MARCO TEÓRICO
2.3. Marco doctrinal de las teorías particulares en el campo de la ciencia en la que se ubica el objeto de estudio.
The enhanced oxidation of the back-door cysteine mutants of Shp2a in cells treated
with H2O2 (Fig. 2) suggests that they are more susceptible to oxidation-mediated inac-
tivation. In Chapter 5 we proposed that after its initial oxidation induced by zebrafish caudal fin amputation, Shp2a would be reduced and its activity required for regenera-
tion of the caudal fin-fold. In Chapter 6, we demonstrate that wild-type Shp2a almost completely rescued caudal fin-fold regeneration in ptpn11a-/-ptpn11b-/- zebrafish
embryos, lacking functional Shp2. We hence used the back-door cysteine mutants of Shp2a to test whether changes in susceptibility to oxidation affect the ability of Shp2a mutants to rescue regeneration in zebrafish embryos lacking functional Shp2.
One-cell stage zebrafish embryos from a ptpn11a+/-ptpn11b-/- in-cross were micro-
injected with synthetic mRNA encoding either Shp2a-C334S, Shp2a-C368S, or Shp2a- C334S-C368S (2CS). The mRNAs encoding mutant Shp2a proteins also encode eGFP linked by a peptide-2a cleavage sequence, resulting in a single polypeptide that is cleaved prior to protein folding (Ryan, King, and Thomas 1991). At 2dpf, eGFP-positive zebrafish embryos were selected and their caudal fin-folds were amputated immedi-
ately posterior to the notochord. Representative pictures of amputated fin-folds at 3 days post amputation (dpa, i.e. 5dpf, 3dpa) of ptpn11a-/-ptpn11b-/- zebrafish embryos
expressing mutant Shp2a protein are depicted (Fig. 3A). Equivalent uncut zebrafish embryo caudal fin-folds (i.e. 5dpf, Uncut) are shown in Fig. 4A. Caudal fin-fold lengths were determined and are presented as percentage caudal fin-fold growth, normalized
to non-injected uncut controls of ptpn11a+/+ptpn11b-/- zebrafish embryos (Fig. 3b). In
contrast to their siblings, ptpn11a-/-ptpn11b-/- zebrafish embryos displayed severely
impaired caudal fin-fold regeneration, which, surprisingly, was rescued by expression of Shp2a-C334S, Shp2a-C368S, or Shp2a-2CS (Fig. 3b). We observed subtle differences
in rescues between the backdoor cysteine mutants, in that Shp2a-C368S rescued to
the least extent, Shp2a-C334S had an intermediate effect and Shp2a-2CS rescued regeneration of ptpn11a-/-ptpn11b-/- zebrafish embryos almost completely to sibling
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ptpn11a ptpn11amRNA n +/+ +/- -/- -/- -/- -/- -/- -/- -/- - - - C334S C368S 2CS D61G A462T RM 52 110 67 33 23 40 35 24 16 n.s. *** *** *** 20 40 60 80 100 120% caudal fin-fold growth
*** *** A B D61G -/- -/- A462T - 5dpf, 3dpa - C334S -/- -/- C368S -/- 2CS +/+ - -/- - - 5dpf, 3dpa - RM -/- H7.3
Fig. 3. impaired caudal fin-fold regeneration in shp2 deficient embryos is rescued by expres- sion of shp2a-C334s, shp2a-C368s, or the double back-door cysteine mutant shp2a-2Cs, and by shp2a-d61g with enhanced catalytic activity or shp2a-A462t with reduced catalytic activity, but
not by the catalytically inactive shp2a-R466m. (A) Embryos from a ptpn11a+/-ptpn11b-/- in-cross
were micro-injected at the one-cell stage with synthetic mRNA encoding Shp2a-C334S, Shp2a- C368S, Shp2a-C334S-C368S (2CS), Shp2a-D61G, Shp2a-A462T, Shp2a-R466M (RM), or were not injected (-). At 2dpf the caudal fin-fold was amputated and regeneration was assessed at 3dpa (i.e. 5dpf, 3dpa). Equivalent uncut controls were included (i.e. 5dpf, uncut) and are shown in Fig. 4. All
embryos were genotyped. Representative images of caudal fin-folds of non-injected embryos, and of embryos injected with Shp2a mutants are shown. (b) The means of caudal fin-fold growth follo-
wing amputation are depicted relative to caudal fin-fold growth of uncut ptpn11a+/+ptpn11b-/- con-
trols. Means of micro-injected amputated ptpn11a-/-ptpn11b-/- embryos were compared to non-
injected amputated ptpn11a-/-ptpn11b-/- embryos using a Mann-Whitney U-test. Significance: ***
p<0.001; error bars indicate standard error of the mean. Data pooled from multiple experiments. Non-injected and Shp2a-R466M injected data re-used from Chapter 5 and Chapter 6, respectively,
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A B - 5dpf, Uncut - C334S -/- -/- C368S -/- 2CS +/+ - -/- - D61G -/- -/- A462T - 5dpf, Uncut - ptpn11a ptpn11amRNA n +/+ +/- -/- -/- -/- -/- -/- -/- -/- - - - C334S C368S 2CS D61G A462T RM 56 118 56 19 14 36 29 18 20 n.s. n.s. n.s. *** 20 40 60 80 100 120% caudal fin-fold growth
RM -/-
H7.4
Fig. 4. Normal caudal fin-fold growth in shp2 deficient embryos expressing shp2a-C334s, shp2a- C368s, the double back-door cysteine mutant shp2a-2Cs, shp2a-d61g with enhanced catalytic
activity, shp2a-A462t with reduced catalytic activity, or the catalytically inactive shp2a-R466m.
(A) Embryos from a ptpn11a+/-ptpn11b-/- in-cross were micro-injected at the one-cell stage with
synthetic mRNA encoding Shp2a-C334S, Shp2a-C368S, Shp2a-C334S-C368S (2CS), Shp2a-D61G, Shp2a-A462T, Shp2a-R466M (RM), or were not injected (-). Caudal fin-fold length was assessed after 5 days (i.e. 5dpf, uncut). All embryos were genotyped. Representative images of caudal fin-folds of non-injected embryos, and of embryos injected with Shp2a mutants are shown. (b) The means of uncut caudal fin-fold growth is depicted relative to caudal fin-fold growth of uncut ptpn11a+/+ptpn11b-/- controls. Means of micro-injected ptpn11a-/-ptpn11b-/- embryos were compa-
red to non-injected ptpn11a-/-ptpn11b-/- embryos. Significance: *** p<0.001; n.s. not significant;
error bars indicate standard error of the mean. Data pooled from multiple experiments. Non- injected and Shp2a-R466M injected data re-used from Chapter 5 and Chapter 6, respectively, for
comparison.
Expression of the back-door cysteine mutants in ptpn11a-/-ptpn11b-/- zebrafish
embryos that were not amputated resulted in normal growth of the caudal fin-fold, with comparable lengths to non-injected zebrafish embryos and siblings (Fig. 4b).
Furthermore, expression of Shp2a-C334S, Shp2a-C368S, or Shp2a-2CS in wild-type
(ptpn11a+/+ptpn11b+/+) zebrafish embryos did not induce any development defects
(Fig. 5). Due to low expression levels of all mutant Shp2a proteins in zebrafish em-
bryos, it was not possible to monitor protein expression in vivo. However, transfection
of constructs encoding the back-door cysteine mutant Shp2a proteins in HEK293T cells
revealed similar expression levels of all mutant Shp2 proteins (Fig. 6). This suggests that the differences in ability to rescue caudal fin-fold regeneration are not due to dif-
ferences in expression or protein stability, but rather to differences in Shp2a function. Collectively, these results suggest that the enhanced susceptibility to oxidation of the
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