Mitochondrial Metabolism and Hif1a induce the Organizer

Mitochondrial leak metabolism induces the Spemann-Mangold Organizer via Hif-1α in Xenopus

Alexandra MacColl Garfinkel, Nelli Mnatsakanyan, Jeet H Patel, Andrea E Wills, Amy Shteyman, Peter J S Smith, Kambiz N Alavian, Elizabeth Ann Jonas, Mustafa K Khokha

Dev Cell. 2023 Sep 1;S1534-5807(23)00411-2. doi: 10.1016/j.devcel.2023.08.015.

PMID: 37673063 DOI: 10.1016/j.devcel.2023.08.015

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Abstract

An instructive role for metabolism in embryonic patterning is emerging, although a role for mitochondria is poorly defined. We demonstrate that mitochondrial oxidative metabolism establishes the embryonic patterning center, the Spemann-Mangold Organizer, via hypoxia-inducible factor 1α (Hif-1α) in Xenopus. Hypoxia or decoupling ATP production from oxygen consumption expands the Organizer by activating Hif-1α. In addition, oxygen consumption is 20% higher in the Organizer than in the ventral mesoderm, indicating an elevation in mitochondrial respiration. To reconcile increased mitochondrial respiration with activation of Hif-1α, we discovered that the "free" c-subunit ring of the F1Fo ATP synthase creates an inner mitochondrial membrane leak, which decouples ATP production from respiration at the Organizer, driving Hif-1α activation there. Overexpression of either the c-subunit or Hif-1α is sufficient to induce Organizer cell fates even when β-catenin is inhibited. We propose that mitochondrial leak metabolism could be a general mechanism for activating Hif-1α and Wnt signaling.

Figure 2. Hif-1α is sufficient to induce ectopic Organizer gene expression

(A) Representative images showing that hif-1α mRNA overexpression induces ectopic chordin at NF stage 10.

(B) Compared with the WT, lrpprc CRISPR mutants (lrpprc CR) have expanded gsc and chordin and reduced vent2; hif-1α MOATG rescues the expression to WT levels.

Images are representative; vegetal view, dorsal at top; n numbers noted are on graphs; minimum three biological replicates per condition (∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05). See also Figure S2. Data are presented as mean ± SD.

Figure 3. Hif-1α drives dorsal gene expression in β-catenin KD animals

(A) hif-1α mRNA is sufficient to rescue gsc (WT, n = 31; β-catenin MOATG, n = 22; hif-1α mRNA + β-catenin MOATG, n = 39), chordin (WT, n = 34; β-catenin MOATG, n = 19; hif-1α mRNA + β-catenin MOATG, n = 36), and vent2 (WT, n = 15; β-catenin MOATG, n = 10; hif-1α mRNA + β-catenin MOATG, n = 21) expression in β-catenin MOATG KD embryos.

(B) Hypoxia is also sufficient to rescue gsc (WT, n = 33; β-catenin MOATG, n = 39; hypoxia + β-catenin MOATG, n = 38), chordin (WT, n = 44; β-catenin MOATG, n = 34; hypoxia + β-catenin MOATG, n = 32), and vent2 (WT, n = 36; β-catenin MOATG, n = 31; hypoxia + β-catenin MOATG, n = 29) expression in β-catenin MOATG KD embryos.

(C) Schematic representation of a cross between heterozygous Tg(pbin7Lef-dGFP) males and WT females yielding a 50% WT and 50% transgenic embryo clutch.

(D) Representative images of embryos at NF stage 8 stained for gfp expression via whole-mount in situ hybridization. Those injected with NLS-β-catenin or hif-1α mRNA show higher gfp expression than control embryos (n numbers noted in the images).

(E) Western blot confirms that β-catenin protein levels remain reduced in β-catenin KD embryos injected with hif-1α mRNA.

(F) Representative images of embryos at NF stage 8. Embryos injected with β-catenin MO have lower gfp expression than controls; hif-1α mRNA can rescue gfp expression in β-catenin-depleted embryos.

Minimum three biological replicates per condition for all experiments; ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗p < 0.05). See also Figure S3. Data are presented as mean ± SD.

Figure 6. ATP synthase c-subunit induces the Organizer via Hif-1α

(A) mRNA overexpression of the ATP synthase c-subunit (atp5g3) is sufficient to induce dose-dependent ectopic dorsal and neural structures; arrows denote cement glands (a transient organ that appears at the rostral end of developing facial structures).

(B) Ectopic expression of chordin detected via whole-mount in situ hybridization also occurs in a dose-dependent response to overexpression of c-subunit (atp5g3) mRNA at the 1-cell stage (n numbers noted in the images).

(C and D) Embryos were injected with both Rose-β-D-Gal and c-subunit (atp5g3; n = 125) or hif-1α mRNA (n = 161) at the 16- to 32-cell stage and were scored for regional colocalization of Rose-β-D-Gal and chordin signal in dorsal, mediolateral, and ventral regions of the 3 germ layers (pink arrows, visible Rose-β-D-Gal signal; purple arrows, visible chordin signal; two-way ANOVA, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p = 0.0001). Overexpression of c-subunit mRNA results in dorsal and ventral exogenous chordin expression, whereas the effect of overexpression of hif-1α mRNA is regionally restricted.

(E) Embryos were injected with both c-subunit (atp5g3) mRNA and hif-1α MOATG. hif-1α MOATG prevents the c-subunit-dependent ectopic Organizer phenotype.

(F) Embryos injected with β-catenin MOATG with or without atp5g3 mRNA. c-subunit overexpression is sufficient to rescue chordin and establish a putative Organizer in β-catenin MO knockdown embryos (n numbers are noted in the images; at least three biological replicates per condition). Embryos are positioned with the dorsal pole at the top in all panels. Data are presented as mean ± SD.

Adapted with permission from Science Direct on behalf of Developmental Cell: MacColl-Garfinkel et al. (2023). Mitochondrial leak metabolism induces the Spemann-Mangold Organizer via Hif-1α in Xenopus. Dev Cell. 2023 Sep 1;S1534-5807(23)00411-2. doi: 10.1016/j.devcel.2023.08.015.