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A vertebrate-conserved cis-regulatory module for targeted expression in the main hypothalamic regulatory region for the stress response.

Gutierrez-Triana JA, Herget U, Lichtner P, Castillo-Ramírez LA, Ryu S - BMC Dev. Biol. (2014)

Bottom Line: To understand how this widely expressed transcription factor accomplishes hypothalamus-specific functions, we performed a comprehensive screening of otp cis-regulatory regions in zebrafish.Lastly, expression of the bacterial nitroreductase gene under this specific enhancer allowed pharmacological attenuation of the stress response in zebrafish larvae.Vertebrates share many cellular and molecular components of the stress response and our work identified a striking conservation at the cis-regulatory level of a key hypothalamic developmental gene.

View Article: PubMed Central - PubMed

Affiliation: Developmental Genetics of the Nervous System, Max Planck Institute for Medical Research, Jahnstrasse 29, D-69120, Heidelberg, Germany. arturo.gutierrez@cos.uni-heidelberg.de.

ABSTRACT

Background: The homeodomain transcription factor orthopedia (Otp) is an evolutionarily conserved regulator of neuronal fates. In vertebrates, Otp is necessary for the proper development of different regions of the brain and is required in the diencephalon to specify several hypothalamic cell types, including the cells that control the stress response. To understand how this widely expressed transcription factor accomplishes hypothalamus-specific functions, we performed a comprehensive screening of otp cis-regulatory regions in zebrafish.

Results: Here, we report the identification of an evolutionarily conserved vertebrate enhancer module with activity in a restricted area of the forebrain, which includes the region of the hypothalamus that controls the stress response. This region includes neurosecretory cells producing Corticotropin-releasing hormone (Crh), Oxytocin (Oxt) and Arginine vasopressin (Avp), which are key components of the stress axis. Lastly, expression of the bacterial nitroreductase gene under this specific enhancer allowed pharmacological attenuation of the stress response in zebrafish larvae.

Conclusion: Vertebrates share many cellular and molecular components of the stress response and our work identified a striking conservation at the cis-regulatory level of a key hypothalamic developmental gene. In addition, this enhancer provides a useful tool to manipulate and visualize stress-regulatory hypothalamic cells in vivo with the long-term goal of understanding the ontogeny of the stress axis in vertebrates.

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A group of cells labeled by the activity of theotpECR6 enhancer project to the pituitary. A-D, Costaining of RFP with Avp (A) or Oxt (C) as typical hypophysiotropic cell types shows a high degree of overlap of fibers reaching the pituitary (magnified views in B and D). E, Costaining of RFP with Avp and Oxt combined (magnified view in F). G, Costaining with ACTH as a pituitary marker confirms the dense RFP bundles as part of the pituitary (magnified view in H). Abbreviations: r, rostral; l, lateral. Scale bars: 100 μm.
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Fig5: A group of cells labeled by the activity of theotpECR6 enhancer project to the pituitary. A-D, Costaining of RFP with Avp (A) or Oxt (C) as typical hypophysiotropic cell types shows a high degree of overlap of fibers reaching the pituitary (magnified views in B and D). E, Costaining of RFP with Avp and Oxt combined (magnified view in F). G, Costaining with ACTH as a pituitary marker confirms the dense RFP bundles as part of the pituitary (magnified view in H). Abbreviations: r, rostral; l, lateral. Scale bars: 100 μm.

Mentions: To characterize the cell identity of the GFP-expressing cells driven by otpECR6, we performed immunohistochemistry and fluorescent in situ hybridization (FISH) using probes and antibodies against different neuropeptidergic markers previously described to be present in this region [28]. GFP cells in the NPO colocalized with neurosecretory cells expressing crh, avp, oxt, or proenkephalin a (penka) (Figure 4). 89% of the oxt–positive cells, and 81% of the avp–positive cells (based on IHC) showed colocalization with GFP; while 62% of the crh-positive cells, and 22% of the penka–positive cells (based on FISH) showed colocalization with GFP. In contrast, diencephalic DA cells and their projections were not included in the GFP expression domain (Figure 4). There was no colocalization with cells producing cholecystokinin (cck), vip, or proenkephalin b (penkb), and low colocalization with cells producing somatostatin (sst1.1) or neurotensin (nts). Because cck, vip, penkb, and nts cluster at the border of the NPO, we concluded that otpECR6 expression overlaps more with the central core region of the NPO, where crh, avp, oxt, and penka are clustered. A morphological hallmark of hypothalamic neurosecretory cells is their projection to the pituitary. To confirm the neurosecretory nature of the cells targeted using otpECR6, we generated a transgenic line expressing a membrane-tagged RFP to visualize cell projections. Indeed, we observed two sets of bilateral projections in Tg(otpECR6-E1b:RFP-CAAX). One set projects caudally, ending halfway along the spinal cord (not shown), whereas the other projects to the pituitary, as shown by co-staining with an ACTH antibody (Figure 5). The RFP projections toward the pituitary colocalize with Oxt and Avp projections. Taken together, our results indicate that otpECR6 is a functional regulatory element with activity in key neurosecretory cells of the NPO.Figure 4


A vertebrate-conserved cis-regulatory module for targeted expression in the main hypothalamic regulatory region for the stress response.

Gutierrez-Triana JA, Herget U, Lichtner P, Castillo-Ramírez LA, Ryu S - BMC Dev. Biol. (2014)

A group of cells labeled by the activity of theotpECR6 enhancer project to the pituitary. A-D, Costaining of RFP with Avp (A) or Oxt (C) as typical hypophysiotropic cell types shows a high degree of overlap of fibers reaching the pituitary (magnified views in B and D). E, Costaining of RFP with Avp and Oxt combined (magnified view in F). G, Costaining with ACTH as a pituitary marker confirms the dense RFP bundles as part of the pituitary (magnified view in H). Abbreviations: r, rostral; l, lateral. Scale bars: 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4248439&req=5

Fig5: A group of cells labeled by the activity of theotpECR6 enhancer project to the pituitary. A-D, Costaining of RFP with Avp (A) or Oxt (C) as typical hypophysiotropic cell types shows a high degree of overlap of fibers reaching the pituitary (magnified views in B and D). E, Costaining of RFP with Avp and Oxt combined (magnified view in F). G, Costaining with ACTH as a pituitary marker confirms the dense RFP bundles as part of the pituitary (magnified view in H). Abbreviations: r, rostral; l, lateral. Scale bars: 100 μm.
Mentions: To characterize the cell identity of the GFP-expressing cells driven by otpECR6, we performed immunohistochemistry and fluorescent in situ hybridization (FISH) using probes and antibodies against different neuropeptidergic markers previously described to be present in this region [28]. GFP cells in the NPO colocalized with neurosecretory cells expressing crh, avp, oxt, or proenkephalin a (penka) (Figure 4). 89% of the oxt–positive cells, and 81% of the avp–positive cells (based on IHC) showed colocalization with GFP; while 62% of the crh-positive cells, and 22% of the penka–positive cells (based on FISH) showed colocalization with GFP. In contrast, diencephalic DA cells and their projections were not included in the GFP expression domain (Figure 4). There was no colocalization with cells producing cholecystokinin (cck), vip, or proenkephalin b (penkb), and low colocalization with cells producing somatostatin (sst1.1) or neurotensin (nts). Because cck, vip, penkb, and nts cluster at the border of the NPO, we concluded that otpECR6 expression overlaps more with the central core region of the NPO, where crh, avp, oxt, and penka are clustered. A morphological hallmark of hypothalamic neurosecretory cells is their projection to the pituitary. To confirm the neurosecretory nature of the cells targeted using otpECR6, we generated a transgenic line expressing a membrane-tagged RFP to visualize cell projections. Indeed, we observed two sets of bilateral projections in Tg(otpECR6-E1b:RFP-CAAX). One set projects caudally, ending halfway along the spinal cord (not shown), whereas the other projects to the pituitary, as shown by co-staining with an ACTH antibody (Figure 5). The RFP projections toward the pituitary colocalize with Oxt and Avp projections. Taken together, our results indicate that otpECR6 is a functional regulatory element with activity in key neurosecretory cells of the NPO.Figure 4

Bottom Line: To understand how this widely expressed transcription factor accomplishes hypothalamus-specific functions, we performed a comprehensive screening of otp cis-regulatory regions in zebrafish.Lastly, expression of the bacterial nitroreductase gene under this specific enhancer allowed pharmacological attenuation of the stress response in zebrafish larvae.Vertebrates share many cellular and molecular components of the stress response and our work identified a striking conservation at the cis-regulatory level of a key hypothalamic developmental gene.

View Article: PubMed Central - PubMed

Affiliation: Developmental Genetics of the Nervous System, Max Planck Institute for Medical Research, Jahnstrasse 29, D-69120, Heidelberg, Germany. arturo.gutierrez@cos.uni-heidelberg.de.

ABSTRACT

Background: The homeodomain transcription factor orthopedia (Otp) is an evolutionarily conserved regulator of neuronal fates. In vertebrates, Otp is necessary for the proper development of different regions of the brain and is required in the diencephalon to specify several hypothalamic cell types, including the cells that control the stress response. To understand how this widely expressed transcription factor accomplishes hypothalamus-specific functions, we performed a comprehensive screening of otp cis-regulatory regions in zebrafish.

Results: Here, we report the identification of an evolutionarily conserved vertebrate enhancer module with activity in a restricted area of the forebrain, which includes the region of the hypothalamus that controls the stress response. This region includes neurosecretory cells producing Corticotropin-releasing hormone (Crh), Oxytocin (Oxt) and Arginine vasopressin (Avp), which are key components of the stress axis. Lastly, expression of the bacterial nitroreductase gene under this specific enhancer allowed pharmacological attenuation of the stress response in zebrafish larvae.

Conclusion: Vertebrates share many cellular and molecular components of the stress response and our work identified a striking conservation at the cis-regulatory level of a key hypothalamic developmental gene. In addition, this enhancer provides a useful tool to manipulate and visualize stress-regulatory hypothalamic cells in vivo with the long-term goal of understanding the ontogeny of the stress axis in vertebrates.

Show MeSH
Related in: MedlinePlus