Korotkova DD et al. (2022), Insulin Receptor-Related Receptor Regulates the...

XB-ART-59282

Int J Mol Sci 2022 Aug 17;2316:. doi: 10.3390/ijms23169250.

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Insulin Receptor-Related Receptor Regulates the Rate of Early Development in Xenopus laevis.

Korotkova DD , Gantsova EA , Goryashchenko AS , Eroshkin FM , Serova OV , Sokolov AS , Sharko F , Zhenilo SV , Martynova NY , Petrenko AG , Zaraisky AG , Deyev IE .

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The orphan insulin receptor-related receptor (IRR) encoded by insrr gene is the third member of the insulin receptor family, also including the insulin receptor (IR) and the insulin-like growth factor receptor (IGF-1R). IRR is the extracellular alkaline medium sensor. In mice, insrr is expressed only in small populations of cells in specific tissues, which contain extracorporeal liquids of extreme pH. In particular, IRR regulates the metabolic bicarbonate excess in the kidney. In contrast, the role of IRR during Xenopus laevis embryogenesis is unknown, although insrr is highly expressed in frog embryos. Here, we examined the insrr function during the Xenopus laevis early development by the morpholino-induced knockdown. We demonstrated that insrr downregulation leads to development retardation, which can be restored by the incubation of embryos in an alkaline medium. Using bulk RNA-seq of embryos at the middle neurula stage, we showed that insrr downregulation elicited a general shift of expression towards genes specifically expressed before and at the onset of gastrulation. At the same time, alkali treatment partially restored the expression of the neurula-specific genes. Thus, our results demonstrate the critical role of insrr in the regulation of the early development rate in Xenopus laevis.

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Species referenced: Xenopus laevis
Genes referenced: des des.2 eomes foxg1 ins insrr kit myc myl1 pax6 pitx2 twsg1
GO keywords: insulin receptor binding [+]
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	Figure 1. Downregulation of insrr by anti-sense morpholino injection retards the Xenopus laevis embryosâ€™ development, whereas alkaline pH prevents this effect. (A). Embryos after injection of insrr MO1 at pH 7.2. (B). Embryos after injection of control MO at pH 7.2. (C). Embryos after injection of insrr MO1 at pH 8.5. (D). Embryos after injection of control MO at pH 8.5. The diagrams on (Aâ€™â€“Dâ€™) demonstrate the percentage distribution of embryos, some of which are shown on (Aâ€™â€“Dâ€™), by stage of development. As one may see, knockdown of insrr by insrr MO1 significantly retards the developmental rate of embryos comparing to their control siblings, and also distribution of the knocked-out embryos developed at pH 8.5 is significantly shifted towards the distribution seen in the wild-type siblings developed at pH 7.2 and pH 8.5.
	Figure 2. (A). PCA analysis of RNA-seq data of control embryos and insrr MO1 knockdown embryos after incubation in pH 7.2 or 8.5 media. (B). Representation of upregulated and downregulated DEGs (padj < 0.05) of control embryos and insrr MO1 knockdown embryos after incubation in pH 7.2 or 8.5 media. (C). The density plot of genes on a field of log2(FoldChange) vs. log10(baseMean) was plotted after differential expression analysis of transcriptomes between control and insrr MO1 knockdown embryos. DEGs with padj < 0.05 are colored in red and other genes are colored in grey. (D). Same plot with multicolor indication of DEGs (padj < 0.05) which were downregulated after alkali treatment of insrr MO1-injected embryos. (E). Same plot with multicolor indication of DEGs (padj < 0.05), which were upregulated after alkali treatment of insrr MO1-injected embryos.
	Figure 3. (A). Example of the temporal expression profiles of eomes.S and des.1.S genes. Data from [19] and Xenbase.org. (B). The intersection of expressed genes from previously published Xenopus laevis stage gene expression analysis at 10 and 15 stages and DEGs between transcriptomes of the control and insrr MO1-injected embryos at pH 7.2. (C). Graphical plot log2 (Fold change insrr MO 7.2/control MO 7.2) vs. plotted log2 (FoldChange stage10/stage15) for each 1465 DEGs. Red color indicated only DEGs between stage 10 and stage 15.
	Figure 4. A model of the regulation of embryonic development by IRR. (A). In embryos injected with the control MO and at the neutral pH (7.2), the level of IRR signaling is enough to ensure the wild-type rate of embryonic development. (B). If insrr is downregulated by injection of insrr MO, the concentration of IRR molecules significantly but not completely decreases. As a result, at neutral pH (7.2) a decreased level of IRR signaling leads to the retardation of development. (C). At alkaline pH (8.5), despite the concentration of IRR molecules being significantly decreased in embryos injected with insrr MO, the level of the IRR signaling enhances, which ensures the wild-type rate of embryonic development.
	Supplement Figure S1. Testing of insrr MO2. A. Embryos injected with insrr MO2 at pH 7.2 and 8.5. B. Embryos injected with control MO at pH 7.2. C. Embryos injected with control MO at pH 8.5. The diagrams on A', B', and C' demonstrate the percentage distribution of embryos, some of which are shown on A, B, and C, by stage of development.
	Supplement Figure S2. Rescue of insrr MO1 effect by the co-injected insrr mRNA. A-C. Embryos injected with control MO (A), insrr MO1 (B), and the mixture of insrr MO1 and insrr mRNA (C) at pH 7.2. The diagrams on A', B', and C' demonstrate the percentage distribution of embryos, some of which are shown on A, B, and C, by stage of development.
	Supplement Figure S3. Analysis of MO specificity using myc-tagged fragments of xIRR cDNA. The obtained cDNA fragments were sub-cloned into pCS2-twsg1-myc plasmid instead of twsg1 coding frame. To obtain capped mRNA, these plasmids were linearized and mRNAs were synthesized using mMESSAGEmMACHINE kit. For injection experiments MOs were diluted to the final concentration 0.3 mM; mRNAs were diluted to the final concentration 25ng/Âµl. For injections, mRNA or MO solutions were mixed with FLD (Fluorescein Lysinated Dextran, 40 kDa, 5 mg/mL, Invitrogen) and 4â€“5 nL of the mixture were injected into single blastomeres at two-cell stage. Injected embryos were cultivated until stage 12, then lysed and expression was analyzed by Western blotting. Coomassie stained gels were used as loading controls.
	Supplement Figure S4. A. Annotated list of DEGs (padj<0.05) between insrr MO and control MO injected embryos at pH 7.2. B. Annotated list of DEGs (padj<0.05) between insrr MO injected embryos at pH 8.5 and pH 7.2.
	Supplement Figure S5. A. The intersection of DEGs (929 DEGs, padj<0.01) between transcriptomes of insrr MO and control MO injected embryos at pH 7.2, and DEGs (634 DEGs, padj<0.01) between transcriptomes of insrr MO injected embryos at pH 8.5 and pH 7.2. B. Analysis of the fold changes of DEGs intersection between trancriptomes of insrr MO and control MO injected embryos at pH 7.2 (red color) and between transcriptomes of insrr MOinjected embryos at pH 8.5 and pH 7.2 (blue color). X axis indicates number of gene and genes were sorted by increasing of the FoldChange between transcriptomes of insrr MO injected embryos at pH 8.5 and pH 7.2. C. Annotated list of 331 DEGs intersection.
	Supplement Figure S6. A. Plot of changing the expression modulus DEGs (929 DEGs, padj<0.01) between transcriptomes of insrr MO and control MO injected embryos at pH 7.2. Intersection DEGs (331 genes) indicated by blue color and other genes by orange color. B. Plot of changing the expression modulus DEGs (634 DEGs, padj<0.01) between transcriptomes of insrrMO injected embryos at pH 8.5 and pH 7.2. Intersection DEGs (331 genes) indicated by blue color and other genes by orange color .

References [+] :

Almouzni, Histone acetylation influences both gene expression and development of Xenopus laevis. 1994, Pubmed, Xenbase