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Sprouty2 mediated tuning of signalling is essential for somite myogenesis.

Abu-Elmagd M, Goljanek Whysall K, Wheeler G, M√ľnsterberg A - BMC Med Genomics (2015)

Bottom Line: Overexpression and dominant-negative interference showed that Spry2 plays a crucial role in regulating chick myogenesis by fine tuning of FGF signaling through a negative feedback loop.We also propose that mir-23, mir-27 and mir-128 could be part of the negative feedback loop mechanism.Our analysis is the first to shed some light on in vivo Spry2 function during chick somite myogenesis.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Negative regulators of signal transduction cascades play critical roles in controlling different aspects of normal embryonic development. Sprouty2 (Spry2) negatively regulates receptor tyrosine kinases (RTK) and FGF signalling and is important in differentiation, cell migration and proliferation. In vertebrate embryos, Spry2 is expressed in paraxial mesoderm and in forming somites. Expression is maintained in the myotome until late stages of somite differentiation. However, its role and mode of action during somite myogenesis is still unclear.

Results: Here, we analysed chick Spry2 expression and showed that it overlaps with that of myogenic regulatory factors MyoD and Mgn. Targeted mis-expression of Spry2 led to inhibition of myogenesis, whilst its C-terminal domain led to an increased number of myogenic cells by stimulating cell proliferation.

Conclusions: Spry2 is expressed in somite myotomes and its expression overlaps with myogenic regulatory factors. Overexpression and dominant-negative interference showed that Spry2 plays a crucial role in regulating chick myogenesis by fine tuning of FGF signaling through a negative feedback loop. We also propose that mir-23, mir-27 and mir-128 could be part of the negative feedback loop mechanism. Our analysis is the first to shed some light on in vivo Spry2 function during chick somite myogenesis.

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Spry2 gain-of-function inhibits somite myogenesis. Electroporation of pCAB-Spry2 full length or RFP-MKP3 expression constructs into epithelial somites at HH16, as indicated, followed by 24-hour incubation to HH21/22. (A-C) Whole mount in situ hybridisation for MyoD (purple) and GFP or RFP (red), (Ai) is a section of embryo in (A). (A, Ai) Spry2 expression led to loss of MyoD in transfected cells, arrows in (A) indicate the targeted electroporated somites and lines indicate the level of sectioning in (Ai). (B) Electroporation of an empty pCAB-IRES-GFP expression vector (used as a control) into somites which showed normal MyoD expression (arrows). (C) Loss of MyoD was observed in cells electroporated with RFP-Mkp3 (arrows); (Ci) RFP detected by fluorescent filter Alexa-Fluor-465 in the same electroporated somites in (C). (D) Control of unelectroporated embryo showing normal MyoD expression. Magnifications: 20x in (A, B, C & D), 24x in (Ci), 200x in (Ai).
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Figure 3: Spry2 gain-of-function inhibits somite myogenesis. Electroporation of pCAB-Spry2 full length or RFP-MKP3 expression constructs into epithelial somites at HH16, as indicated, followed by 24-hour incubation to HH21/22. (A-C) Whole mount in situ hybridisation for MyoD (purple) and GFP or RFP (red), (Ai) is a section of embryo in (A). (A, Ai) Spry2 expression led to loss of MyoD in transfected cells, arrows in (A) indicate the targeted electroporated somites and lines indicate the level of sectioning in (Ai). (B) Electroporation of an empty pCAB-IRES-GFP expression vector (used as a control) into somites which showed normal MyoD expression (arrows). (C) Loss of MyoD was observed in cells electroporated with RFP-Mkp3 (arrows); (Ci) RFP detected by fluorescent filter Alexa-Fluor-465 in the same electroporated somites in (C). (D) Control of unelectroporated embryo showing normal MyoD expression. Magnifications: 20x in (A, B, C & D), 24x in (Ci), 200x in (Ai).

Mentions: Next we wanted to examine the role of Spry2 in somite myogenesis using a gain-of-function and a functional interference approach. First, we used microinjection and electroporation of a plasmid encoding full length Spry2 and GFP from the same vector backbone (pCAB-Spry2-IRES-GFP). Epithelial somites of HH16-17 embryos were targeted. Embryos were harvested 24- or 48-hours after electroporation after which transfected somites were identified by GFP fluorescence and effects on myogenesis were examined by analysing changes in MyoD expression using in situ hybridisation. This revealed a loss of MyoD expression in regions of somite transfected with pCAB-Spry2-IRES-GFP (n=19) (Figure 3A, Ai). There was no difference in the effect of Spry2 misexpression on MyoD expression if electroporation was in dermomyotomes or myotomes. Control embryos electroporated with pCAB-IRES-GFP showed normal MyoD expression (Figure 3B). We compared this phenotype to that obtained with a different antagonist of FGF signalling, which we had previously characterized [9]. We electroporated expression constructs encoding Mkp3, a dual-specific phosphatase, which inactivates ERK. Consistent with previous observations the electroporation of Mkp3-RFP led to localized loss of MyoD expression in transfected somites (Figure 3C-Ci) (n=13, see also [9]). Un-electroporated control embryos showed normal MyoD expression (Figure 3D). Conversely, electroporation of a truncated form of Spry2 which only contains the carboxy-terminus, led to promotion of myogenesis indicated by an increase in MyoD expression (n=27) (Figure 4). This was detectable after 6 hours of electroporation (Figure 4A). Increased expression of MyoD was also observed after 11, 24 and 48 hours (Figure 4B-Di). We also noticed an increase in somite size after longer incubation for 48 hours, when comparing electroporated somites with those on the opposite side (Figure 4D, Di).


Sprouty2 mediated tuning of signalling is essential for somite myogenesis.

Abu-Elmagd M, Goljanek Whysall K, Wheeler G, M√ľnsterberg A - BMC Med Genomics (2015)

Spry2 gain-of-function inhibits somite myogenesis. Electroporation of pCAB-Spry2 full length or RFP-MKP3 expression constructs into epithelial somites at HH16, as indicated, followed by 24-hour incubation to HH21/22. (A-C) Whole mount in situ hybridisation for MyoD (purple) and GFP or RFP (red), (Ai) is a section of embryo in (A). (A, Ai) Spry2 expression led to loss of MyoD in transfected cells, arrows in (A) indicate the targeted electroporated somites and lines indicate the level of sectioning in (Ai). (B) Electroporation of an empty pCAB-IRES-GFP expression vector (used as a control) into somites which showed normal MyoD expression (arrows). (C) Loss of MyoD was observed in cells electroporated with RFP-Mkp3 (arrows); (Ci) RFP detected by fluorescent filter Alexa-Fluor-465 in the same electroporated somites in (C). (D) Control of unelectroporated embryo showing normal MyoD expression. Magnifications: 20x in (A, B, C & D), 24x in (Ci), 200x in (Ai).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Spry2 gain-of-function inhibits somite myogenesis. Electroporation of pCAB-Spry2 full length or RFP-MKP3 expression constructs into epithelial somites at HH16, as indicated, followed by 24-hour incubation to HH21/22. (A-C) Whole mount in situ hybridisation for MyoD (purple) and GFP or RFP (red), (Ai) is a section of embryo in (A). (A, Ai) Spry2 expression led to loss of MyoD in transfected cells, arrows in (A) indicate the targeted electroporated somites and lines indicate the level of sectioning in (Ai). (B) Electroporation of an empty pCAB-IRES-GFP expression vector (used as a control) into somites which showed normal MyoD expression (arrows). (C) Loss of MyoD was observed in cells electroporated with RFP-Mkp3 (arrows); (Ci) RFP detected by fluorescent filter Alexa-Fluor-465 in the same electroporated somites in (C). (D) Control of unelectroporated embryo showing normal MyoD expression. Magnifications: 20x in (A, B, C & D), 24x in (Ci), 200x in (Ai).
Mentions: Next we wanted to examine the role of Spry2 in somite myogenesis using a gain-of-function and a functional interference approach. First, we used microinjection and electroporation of a plasmid encoding full length Spry2 and GFP from the same vector backbone (pCAB-Spry2-IRES-GFP). Epithelial somites of HH16-17 embryos were targeted. Embryos were harvested 24- or 48-hours after electroporation after which transfected somites were identified by GFP fluorescence and effects on myogenesis were examined by analysing changes in MyoD expression using in situ hybridisation. This revealed a loss of MyoD expression in regions of somite transfected with pCAB-Spry2-IRES-GFP (n=19) (Figure 3A, Ai). There was no difference in the effect of Spry2 misexpression on MyoD expression if electroporation was in dermomyotomes or myotomes. Control embryos electroporated with pCAB-IRES-GFP showed normal MyoD expression (Figure 3B). We compared this phenotype to that obtained with a different antagonist of FGF signalling, which we had previously characterized [9]. We electroporated expression constructs encoding Mkp3, a dual-specific phosphatase, which inactivates ERK. Consistent with previous observations the electroporation of Mkp3-RFP led to localized loss of MyoD expression in transfected somites (Figure 3C-Ci) (n=13, see also [9]). Un-electroporated control embryos showed normal MyoD expression (Figure 3D). Conversely, electroporation of a truncated form of Spry2 which only contains the carboxy-terminus, led to promotion of myogenesis indicated by an increase in MyoD expression (n=27) (Figure 4). This was detectable after 6 hours of electroporation (Figure 4A). Increased expression of MyoD was also observed after 11, 24 and 48 hours (Figure 4B-Di). We also noticed an increase in somite size after longer incubation for 48 hours, when comparing electroporated somites with those on the opposite side (Figure 4D, Di).

Bottom Line: Overexpression and dominant-negative interference showed that Spry2 plays a crucial role in regulating chick myogenesis by fine tuning of FGF signaling through a negative feedback loop.We also propose that mir-23, mir-27 and mir-128 could be part of the negative feedback loop mechanism.Our analysis is the first to shed some light on in vivo Spry2 function during chick somite myogenesis.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Negative regulators of signal transduction cascades play critical roles in controlling different aspects of normal embryonic development. Sprouty2 (Spry2) negatively regulates receptor tyrosine kinases (RTK) and FGF signalling and is important in differentiation, cell migration and proliferation. In vertebrate embryos, Spry2 is expressed in paraxial mesoderm and in forming somites. Expression is maintained in the myotome until late stages of somite differentiation. However, its role and mode of action during somite myogenesis is still unclear.

Results: Here, we analysed chick Spry2 expression and showed that it overlaps with that of myogenic regulatory factors MyoD and Mgn. Targeted mis-expression of Spry2 led to inhibition of myogenesis, whilst its C-terminal domain led to an increased number of myogenic cells by stimulating cell proliferation.

Conclusions: Spry2 is expressed in somite myotomes and its expression overlaps with myogenic regulatory factors. Overexpression and dominant-negative interference showed that Spry2 plays a crucial role in regulating chick myogenesis by fine tuning of FGF signaling through a negative feedback loop. We also propose that mir-23, mir-27 and mir-128 could be part of the negative feedback loop mechanism. Our analysis is the first to shed some light on in vivo Spry2 function during chick somite myogenesis.

Show MeSH