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Biochem Biophys Res Commun
2016 Oct 14;4792:238-244. doi: 10.1016/j.bbrc.2016.09.051.
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NSrp70 is significant for embryonic growth and development, being a crucial factor for gastrulation and mesoderm induction.
Lee SH
,
Kim C
,
Lee HK
,
Kim YK
,
Ismail T
,
Jeong Y
,
Park K
,
Park MJ
,
Park DS
,
Lee HS
.
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NSrp70 (nuclear speckle-related protein 70), a recently discovered protein and it belongs to the serine/arginine (SR) rich related protein family. NSrp70 is recognized as an important splicing factor comprising RNA recognition motif (RRM) and arginine/serine (RS)-like regions at the N- and C-terminus respectively, along with two coiled coil domains at each terminus. However, other functions of NSrp70 remain unelucidated. In this study, we investigated the role of NSrp70 in Xenopus embryogenesis and found that its maternal expression plays a critical role in embryonic development. Knockdown of NSrp70 resulted in dramatic reduction in the length of developing tadpoles and mild to severe malformation in Xenopus embryos. In addition, knockdown of NSrp70 resulted in an extremely short axis by blocking gastrulation and convergent extension. Further, animal cap assays along with activin A treatment revealed that NSrp70 is an essential factor for dorsal mesoderm induction as knockdown of NSrp70 caused a dramatic down-regulation of dorsal mesoderm specific genes and its loss significantly shortened the elongation region of animal caps. In conclusion, NSrp70 is crucial for early embryonic development, influencing gastrulation and mesoderm induction.
Fig. 1.
NSrp70 is a maternal gene enriched in dorso-anterior regions during embryogenesis. A. Whole-mount in-situ hybridization revealed that NSrp70 was expressed in the dorso-anterior regions. B. RT-PCR was performed using ODC as an internal gene marker. NSrp70 is a maternal gene strongly expressed throughout all stages of embryo development.
Fig. 2.
Loss of NSrp70 caused dramatic length reduction and inhibition of dorso-anterior structures. A. NSrp70 gene function was analyzed using NSrp70 MO to reduce NSrp70 expression. NSrp70 MO at various doses was injected at the 2-cell stage of embryo development. Loss of NSrp70 resulted in length reduction of tadpoles and inhibition of dorso-anterior structures. Malformation induced by knockdown of NSrp70 was dose-dependent. B. Graphical representation of abnormal phenotypes induced by microinjection of NSrp70 MO and control MO. Percentage of abnormal phenotypes was significantly higher in NSrp70 MO-injected embryos than in control MOs.
Fig. 3.
Knockdown of NSrp70 blocked gastrulation movements. A. Loss of NSrp70 caused large blastopores and yolk plugs in NSrp70 MO-injected embryos as compared to those in control embryos. This indicated that NSrp70 is essential for gastrulation movements. B. Graph showing the percentage of gastrulation defects in NSrp70 MO-injected versus control embryos. Number of gastrulation defects was considerably higher in NSrp70 MO-injected embryos than in the control group.
Fig. 4.
NSrp70 is a crucial component of dorsal mesoderm induction through modulating the expression of dorsal-specific genes. A. Animal caps were isolated from stage 8.5–9 embryos injected with NSrp70 MOs and control MOs and then treated with activin A. The length of the elongation region was reduced in the NSRP70-injected animal caps. B. Graphical representation of relative length of elongated animal caps in embryos treated with control MOs, control MOs plus activin, and NSrp70 MOs plus activin. The elongation region was significantly shorter in animal caps of the NSrp70-downregulated group than in the activin-treated control MOs. C. Activin treatment led to expression of dorsal mesoderm markers. ODC was used as an internal control marker of gene expression. Goosecoid and chordin are mesoderm-specific markers. RT-PCR results revealed a strong reduction in the expression of these dorsal mesoderm-specific markers in NSrp70 MO-injected embryos, whereas in control embryos, expression remained normal. Abbreviations: WE, Whole embryo; CTL MO, control MO-injected animal caps. D. Graphs showing the expression of goosecoid and chordin in embryos treated with control MOs, control MOs plus activin, and NSrp70 MOs plus activin. The relative goosechoid expression was reduced in NSrp70 MO-injected embryos compared with that control MO-injected embryos; but chordin expression was negligible in NSrp70 MO-injected embryos as compared with that in control MO plus activin-treated embryos.
