MondoA regulates gene expression in cholesterol biosynthesis-associated pathways required for zebrafish epiboly

Abstract
The glucose-sensing Mondo pathway regulates expression of metabolic genes in mammals. Here, we characterized its function in the zebrafish and revealed an unexpected role of this pathway in vertebrate embryonic development. We showed that knockdown of mondoa impaired the early morphogenetic movement of epiboly in zebrafish embryos and caused microtubule defects. Expression of genes in the terpenoid backbone and sterol biosynthesis pathways upstream of pregnenolone synthesis was coordinately downregulated in these embryos, including the most downregulated gene nsdhl. Loss of Nsdhl function likewise impaired epiboly, similar to MondoA loss of function. Both epiboly and microtubule defects were partially restored by pregnenolone treatment. Maternal-zygotic mutants of mondoa showed perturbed epiboly with low penetrance and compensatory changes in the expression of terpenoid/sterol/steroid metabolism genes. Collectively, our results show a novel role for MondoA in the regulation of early vertebrate development, connecting glucose, cholesterol and steroid hormone metabolism with early embryonic cell movements.

eLife digest

In most animals, a protein called MondoA closely monitors the amount of glucose in the body, as this type of sugar is the fuel required for many life processes. Glucose levels also act as a proxy for the availability of other important nutrients. Once MondoA has detected glucose molecules, it turns genetic programmes on and off depending on the needs of the cell. So far, these mechanisms have mainly been studied in adult cells.
However, recent studies have shown that proteins that monitor nutrient availability, and their associated pathways, can control early development. MondoA had not been studied in this context before, so Weger et al. decided to investigate its role in embryonic development. The experiments used embryos from zebrafish, a small freshwater fish whose early development is easily monitored and manipulated in the laboratory.
Inhibiting production of the MondoA protein in zebrafish embryos prevented them from maturing any further, stopping their development at an early key stage. This block was caused by defects in microtubules, the tubular molecules that act like a microscopic skeleton to provide structural support for cells and guide transport of cell components.
In addition, the pathway involved in the production of cholesterol and cholesterol-based hormones was far less active in embryos lacking MondoA. Treating MondoA-deficient embryos with one of these hormones corrected the microtubule defects and let the embryos progress to more advanced stages of development.
These results reveal that, during development, the glucose sensor MondoA also controls pathways involved in the creation of cholesterol and associated hormones. These new insights into the metabolic regulation of development could help to understand certain human conditions; for example, certain patients with defective cholesterol pathway genes also show developmental perturbations. In addition, the work highlights a biological link between cholesterol production and cellular responses to glucose, which Weger et al. hope could one day help to identify new cholesterol-lowering drugs.

Introduction

The glucose-sensing Mondo pathway is a key regulator of energy metabolism (Abdul-Wahed et al., 2017; Richards et al., 2017). It consists of three basic helix-loop-helix/leucine zipper (bHLH/Zip) family transcription factors (TFs): the two Mondo family factors ‘Mlx interacting protein’ (Mlxip; MondoA) and ‘Mlx interacting protein-like’ (Mlxipl; or ChREBP for ‘carbohydrate response element binding protein’) and their binding partner ‘Max-like protein X’ (Mlx) (Havula and Hietakangas, 2018; Richards et al., 2017). After their activation by glucose, likely via glucose-6-phosphate (G6P) (Dentin et al., 2012; Li et al., 2010b; Stoltzman et al., 2008), the Mondo family factors regulate gene expression as MondoA:Mlx or ChREBP:Mlx heterodimers by binding E-box type enhancer elements (‘carbohydrate response elements’, ‘ChoREs’) in the regulatory regions of their target genes (Billin et al., 2000; Jeong et al., 2011; Koo and Towle, 2000; Ma et al., 2006; Poungvarin et al., 2015; Yamashita et al., 2001).
While recent studies have highlighted a crucial role for metabolism and metabolic signaling in embryonic development (Miyazawa and Aulehla, 2018), research on Mondo pathway function has concentrated on adult animals (Hunt et al., 2015; Iizuka et al., 2004; Imamura et al., 2014; Song et al., 2019). As it is currently unknown if and how MondoA, ChREBP or Mlx contribute to metabolic regulation of embryogenesis, we investigated a potential developmental role of the Mondo pathway in zebrafish, a model for early vertebrate development (Nüsslein-Volhard, 2002). After fertilization, a cap of cells (the blastoderm) forms on top of the yolk cell, which it subsequently engulfs in a morphogenetic movement called epiboly (Bruce, 2016). Mechanistically, epiboly involves processes both in the enveloping layer (EVL) as well as the deep cell layer (DEL) of the blastoderm and in a teleost specific structure of the yolk cell, the yolk syncytial layer (YSL). By the end of epiboly, germ layers and body axes have been formed. Most developmental pathways as well as many facets of metabolism and its regulation are highly conserved between mammals and zebrafish (Gut et al., 2017; Schier and Talbot, 2005; Solnica-Krezel and Sepich, 2012). However, studies addressing Mondo pathway function in the zebrafish are still lacking.
Herein, we uncover a role of the Mondo pathway in vertebrate development. Mondo pathway gene expression and function was characterized in zebrafish embryos and cultured cells. Morpholino oligonucleotide (MO) mediated loss-of-function of MondoA severely impaired epiboly movements. Subsequent transcriptome analysis revealed genes deregulated upon loss of MondoA function and highlighted an enzyme involved in cholesterol biosynthesis, Nsdhl (NAD(P) dependent steroid dehydrogenase-like), as a potential key mediator of MondoA function in epiboly. Functional analysis of Nsdhl suggests that Nsdhl-mediated cholesterol synthesis downstream of MondoA is required for the synthesis of sufficient levels of the steroid hormone pregnenolone, which stabilizes YSL microtubules necessary for epiboly. Maternal-zygotic (MZ) mutant embryos homozygous for a small deletion allele of mondoa leading to a premature stop codon showed a severe aberrant epiboly phenotype, albeit with low penetrance that our transcriptome analysis indicates to result from compensatory changes in the expression of cholesterol and steroid biosynthesis genes.

Results

Glucose signaling by the Mondo pathway is conserved in zebrafish

For each of the Mondo pathway members MondoA, ChREBP and Mlx one single orthologue is present in the zebrafish genome (GRCz11/danRer11). We cloned the full cDNAs for zebrafish MondoA and ChREBP (GenBank Accession KF713493 [mondoa], KF713494 [chrebp]) based on the (partially) predicted sequences. Phylogenetic analysis showed that zebrafish mondoa, chrebp and mlx cluster with their mammalian and chicken homologs (Figure 1A) and revealed a high level of protein sequence conservation between zebrafish and human orthologs (Figure 1B), especially of the glucose-sensing module (GSM) specific to the Mondo family and of the DNA binding bHLH/Zip domains. This finding suggests that the functions of these proteins are conserved.

The glucose-sensing Mondo pathway in zebrafish.

(A) Phylogenetic tree of ChREBP, MondoA and Mlx proteins. h, human, m, mouse, b, bovine, g, chicken, zf, zebrafish. Outgroup: zf Neurogenin 1 (Neurog 1). Scale bar: 0.1 estimated amino acid substitutions per site. (B) Amino acid identities in % between zebrafish and human domains of ChREBP, MondoA and Mlx: ‘Mondo conserved regions/glucose-sensing module’ (MCR/GSM); ‘low-glucose inhibitory domain’ (LID; light blue); ‘glucose-response activation conserved element’ (GRACE; dark blue); ‘basic-helix-loop-helix/leucine zipper’ (bHLH/ZIP; green); ‘dimerization and cytoplasmic localization domain’ (DCD; red). (C, D) Bioluminescence levels after 24 hr of glucose treatment of PAC2 cells transiently transfected with the 2xChoRE reporter consisting of a luciferase reporter gene (yellow) regulated by a minimal promoter (TATA; arrow) and two carbohydrate response elements (ChoREs; each with the sequence 5’-CACGCG-N5-CTCGTG-3’; pA for polyadenylation site; n = 4, (C) or with the constitutively expressed pGL3-Control reporter construct (D, n = 4). (E, F) Bioluminescence levels in 2xChoRE reporter expressing PAC2 cells upon 24 hr of treatment with 0.3 mM (white bars) or 12 mM (black bars) glucose after overexpression of MondoA and/or Mlx (E, n = 4) or transfection with mondoa-mo or mondoa-mis (F, n = 8). Data were normalized to Renilla luciferase activity (Norm. bioluminescence). (G–I) mRNA expression profiles of mondoa (G), chrebp (H) and mlx (I) during zebrafish developmental stages from zygote to larval stage five extracted from a published dataset (White et al., 2017). n = 20; FKPM, Fragments Per Kilobase of transcript per Million mapped reads. (J) WISH of mondoa transcripts at zygote (maternal), 50% epiboly (50% epi.) and 18-somite stages. as, antisense probe, ss, sense probe. Scale bar: 0.2 mm. (K) Epon sections of 50% epi. embryos showed mondoa expression in the enveloping layer (EVL), the deep cell layer (DEL) of the blastoderm (Bl) and the yolk syncyctial layer (YSL). Scale bar: 0.2 mm, for higher magnification 50 µm. (L–O) Glucose induction of Mondo pathway target gene expression in early zebrafish embryos. Embryos were injected with the glucose analog 2-deoxy-D-glucose (2-DG; black bars) or with water (white bars) as a control. RNA was extracted at the sphere stage to perform RT-qPCR of genes known to be Mondo pathway targets in mammals: hexokinase 2 (hk2, L), fatty acid synthase (fasn, M), thioredoxin-interacting protein a (txnipa, N), eukaryotic translation elongation factor 1 alpha 1 (ef1a, O) (n = 9). (P) Embryos (n ≥ 72) injected with the 2xChoRE reporter showed increased bioluminescence at sphere stage when co-injected with 2-DG. Error bars represent SEM; *, p≤0.05; **, p≤0.01; ***, p≤0.001.

To examine whether the zebrafish Mondo pathway factors function similarly in the regulation of gene transcription as their mammalian orthologs, we studied the pathway in zebrafish PAC2 cells (Lin et al., 1994). All three Mondo pathway factors are expressed in these cells (Figure 1—figure supplement 1A). To monitor Mondo signaling, we generated a luciferase reporter gene construct driven by two ChoREs fitting the mammalian consensus (Ma et al., 2006) and a TATA box minimal promoter (Figure 1C). This construct was active in HepG2 cells, a mammalian cell culture model commonly used for Mondo pathway studies (Kim et al., 1996; Yu and Luo, 2009; Figure 1—figure supplement 1B). In PAC2 cells, bioluminescence equally increased in a dose-dependent manner upon glucose treatment (Figure 1C). No significant changes in bioluminescence levels were detected upon glucose treatment in cells expressing a constitutively active luciferase reporter (pGL3-Control; Figure 1D), excluding a general unspecific increase in transcriptional activity by glucose treatment.
We next tested whether overexpression of Mondo pathway members enhances glucose induced pathway activity. Transient overexpression of MondoA and Mlx activated transcription from the reporter also under low glucose conditions (Figure 1E), as shown by the increased bioluminescence compared with transfection of the 2xChoRE reporter alone (p≤0.001). Overexpression of the MondoA and Mlx factors together led to an even more pronounced effect on bioluminescence (p≤0.01), revealing synergistic effects. High glucose levels caused significant reporter gene induction in control cells (p≤0.01; Figure 1E). Importantly, strong glucose induction of the reporter was also shown by cells overexpressing either Mlx (p≤0.01) or MondoA (p≤0.001) alone as well as both factors together (p≤0.001; Figure 1E). An unrelated control reporter construct (pGRE-Luc, Weger et al., 2012) was not responsive to any of the treatments (Figure 1—figure supplement 1C). Together, the data indicate that limited availability of endogenous pathway components restrains pathway activity, and that both basal activity and the response to higher glucose levels are potentiated when more sensor proteins are available.
In addition, we explored the effect of morpholino oligonucleotide (MO) mediated loss-of-function of mondoa to confirm that Mondo pathway function is required for glucose induction of reporter expression. Cells transfected with a control 5 bp mismatch MO (mondoa-mis) showed a 2.0-fold induction of bioluminescence from the 2xChoRE reporter plasmid by high glucose levels, while in cells transfected with a MO directed against the translation start site (mondoa-mo) this glucose response was abolished (Figure 1F). Taken together, our data demonstrate that ChoRE mediated glucose induction of gene expression is regulated by the Mondo pathway also in zebrafish.

The zebrafish Mondo pathway is present in early embryos

After confirming the similarity of zebrafish Mondo pathway function to mammals in cultured cells, we studied its role during development. We reanalyzed a previously published zebrafish developmental transcriptome dataset (White et al., 2017, ENA accession number ERP014517) and detected maternal transcripts of mondoa, chrebp and mlx as well as expression of the three genes throughout development, following distinct temporal patterns (Figure 1G–I). Whole mount in situ hybridization analysis (WISH) for mondoa revealed a ubiquitous expression pattern (Figure 1J). Similar results were observed for chrebp and mlx (Figure 1—figure supplement 1D,E). At the 50% epiboly stage, all three genes are expressed throughout the embryo (Figure 1K; Figure 1—figure supplement 1F).
Next, we tested whether glucose treatment increases the expression of mammalian Mondo pathway target gene homologs in early zebrafish embryos, namely hexokinase 2 (hk2) (Sans et al., 2006), fatty acid synthase (fasn) (Ma et al., 2006) and thioredoxin-interacting protein a (txnipa) (Stoltzman et al., 2008). We injected the glucose analogue 2-deoxy-glucose (2-DG), which is metabolized to the G6P analogue 2-DG6P but not further (Chi et al., 1987; Stoltzman et al., 2008), thereby avoiding activation of other pathways relying on downstream metabolization of glucose. This treatment significantly increased expression of hk2 (p
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