Duncan AR et al. (2019), Alkylglycerol monooxygenase, a heterotaxy candi...

XB-ART-56196

Dev Biol 2019 Dec 01;4561:1-7. doi: 10.1016/j.ydbio.2019.07.019.

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Alkylglycerol monooxygenase, a heterotaxy candidate gene, regulates left-right patterning via Wnt signaling.

Duncan AR , González DP , Del Viso F , Robson A , Khokha MK , Griffin JN .

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Congenital heart disease (CHD) is a major cause of morbidity in the pediatric population yet its genetic and molecular causes remain poorly defined. Previously, we identified AGMO as a candidate heterotaxy disease gene, a disorder of left-right (LR) patterning that can have a profound effect on cardiac function. AGMO is the only known alkylglycerol monooxygenase, an orphan tetrahydrobiopterin dependent enzyme that cleaves the ether linkage in alkylglycerols. However, whether AGMO plays a role in LR patterning was unexplored. Here we reveal that Agmo is required for correct development of the embryonic LR axis in Xenopus embryos recapitulating the patient's heterotaxy phenotype. Mechanistically, we demonstrate that Agmo is a regulator of canonical Wnt signaling, required during gastrulation for normal formation of the left - right organizer. Mutational analysis demonstrates that this function is dependent on Agmo's alkylglycerol monooxygenase activity. Together, our findings identify Agmo as a regulator of canonical Wnt signaling, demonstrate a role for Agmo in embryonic axis formation, and provide insight into the poorly understood developmental requirements for ether lipid cleavage.

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Species referenced: Xenopus
Genes referenced: agmo ctnnb1 dand5 gdf3 nodal1
GO keywords: Wnt signaling pathway

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	Fig. 1. agmo is required for L-R development. A) Depletion of Agmo disrupts cardiac looping. Lower images are pseudo-colored to highlight cardiac looping. B) Expression of pitx2c, which is normally only in left side mesoderm (upper row), is impaired in Agmo morphants (typically absent, lower row). abn.; abnormal C) coco expression is greatly reduced in the LRO from early in its development (before the initiation of flow, stage 14). LROs are dissected out and imaged ventrally. A; anterior, P; posterior, L; left, R; right. D) Expression of xnr1 and E) gdf3 is reduced in the left – right organizer of agmo morphants. Figure shows three developmental stages: stage 14 (pre-directional fluid flow) and stage 16 and 19. Graphs represent the combined results of six independent experiments for cardiac looping and xnr1, three repeats for pitx2, five repeats for coco, and 3 independent repeats for gdf3. UC; uninjected control, MO; morphant, ns; non-significant, p < 0.05, p < 0.01, ***p < 0.0001 by Chi-square analysis.
	Fig. 2. Depletion of agmo impairs gastrulation. A) Time lapse images of gastrulation in uninjected control (UC, upper row), embryos injected with 1 ng MOATG (middle row) and embryos injected with 2 ng MOATG (lower row). UC embryos gastrulate normally, successfully closing the blastopore and forming the neural plate (green arrowheads). 1 ng MOATG embryos typically require several attempts to fully close the blastopore, and occasionally fail to complete gastrulation (blue arrowhead). 2 ng MOATG embryos never complete gastrulation despite multiple attempts (red arrowheads). B) The agmo morphant gastrulation phenotype can be rescued by co-injection of human AGMO mRNA (hRNA). Mini-ruby traces MOATG injection and GFP traces hRNA. Note that MOATG only embryos fail to gastrulate while co-injection of hRNA rescues the phenotype. Graph on right displays quantification of the human AGMO mRNA rescue. Data represent the combined result of three independent experiments. UC; uninjected control, MO; morphant. ****p < 0.0001 by Chi-square analysis.
	Fig. 3. agmo mediates canonical Wnt signaling. A) TOPflash assays reveal that Wnt driven luciferase production is reduced in agmo morphants throughout gastrulation (stages 9, 10, 12). B) Injection of agmo mRNA alone had no significant effect on luciferase activity, while co-injection of agmo mRNA and β-catenin mRNA increased Wnt activity significantly relative to injection of β-catenin alone. In A and B 10 embryos were pooled in each condition, and graphs represent the combined results of three biological and technical replicates. C) β-catenin protein levels are reduced in morphants at stage 10 and 12 as assayed by Western blot. D) Western blot analysis reveals that β-catenin protein levels are reduced in both the cytoplasmic (Cyto) and nuclear (Nuc) fractions of agmo morphants. The graph represents the combined result of four independent experiments. AGMO, AGMO mRNA; Top, TOPFlash reporter plasmid; Fop, FOPFlash negative control; BC, TOPFlash reporter plasmid + β-catenin mRNA; BC + MO, TOPFlash reporter plasmid + β-catenin mRNA + agmo morpholino; UC, uninjected control. All error bars represent ± sd. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 determined by students T-test.
	Fig. 4. Agmo mediates Wnt activity via its alkylglycerol monooxygenase activity. A) Injection of AGMO RNA alone can induce secondary axes, while injection of the catalytically dead H136A and H149A mutants cannot. Photos are representative examples of single (green dotted line) and AGMO induced double axes (red dotted lines) as seen at stage 19. Graph displays percentage of double axes in uninjected control embryos (UC), in embryos injected with wild type AGMO mRNA, and embryos injected with the H136A or H149A AGMO mutant mRNA. Data represents the combined outcome of three independent experiments. p < 0.05, **p < 0.001 by students T-test.

References [+] :

Alrayes, The alkylglycerol monooxygenase (AGMO) gene previously involved in autism also causes a novel syndromic form of primary microcephaly in a consanguineous Saudi family. 2016, Pubmed

	Fig. 1. agmo is required for L-R development. A) Depletion of Agmo disrupts cardiac looping. Lower images are pseudo-colored to highlight cardiac looping. B) Expression of pitx2c, which is normally only in left side mesoderm (upper row), is impaired in Agmo morphants (typically absent, lower row). abn.; abnormal C) coco expression is greatly reduced in the LRO from early in its development (before the initiation of flow, stage 14). LROs are dissected out and imaged ventrally. A; anterior, P; posterior, L; left, R; right. D) Expression of xnr1 and E) gdf3 is reduced in the left – right organizer of agmo morphants. Figure shows three developmental stages: stage 14 (pre-directional fluid flow) and stage 16 and 19. Graphs represent the combined results of six independent experiments for cardiac looping and xnr1, three repeats for pitx2, five repeats for coco, and 3 independent repeats for gdf3. UC; uninjected control, MO; morphant, ns; non-significant, p < 0.05, p < 0.01, ***p < 0.0001 by Chi-square analysis.
	Fig. 2. Depletion of agmo impairs gastrulation. A) Time lapse images of gastrulation in uninjected control (UC, upper row), embryos injected with 1 ng MOATG (middle row) and embryos injected with 2 ng MOATG (lower row). UC embryos gastrulate normally, successfully closing the blastopore and forming the neural plate (green arrowheads). 1 ng MOATG embryos typically require several attempts to fully close the blastopore, and occasionally fail to complete gastrulation (blue arrowhead). 2 ng MOATG embryos never complete gastrulation despite multiple attempts (red arrowheads). B) The agmo morphant gastrulation phenotype can be rescued by co-injection of human AGMO mRNA (hRNA). Mini-ruby traces MOATG injection and GFP traces hRNA. Note that MOATG only embryos fail to gastrulate while co-injection of hRNA rescues the phenotype. Graph on right displays quantification of the human AGMO mRNA rescue. Data represent the combined result of three independent experiments. UC; uninjected control, MO; morphant. ****p < 0.0001 by Chi-square analysis.
	Fig. 3. agmo mediates canonical Wnt signaling. A) TOPflash assays reveal that Wnt driven luciferase production is reduced in agmo morphants throughout gastrulation (stages 9, 10, 12). B) Injection of agmo mRNA alone had no significant effect on luciferase activity, while co-injection of agmo mRNA and β-catenin mRNA increased Wnt activity significantly relative to injection of β-catenin alone. In A and B 10 embryos were pooled in each condition, and graphs represent the combined results of three biological and technical replicates. C) β-catenin protein levels are reduced in morphants at stage 10 and 12 as assayed by Western blot. D) Western blot analysis reveals that β-catenin protein levels are reduced in both the cytoplasmic (Cyto) and nuclear (Nuc) fractions of agmo morphants. The graph represents the combined result of four independent experiments. AGMO, AGMO mRNA; Top, TOPFlash reporter plasmid; Fop, FOPFlash negative control; BC, TOPFlash reporter plasmid + β-catenin mRNA; BC + MO, TOPFlash reporter plasmid + β-catenin mRNA + agmo morpholino; UC, uninjected control. All error bars represent ± sd. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001 determined by students T-test.
	Fig. 4. Agmo mediates Wnt activity via its alkylglycerol monooxygenase activity. A) Injection of AGMO RNA alone can induce secondary axes, while injection of the catalytically dead H136A and H149A mutants cannot. Photos are representative examples of single (green dotted line) and AGMO induced double axes (red dotted lines) as seen at stage 19. Graph displays percentage of double axes in uninjected control embryos (UC), in embryos injected with wild type AGMO mRNA, and embryos injected with the H136A or H149A AGMO mutant mRNA. Data represents the combined outcome of three independent experiments. p < 0.05, **p < 0.001 by students T-test.