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The mevalonate pathway regulates primitive streak formation via protein farnesylation

View Article: PubMed Central - PubMed

ABSTRACT

The primitive streak in peri-implantation embryos forms the mesoderm and endoderm and controls cell differentiation. The metabolic cues regulating primitive streak formation remain largely unknown. Here we utilised a mouse embryonic stem (ES) cell differentiation system and a library of well-characterised drugs to identify these metabolic factors. We found that statins, which inhibit the mevalonate metabolic pathway, suppressed primitive streak formation in vitro and in vivo. Using metabolomics and pharmacologic approaches we identified the downstream signalling pathway of mevalonate and revealed that primitive streak formation requires protein farnesylation but not cholesterol synthesis. A tagging-via-substrate approach revealed that nuclear lamin B1 and small G proteins were farnesylated in embryoid bodies and important for primitive streak gene expression. In conclusion, protein farnesylation driven by the mevalonate pathway is a metabolic cue essential for primitive streak formation.

No MeSH data available.


The effects of statins on primitive streak formation in mouse EBs.(a) Microarray results for the top 35 downregulated genes in EBs treated with ATV during days 1–4. Data were compared with control EBs and analysed on days 3 and 4. Gene expression levels in ATV-treated EBs are expressed as Log2 (fold change) values relative to control EBs. (b) In situ hybridisation to detect T and Sox2 in EBs treated with or without ATV on days 1–6 and collected at the indicated times. Results are representative of >100 EBs/group. (c) Real-time PCR of T and Sox2 in EBs treated with or without ATV and/or MVA during days 1–4 and collected on day 4. Results were analysed as in Fig. 1b. *P < 0.05, **P < 0.0001.
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f2: The effects of statins on primitive streak formation in mouse EBs.(a) Microarray results for the top 35 downregulated genes in EBs treated with ATV during days 1–4. Data were compared with control EBs and analysed on days 3 and 4. Gene expression levels in ATV-treated EBs are expressed as Log2 (fold change) values relative to control EBs. (b) In situ hybridisation to detect T and Sox2 in EBs treated with or without ATV on days 1–6 and collected at the indicated times. Results are representative of >100 EBs/group. (c) Real-time PCR of T and Sox2 in EBs treated with or without ATV and/or MVA during days 1–4 and collected on day 4. Results were analysed as in Fig. 1b. *P < 0.05, **P < 0.0001.

Mentions: To gain initial insight into the mechanism by which statins affect early embryogenesis, we performed microarray analysis of EBs during days 3–4, which is the most sensitive two day period for ATV when compared to days 1–2 and days 5–6. ATV reduced the expression of 417 genes by 50% in EBs on days 3 and 4 (Fig. 2a). Ontology analysis revealed that many of these genes are involved in early embryogenesis, particularly gastrulation (GO: 0007369, P = 5.26 × 10−31) (Supplementary Table 2). Consistent with a role specifically in primitive streak formation, ATV markedly suppressed primitive streak and mesodermal markers (Lhx1, Wnt3, Wnt8, GSC, Fgf10 and Fgf8) as well as endodermal markers (Sox17 and Gata6) on day 4, which was reversed by MVA addition (Supplementary Figure 2a). In cultured mouse EBs, a primitive streak-like domain initiates a gastrulation process driven by many genes (Supplementary Figure 2b)17. We used real-time PCR and in situ hybridisation to examine the expression of the primitive streak marker Brachyury T (T) and the early ectodermal marker Sox2 in mouse EBs treated with ATV. As expected, T was expressed at high levels in a localised region in control EBs, however levels were significantly reduced by ATV treatment (Fig. 2b,c). This effect was partially rescued by MVA (Fig. 2c and Supplementary Figure 2c). In contrast, Sox2 levels increased in ATV-treated EBs compared with controls, and this was again reversed by MVA addition. Together, these results indicate that the inhibition of the mevalonate pathway by ATV blocks primitive streak formation. Instead, ATV-treated epiblast cells differentiate into ectoderm that gives rise to neurons (Supplementary Figure 2d).


The mevalonate pathway regulates primitive streak formation via protein farnesylation
The effects of statins on primitive streak formation in mouse EBs.(a) Microarray results for the top 35 downregulated genes in EBs treated with ATV during days 1–4. Data were compared with control EBs and analysed on days 3 and 4. Gene expression levels in ATV-treated EBs are expressed as Log2 (fold change) values relative to control EBs. (b) In situ hybridisation to detect T and Sox2 in EBs treated with or without ATV on days 1–6 and collected at the indicated times. Results are representative of >100 EBs/group. (c) Real-time PCR of T and Sox2 in EBs treated with or without ATV and/or MVA during days 1–4 and collected on day 4. Results were analysed as in Fig. 1b. *P < 0.05, **P < 0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5121603&req=5

f2: The effects of statins on primitive streak formation in mouse EBs.(a) Microarray results for the top 35 downregulated genes in EBs treated with ATV during days 1–4. Data were compared with control EBs and analysed on days 3 and 4. Gene expression levels in ATV-treated EBs are expressed as Log2 (fold change) values relative to control EBs. (b) In situ hybridisation to detect T and Sox2 in EBs treated with or without ATV on days 1–6 and collected at the indicated times. Results are representative of >100 EBs/group. (c) Real-time PCR of T and Sox2 in EBs treated with or without ATV and/or MVA during days 1–4 and collected on day 4. Results were analysed as in Fig. 1b. *P < 0.05, **P < 0.0001.
Mentions: To gain initial insight into the mechanism by which statins affect early embryogenesis, we performed microarray analysis of EBs during days 3–4, which is the most sensitive two day period for ATV when compared to days 1–2 and days 5–6. ATV reduced the expression of 417 genes by 50% in EBs on days 3 and 4 (Fig. 2a). Ontology analysis revealed that many of these genes are involved in early embryogenesis, particularly gastrulation (GO: 0007369, P = 5.26 × 10−31) (Supplementary Table 2). Consistent with a role specifically in primitive streak formation, ATV markedly suppressed primitive streak and mesodermal markers (Lhx1, Wnt3, Wnt8, GSC, Fgf10 and Fgf8) as well as endodermal markers (Sox17 and Gata6) on day 4, which was reversed by MVA addition (Supplementary Figure 2a). In cultured mouse EBs, a primitive streak-like domain initiates a gastrulation process driven by many genes (Supplementary Figure 2b)17. We used real-time PCR and in situ hybridisation to examine the expression of the primitive streak marker Brachyury T (T) and the early ectodermal marker Sox2 in mouse EBs treated with ATV. As expected, T was expressed at high levels in a localised region in control EBs, however levels were significantly reduced by ATV treatment (Fig. 2b,c). This effect was partially rescued by MVA (Fig. 2c and Supplementary Figure 2c). In contrast, Sox2 levels increased in ATV-treated EBs compared with controls, and this was again reversed by MVA addition. Together, these results indicate that the inhibition of the mevalonate pathway by ATV blocks primitive streak formation. Instead, ATV-treated epiblast cells differentiate into ectoderm that gives rise to neurons (Supplementary Figure 2d).

View Article: PubMed Central - PubMed

ABSTRACT

The primitive streak in peri-implantation embryos forms the mesoderm and endoderm and controls cell differentiation. The metabolic cues regulating primitive streak formation remain largely unknown. Here we utilised a mouse embryonic stem (ES) cell differentiation system and a library of well-characterised drugs to identify these metabolic factors. We found that statins, which inhibit the mevalonate metabolic pathway, suppressed primitive streak formation in vitro and in vivo. Using metabolomics and pharmacologic approaches we identified the downstream signalling pathway of mevalonate and revealed that primitive streak formation requires protein farnesylation but not cholesterol synthesis. A tagging-via-substrate approach revealed that nuclear lamin B1 and small G proteins were farnesylated in embryoid bodies and important for primitive streak gene expression. In conclusion, protein farnesylation driven by the mevalonate pathway is a metabolic cue essential for primitive streak formation.

No MeSH data available.