a and nkx2.2b are stained in dark blue, reporter egfp stained in red. (C-D) SU5402 treatment severely reduces CNS2 activity. Vehicle DMSO treated embryos show restricted hypothalamic EGFP reporter expression (pointed by the arrow in C). Embryos treated with the FGF signaling inhibitor SU5402 during the segmentation stage (10-24hpf) show minimal EGFP signals in the hypothalamic region (arrow in D, n = 48/55). (E-F) SU5402 treatment down-regulates endogenous lhx5 expression in the hypothalamic region. Endogenous lhx5 shows robust expression in the hypothalamic region (pointed by the arrow in E). SU5402 treatment during the segmentation stage down-regulates lhx5 expression in the hypothalamic region (arrow in F, n = 25/28). (G) Multiple sequence alignments of the CNS2 region in the five teleost species. The identified FGF downstream factor Pea3 binding site is highlighted in blue. Lateral view of the forebrain regions of embryos at 24 hpf (A-F), anterior to the left. Scale bar: 40μm in A-B; 50μm in C-D.
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pone.0132525.g003: CNS2 contains hypothalamic enhancer activity and responses to FGF signaling.(A-B) Double in situ hybridization results indicate CNS2 contains hypothalamic enhancer activity. The hypothalamic marker nkx2.1a and nkx2.2b are stained in dark blue, reporter egfp stained in red. (C-D) SU5402 treatment severely reduces CNS2 activity. Vehicle DMSO treated embryos show restricted hypothalamic EGFP reporter expression (pointed by the arrow in C). Embryos treated with the FGF signaling inhibitor SU5402 during the segmentation stage (10-24hpf) show minimal EGFP signals in the hypothalamic region (arrow in D, n = 48/55). (E-F) SU5402 treatment down-regulates endogenous lhx5 expression in the hypothalamic region. Endogenous lhx5 shows robust expression in the hypothalamic region (pointed by the arrow in E). SU5402 treatment during the segmentation stage down-regulates lhx5 expression in the hypothalamic region (arrow in F, n = 25/28). (G) Multiple sequence alignments of the CNS2 region in the five teleost species. The identified FGF downstream factor Pea3 binding site is highlighted in blue. Lateral view of the forebrain regions of embryos at 24 hpf (A-F), anterior to the left. Scale bar: 40μm in A-B; 50μm in C-D.

Mentions: We used molecular markers to further examine the reporter EGFP expression in animals carrying the CNS2 enhancer element. nkx2.1a and nkx2.2b are two transcription factors that are specifically expressed in the ventral forebrain, in the hypothalamic regions at 24 hpf. Two-color in situ hybridization results showed reporter EGFP and the two nkx markers were co-expressed in the hypothalamic regions. The region expressing nkx2.1a was broader than the EGFP expressing region, whilst the nkx2.2b expressing region almost overlapped the EGFP expressing region in the hypothalamus (Fig 3A and 3B).


Conserved Noncoding Sequences Regulate lhx5 Expression in the Zebrafish Forebrain.

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

CNS2 contains hypothalamic enhancer activity and responses to FGF signaling.(A-B) Double in situ hybridization results indicate CNS2 contains hypothalamic enhancer activity. The hypothalamic marker nkx2.1a and nkx2.2b are stained in dark blue, reporter egfp stained in red. (C-D) SU5402 treatment severely reduces CNS2 activity. Vehicle DMSO treated embryos show restricted hypothalamic EGFP reporter expression (pointed by the arrow in C). Embryos treated with the FGF signaling inhibitor SU5402 during the segmentation stage (10-24hpf) show minimal EGFP signals in the hypothalamic region (arrow in D, n = 48/55). (E-F) SU5402 treatment down-regulates endogenous lhx5 expression in the hypothalamic region. Endogenous lhx5 shows robust expression in the hypothalamic region (pointed by the arrow in E). SU5402 treatment during the segmentation stage down-regulates lhx5 expression in the hypothalamic region (arrow in F, n = 25/28). (G) Multiple sequence alignments of the CNS2 region in the five teleost species. The identified FGF downstream factor Pea3 binding site is highlighted in blue. Lateral view of the forebrain regions of embryos at 24 hpf (A-F), anterior to the left. Scale bar: 40μm in A-B; 50μm in C-D.
© Copyright Policy
Related In: Results  -  Collection

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pone.0132525.g003: CNS2 contains hypothalamic enhancer activity and responses to FGF signaling.(A-B) Double in situ hybridization results indicate CNS2 contains hypothalamic enhancer activity. The hypothalamic marker nkx2.1a and nkx2.2b are stained in dark blue, reporter egfp stained in red. (C-D) SU5402 treatment severely reduces CNS2 activity. Vehicle DMSO treated embryos show restricted hypothalamic EGFP reporter expression (pointed by the arrow in C). Embryos treated with the FGF signaling inhibitor SU5402 during the segmentation stage (10-24hpf) show minimal EGFP signals in the hypothalamic region (arrow in D, n = 48/55). (E-F) SU5402 treatment down-regulates endogenous lhx5 expression in the hypothalamic region. Endogenous lhx5 shows robust expression in the hypothalamic region (pointed by the arrow in E). SU5402 treatment during the segmentation stage down-regulates lhx5 expression in the hypothalamic region (arrow in F, n = 25/28). (G) Multiple sequence alignments of the CNS2 region in the five teleost species. The identified FGF downstream factor Pea3 binding site is highlighted in blue. Lateral view of the forebrain regions of embryos at 24 hpf (A-F), anterior to the left. Scale bar: 40μm in A-B; 50μm in C-D.
Mentions: We used molecular markers to further examine the reporter EGFP expression in animals carrying the CNS2 enhancer element. nkx2.1a and nkx2.2b are two transcription factors that are specifically expressed in the ventral forebrain, in the hypothalamic regions at 24 hpf. Two-color in situ hybridization results showed reporter EGFP and the two nkx markers were co-expressed in the hypothalamic regions. The region expressing nkx2.1a was broader than the EGFP expressing region, whilst the nkx2.2b expressing region almost overlapped the EGFP expressing region in the hypothalamus (Fig 3A and 3B).

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.