Role of the insulin receptor‐related receptor in Xenopus laevis development

Abstract

The orphan insulin receptor‐related receptor (IRR), in contrast to its close homologs, the insulin receptor (IR) and insulin‐like growth factor receptor (IGF‐IR) can be activated by mildly alkaline extracellular medium. Unlike ubiquitously expressed IR and IGF‐IR, IRR is found in specific sets of cells in only some tissues, most of them being exposed to extracorporal liquids of extreme pH. In the evolution, IRR is highly conserved since its divergence from the insulin and insulin‐like growth factor receptors in amphibia. IR and IGF‐IR signaling are important in growth and development in zebrafish or frog embryogenesis. In contrast, the role of IRR activation during embryogenesis is unknown although Xenopus embryo have strong IRR expression. To address this, we examined the function of the IRR during Xenopus laevis development by morpholino‐mediated selective knockdown of IRR. We demonstrated that inhibition of IRR expression in Xenopus laevis leads to delayed development, but this phenotype can be restored by incubation of embryos in alkaline medium. Also, using RNA‐seq of total RNA we showed that IRR inhibition dramatically changed the embryo transcriptome, and partially restored after alkali treatment. We have identified several hundred genes (eomes, frzb, pax6 etc.) whose expression changes after IRR knockdown and restores by alkali exposure. Our results clearly demonstrate that IRR plays previously unidentified important role in frog embryogenesis and growth.Support or Funding InformationThis work was financially supported by the Russian Foundation for Basic Research (grants No.19‐04‐00815, 17‐00‐00486)To understand which role IRR could play in the embryonic development, we downregulated it by injecting anti‐sense morpholino oligonucleotide to IRR mRNA (MO1 xIRR) into the two‐cells embryos (Fig. 1A). As a result, we observed significant retardation of development of these embryos comparing to their siblings injected by the control MO (Fig. 1B,C). As IRR in mouse is known as a receptor which activity depends on pH, we decided to verify if pH different from the neutral one could interfere with the effects observed in embryos with downregulated pH. To this end, we incubated embryos injected with IRR MO and by the control one in solutions with pH 5.5, 7.2 and 8.4.Whereas clear retardation of development was seen in the MO1 xIRR injected embryos incubated in pH 5.2 and 7.2 (Fig, 1B,C,D,E). Surprisingly, the embryos incubated at pH 8.4 developed approximately synchronously with the control ones (Fig. 1F,G). In other words, unexpectedly alkaline pH “rescues” embryos with downregulated IRR from retardation of development.We represented in table 1 top of 15 up‐regulated and 15 down‐regulated genes after MO injection in embryos at pH 7.2, which included several key transcription factors such as eomes, pou5f3.2 (or oct25), hhex and pax6. Also, we see that genes with the largest modified expression are actin or myosin coding genes.Figure 1 gene Fold change MO xIRR 7.2/control 7.2 Fold change MO xIRR 8.4/MO xIRR 7.2 mylpf.L 0,15 2,95 myosin light chain, phosphorylatable, fast skeletal muscle S homeolog(mylpf.S) act3.L 0,15 1,89 actin, alpha skeletal muscle MGC64484 0,15 1,89 Actin, alpha cardiac muscle 1 act2 0,20 2,32 actin, alpha sarcomeric/cardiac myl1.S 0,25 1,93 myosin light chain 1 S homeolog MGC53823 0,25 1,99 Actin, alpha cardiac muscle 2‐like col2a1.L 0,26 2,07 collagen, type II, alpha 1 L homeolog des.1.L 0,26 2,11 desmin, gene 1 L homeolog fbxl22.S 0,30 1,94 F‐box and leucine‐rich repeat protein 22 S homeolog tnnc2.L 0,30 1,68 troponin C type 2 (fast) L homeolog pax6.S 0,30 2,43 paired box 6 S homeolog des.1.S 0,30 1,95 desmin, gene 1 S homeolog smyd1.L 0,31 2,21 SET and MYND domain containing 1 L homeolog tnnt3.L 0,32 2,26 troponin T3, fast skeletal type L homeolog nr2f5.S 0,32 1,87 nuclear receptor subfamily 2, group F, member 5 S homeolog dctn2.L 1,92 0,58 dynactin subunit 2 L homeolog pou5f3.2.L 1,93 0,51 POU class 5 homeobox 3, gene 2 L homeolog ywhag.L 1,93 0,50 tyrosine 3‐monooxygenase/tryptophan 5‐monooxygenase activation protein, gamma L homeolog ppp2r2b.L 1,93 0,52 protein phosphatase 2 regulatory subunit B, beta L homeolog chm.L 1,97 0,56 choroideremia (Rab escort protein 1) L homeolog hhex.L 1,97 0,62 hematopoietically expressed homeobox L homeolog slc22a15.2.S 1,97 0,63 solute carrier family 22, member 15, gene 2 S homeolog gramd1c.L 1,97 0,70 GRAM domain containing 1C L homeolog ccna1.L 1,99 0,48 cyclin A1 L homeolog neu1.L 2,00 0,56 neuraminidase 1 (lysosomal sialidase) L homeolog cdk5r2.S 2,03 0,57 cyclin‐dependent kinase 5, regulatory subunit 2 (p39) S homeolog cer1.S 2,11 0,54 cerberus 1, DAN family BMP antagonist S homeolog xpo6.S 2,19 0,67 exportin 6 S homeolog eomes.S 2,42 0,36 eomesodermin S homeolog frzb.S 2,71 0,39 frizzled‐related protein S homeolog