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Differential requirements for Gli2 and Gli3 in the regional specification of the mouse hypothalamus.

Haddad-Tóvolli R, Paul FA, Zhang Y, Zhou X, Theil T, Puelles L, Blaess S, Alvarez-Bolado G - Front Neuroanat (2015)

Bottom Line: The hypothalamus, a forebrain region regulating vital functions like homeostasis and hormone secretion, shows dynamic and intricate Shh expression as well as complex regional differentiation.Finally, we have mapped our results on a recent model which considers the hypothalamus as a transverse region with alar and basal portions.Our data confirm the model and are explained by it.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Cell Biology and Neuroanatomy, University of Heidelberg Heidelberg, Germany.

ABSTRACT
Secreted protein Sonic hedgehog (Shh) ventralizes the neural tube by modulating the crucial balance between activating and repressing functions (GliA, GliR) of transcription factors Gli2 and Gli3. This balance-the Shh-Gli code-is species- and context-dependent and has been elucidated for the mouse spinal cord. The hypothalamus, a forebrain region regulating vital functions like homeostasis and hormone secretion, shows dynamic and intricate Shh expression as well as complex regional differentiation. Here we asked if particular combinations of Gli2 and Gli3 and of GliA and GliR functions contribute to the variety of hypothalamic regions, i.e., we wanted to approach the question of a possible hypothalamic version of the Shh-Gli code. Based on mouse mutant analysis, we show that: (1) hypothalamic regional heterogeneity is based in part on differentially stringent requirements for Gli2 or Gli3; (2) another source of diversity are differential requirements for Shh of neural vs. non-neural origin; (3) the medial progenitor domain known to depend on Gli2 for its development generates several essential hypothalamic nuclei plus the pituitary and median eminence; (4) the suppression of Gli3R by neural and non-neural Shh is essential for hypothalamic specification. Finally, we have mapped our results on a recent model which considers the hypothalamus as a transverse region with alar and basal portions. Our data confirm the model and are explained by it.

No MeSH data available.


Related in: MedlinePlus

Gli3 promotes proliferation in the mamillary region. (A–C)In situ detection of Gli genes on E12.5 WT mouse brain sections. Arrowheads indicate the mamillary region. (D) LacZ reporter detection on an E12.5 Gli3-nlacZ knock-in mouse brain section; arrowhead indicates the mamillary region. (E) BrdU-labeled cells per bin at E12.5, genotypes as indicated. Unpaired t-test, two-tailed, mean ± SD; ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001. (F)Gli1 expression in the medial domain of the hypothalamus (arrows) on E10.5 horizontal sections of WT (upper panel) and Gli3Xt-J/Xt-J (lower panel) embryos. (G–I) Labeled cells in the mamillary region of WT E18.5 embryos after in utero electroporation with GFP and Ptch-δ-loop-tomato DNA constructs at E12.5. White arrowheads show double-labeled cells. (J) Percent of GFP-expressing cells co-expressing red reporter “tomato” after in utero electroporation of control (white bars) or experimental (black bars) constructs, in two different regions, as indicated. Unpaired t-test, two-tailed, mean ± SD; ∗∗p ≤ 0.01; n.s., non-significant.
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Figure 10: Gli3 promotes proliferation in the mamillary region. (A–C)In situ detection of Gli genes on E12.5 WT mouse brain sections. Arrowheads indicate the mamillary region. (D) LacZ reporter detection on an E12.5 Gli3-nlacZ knock-in mouse brain section; arrowhead indicates the mamillary region. (E) BrdU-labeled cells per bin at E12.5, genotypes as indicated. Unpaired t-test, two-tailed, mean ± SD; ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001. (F)Gli1 expression in the medial domain of the hypothalamus (arrows) on E10.5 horizontal sections of WT (upper panel) and Gli3Xt-J/Xt-J (lower panel) embryos. (G–I) Labeled cells in the mamillary region of WT E18.5 embryos after in utero electroporation with GFP and Ptch-δ-loop-tomato DNA constructs at E12.5. White arrowheads show double-labeled cells. (J) Percent of GFP-expressing cells co-expressing red reporter “tomato” after in utero electroporation of control (white bars) or experimental (black bars) constructs, in two different regions, as indicated. Unpaired t-test, two-tailed, mean ± SD; ∗∗p ≤ 0.01; n.s., non-significant.

Mentions: In parallel, we performed control experiments transfecting a 2:1 mixture of GFP construct and tdTomato construct (without loss-of-function Ptch1 protein) in order to evaluate how many only green and how many green-plus-red neurons we obtain in normal circumstances (i.e., without introducing any dominant loss-of-function). Those are the gray bars in Figure 10J. Additionally, these control experiments remove a possible concern related to the relative brightness of the green and the red reporters. In principle, a green cell could have been transfected also with some red (experimental) constructs in a number to small to be detected (since EmGFP is brighter than tdTomato). This possible source of imprecision can be disregarded since our readout is not absolute but relative (comparison between gray bars and black bars; Figure 10J).


Differential requirements for Gli2 and Gli3 in the regional specification of the mouse hypothalamus.

Haddad-Tóvolli R, Paul FA, Zhang Y, Zhou X, Theil T, Puelles L, Blaess S, Alvarez-Bolado G - Front Neuroanat (2015)

Gli3 promotes proliferation in the mamillary region. (A–C)In situ detection of Gli genes on E12.5 WT mouse brain sections. Arrowheads indicate the mamillary region. (D) LacZ reporter detection on an E12.5 Gli3-nlacZ knock-in mouse brain section; arrowhead indicates the mamillary region. (E) BrdU-labeled cells per bin at E12.5, genotypes as indicated. Unpaired t-test, two-tailed, mean ± SD; ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001. (F)Gli1 expression in the medial domain of the hypothalamus (arrows) on E10.5 horizontal sections of WT (upper panel) and Gli3Xt-J/Xt-J (lower panel) embryos. (G–I) Labeled cells in the mamillary region of WT E18.5 embryos after in utero electroporation with GFP and Ptch-δ-loop-tomato DNA constructs at E12.5. White arrowheads show double-labeled cells. (J) Percent of GFP-expressing cells co-expressing red reporter “tomato” after in utero electroporation of control (white bars) or experimental (black bars) constructs, in two different regions, as indicated. Unpaired t-test, two-tailed, mean ± SD; ∗∗p ≤ 0.01; n.s., non-significant.
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Related In: Results  -  Collection

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Figure 10: Gli3 promotes proliferation in the mamillary region. (A–C)In situ detection of Gli genes on E12.5 WT mouse brain sections. Arrowheads indicate the mamillary region. (D) LacZ reporter detection on an E12.5 Gli3-nlacZ knock-in mouse brain section; arrowhead indicates the mamillary region. (E) BrdU-labeled cells per bin at E12.5, genotypes as indicated. Unpaired t-test, two-tailed, mean ± SD; ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001. (F)Gli1 expression in the medial domain of the hypothalamus (arrows) on E10.5 horizontal sections of WT (upper panel) and Gli3Xt-J/Xt-J (lower panel) embryos. (G–I) Labeled cells in the mamillary region of WT E18.5 embryos after in utero electroporation with GFP and Ptch-δ-loop-tomato DNA constructs at E12.5. White arrowheads show double-labeled cells. (J) Percent of GFP-expressing cells co-expressing red reporter “tomato” after in utero electroporation of control (white bars) or experimental (black bars) constructs, in two different regions, as indicated. Unpaired t-test, two-tailed, mean ± SD; ∗∗p ≤ 0.01; n.s., non-significant.
Mentions: In parallel, we performed control experiments transfecting a 2:1 mixture of GFP construct and tdTomato construct (without loss-of-function Ptch1 protein) in order to evaluate how many only green and how many green-plus-red neurons we obtain in normal circumstances (i.e., without introducing any dominant loss-of-function). Those are the gray bars in Figure 10J. Additionally, these control experiments remove a possible concern related to the relative brightness of the green and the red reporters. In principle, a green cell could have been transfected also with some red (experimental) constructs in a number to small to be detected (since EmGFP is brighter than tdTomato). This possible source of imprecision can be disregarded since our readout is not absolute but relative (comparison between gray bars and black bars; Figure 10J).

Bottom Line: The hypothalamus, a forebrain region regulating vital functions like homeostasis and hormone secretion, shows dynamic and intricate Shh expression as well as complex regional differentiation.Finally, we have mapped our results on a recent model which considers the hypothalamus as a transverse region with alar and basal portions.Our data confirm the model and are explained by it.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Cell Biology and Neuroanatomy, University of Heidelberg Heidelberg, Germany.

ABSTRACT
Secreted protein Sonic hedgehog (Shh) ventralizes the neural tube by modulating the crucial balance between activating and repressing functions (GliA, GliR) of transcription factors Gli2 and Gli3. This balance-the Shh-Gli code-is species- and context-dependent and has been elucidated for the mouse spinal cord. The hypothalamus, a forebrain region regulating vital functions like homeostasis and hormone secretion, shows dynamic and intricate Shh expression as well as complex regional differentiation. Here we asked if particular combinations of Gli2 and Gli3 and of GliA and GliR functions contribute to the variety of hypothalamic regions, i.e., we wanted to approach the question of a possible hypothalamic version of the Shh-Gli code. Based on mouse mutant analysis, we show that: (1) hypothalamic regional heterogeneity is based in part on differentially stringent requirements for Gli2 or Gli3; (2) another source of diversity are differential requirements for Shh of neural vs. non-neural origin; (3) the medial progenitor domain known to depend on Gli2 for its development generates several essential hypothalamic nuclei plus the pituitary and median eminence; (4) the suppression of Gli3R by neural and non-neural Shh is essential for hypothalamic specification. Finally, we have mapped our results on a recent model which considers the hypothalamus as a transverse region with alar and basal portions. Our data confirm the model and are explained by it.

No MeSH data available.


Related in: MedlinePlus