Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Nov 27:14:41.
doi: 10.1186/s12861-014-0041-x.

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

Jose Arturo Gutierrez-Triana  1   2 , Ulrich Herget  3   4 , Patrick Lichtner  5 , Luis A Castillo-Ramírez  6   7 , Soojin Ryu  8
Affiliations

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

Jose Arturo Gutierrez-Triana et al. BMC Dev Biol. .

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Genomic structure and evolutionarily conserved non-coding regions (ECRs) of the otp gene. A, Graphical comparison of the otp locus, according to the ECR browser, showing intergenic ECRs (red) and exons (blue) in the zebrafish (genome assembly danRer7), the human (genome assembly hg19) and the mouse (genome assembly mm10). We used the sequence of the otpa gene from zebrafish as the reference. B, A schematic phylogenetic tree of vertebrates. otp ECRs are evident only in gnathostome vertebrates. We noted that some ECRs were absent in the paralogous gene otpb in zebrafish (see text for more details).
Figure 2
Figure 2
Enhancer activity displayed by stable otp ECR-transgenic lines. A, A schematic depiction of constructs used to generate the transgenic lines. B, Enhancer activity displayed by transgenic lines analyzed at 3-5 dpf. The table shows the GFP levels and expression patterns observed in cardiac-RFP positive founders for the individual otpECRs. C, Numbers of larvae showing GFP+ cells within different brain regions (Data from 3 independent clutches are shown, n = 50 per clutch). In all animals, the caudal telecephalic-preoptic cluster is present, and in many animals, few ectopic cells appear in the tectal-tegmental region. D, Confocal z-stack maximum projection of a dorsal in vivo view of Tg(otpECR6-E1b:mmGFP) expression. GFP is expressed in a dense cluster within the preoptic area and extends into the caudal telencephalon. In many larvae, a few isolated ectopic cells can be detected in the midbrain. E, The evolutionarily conserved region otpECR6 contains predicted binding sites for transcription factors belonging to the families Otx, Pax, Nkx and Zic. The otpECR6 DNA sequences of zebrafish (Dr) and human (Hs) were aligned and analyzed using the multiF program (https://multitf.dcode.org of the ECR browser). Abbreviations: Tel, telencephalon; PO, preoptic area; PT, posterior tuberculum; H, hypothalamus; TeO, optic tectum; T, tegmentum; MO, medulla oblongata; r, rostral; l, lateral. Scale bar: 100 μm.
Figure 3
Figure 3
otp ECR6 displays enhancer activity in the forebrain with expression in cells localizing within the caudal telencephalon and dorsal half of the preoptic Otpa domain. A, Confocal z-stack maximum projection of an immunohistochemically stained 5 dpf Tg(otpECR6-E1b:mmGFP) larva double-labeled for GFP (A’) and Otpa (A”). B, Reconstructed localization and variability of GFP-expressing cells. Colors represent three representative maps derived from three different animals after registration using the outlines of the Otpa signal as references (transparent surface). Abbreviations: Tel, telencephalon; PO, preoptic area; PT, posterior tuberculum; H, hypothalamus; Hb, hindbrain; Pit, pituitary; ac, anterior commissure; TeO, optic tectum; Ha, habenula; PTh, prethalamus; oc, optic chiasm; poc, postoptic commissure; r, rostral; l, lateral; d, dorsal. Scale bars: 100 μm.
Figure 4
Figure 4
NPO cells involved in the HPA axis are labeled in the Tg(otpECR6-E1b:mmGFP) transgenic line. A, There is no overlap of GFP with in situ stained cck+ cells. B, There is some overlap of GFP with penka+ cells. C, There is extensive overlap of GFP with oxt+ cells. D, There is no overlap of GFP with vip+ cells. E, There is a high degree of overlap of GFP with avp+ cells. F, There is some overlap of GFP with sst1.1+ cells. G, There is a high degree of overlap of GFP with crh + cells. H, There is some overlap of GFP with nts+ cells. I, There is no overlap of GFP with penkb + cells. J, There is no overlap of GFP with immunostained TH+ cells. Abbreviations: r, rostral; l, lateral. Scale bars: 50 μm.
Figure 5
Figure 5
A group of cells labeled by the activity of the otp ECR6 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.
Figure 6
Figure 6
Nitroreductase-expressing cells in Tg(otpECR6-E1b:nfsb-GFP) larvae display a reduction in the number of projections to the pituitary and an increase in apoptotic bodies when exposed to Mtz. A, TUNEL staining of the preoptic GFP expression domain in an untreated larva. B, TUNEL staining of the preoptic GFP expression domain in an Mtz-treated larva. C, Pituitary bundles of GFP-stained fibers in a control larva (arrow). D, The pituitary location in an Mtz-treated larva (arrow). Note that in the control larva, the GFP fibers reach the pituitary and no apoptosis is detected, but in the Mtz-treated larva, the pituitary innervation is disrupted and apoptosis is apparent in preoptic cell bodies. Scale bars: 50 μm.
Figure 7
Figure 7
Mtz-induced impairment of the cortisol response to stress in Tg(otpECR6-E1b:nfsb-GFP) larvae. A, A schematic illustration of a functional stress axis (fibers of the hypothalamo-pituitary complex in green, interrenal gland in blue) and an impaired stress axis after Mtz treatment. B-C, Basal and stress-induced cortisol levels in Tg(otpECR6-E1b:mmGFP) and Tg(otpECR6-E1b:nfsb-GFP) larvae, treated or untreated with Mtz.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Acampora D, Postiglione MP, Avantaggiato V, Di Bonito M, Simeone A. The role of Otx and Otp genes in brain development. Int J Dev Biol. 2000;44(6):669–677. - PubMed
    1. Simeone A, D’Apice MR, Nigro V, Casanova J, Graziani F, Acampora D, Avantaggiato V. Orthopedia, a novel homeobox-containing gene expressed in the developing CNS of both mouse and Drosophila. Neuron. 1994;13(1):83–101. doi: 10.1016/0896-6273(94)90461-8. - DOI - PubMed
    1. Wang W, Lufkin T. The murine Otp homeobox gene plays an essential role in the specification of neuronal cell lineages in the developing hypothalamus. Dev Biol. 2000;227(2):432–449. doi: 10.1006/dbio.2000.9902. - DOI - PubMed
    1. Acampora D, Postiglione MP, Avantaggiato V, Di Bonito M, Vaccarino FM, Michaud J, Simeone A. Progressive impairment of developing neuroendocrine cell lineages in the hypothalamus of mice lacking the Orthopedia gene. Genes Dev. 1999;13(21):2787–2800. doi: 10.1101/gad.13.21.2787. - DOI - PMC - PubMed
    1. Blechman J, Borodovsky N, Eisenberg M, Nabel-Rosen H, Grimm J, Levkowitz G. Specification of hypothalamic neurons by dual regulation of the homeodomain protein Orthopedia. Development. 2007;134(24):4417–4426. doi: 10.1242/dev.011262. - DOI - PubMed

Publication types

LinkOut - more resources