Injection of Gadd45γ mRNA augments the cell cycle exit function of p27 Xic

To determine if Gadd45γ was capable of aiding the cell cycle exit function of p27Xic1, in the same manner as has been shown for Gadd45α and p21Cip1, we performed single injections ofGadd45γmRNA andp27Xic1mRNA and analysed how the pronephric phenotypes caused by these single injections compared to embryos co-injected with Gadd45γ mRNA and p27Xic1 mRNA. RNAs were injected into the V2 blastomere of 8-cell stage embryos with either 500 pg Gadd45γ mRNA, 50 pg p27Xic1 mRNA or a combination of 500 pg Gadd45γ mRNA and 50 pg p27Xic1 mRNA. 400 pgβgalmRNA was co-injected to act as a lineage tracer. Embryos were then left to develop to stage 24 and 41 where markers for the pronephros were detected either byin situhybridisation for expression ofLim-1(at stage 24) or whole mount antibody staining using 3G8, which detects the nephrostomes and proximal tubule, and 4A6, which detects the intermediate and distal tubules (at stage 41). All results described in this section have been repeated at least twice, yielding similar phenotypes, and statistically significant phenotypes were determined within 95%

From these injections we expected to observe more severe pronephric phenotypes upon co-injection of Gadd45γ mRNA and p27Xic1 mRNA compared to the single injections of either message. The first indication suggesting our expectations were accurate was observed by the cell cycle effect such injections have at stage 8.5 (Fig 4.4). After injection of p27Xic1 mRNA and translation of the exogenous message, the concentration of p27Xic1 within the cell is sufficient to inhibit cell division, a phenomenon observable by enlarged cells in embryos up to stage 9/ 10. We observed no cell cycle effect after injection of βgal mRNA alone (Fig 4.4A) or in combination with Gadd45γ mRNA (Fig 4.4C). Injection of p27Xic1 mRNA, as previously described, produced a cell cycle effect with enlarged cells around the site of injection (Fig 4.4B). However, co-injection of Gadd45γ mRNA with p27Xic1 mRNA had the most severe cell cycle effect (Fig 4.4D); the injected messages produced substantially larger cells around the site of injection than the single p27Xic1 mRNA injection. Though entirely non-quantitative, this observation suggests the cell cycle exit function of p27Xic1is augmented by Gadd45γ.

p27Xic1over-expression reduced formation of the pronephros anlagen at stage 24 at a similar frequency to the data presented and interpreted in Chapter 3. 51% of embryos had reduced Lim-1 expression, with 20% having completely absent pronephric anlagen (n=49, Fig 4.5B). Injection of Gadd45γ mRNA also decreased pronephros anlagen development in 62% of embryos (n=47, Fig 4.5C). Co-injection of p27Xic1 mRNA and Gadd45γ mRNA significantly increased the prevalence of pronephric phenotypes at stage 24 with 84% of embryos having reduced Lim-1 expression and 50% having completely absentLim-1expression (n=31, Fig 4.5D). In conclusion early pronephrogenesis is inhibited by over-expression of eitherp27Xic1or

Figure 4.4 St 8.5 embryos displaying cell cycle effects after injection ofp27Xic1

mRNAInjection of theβgalmRNA lineage tracer into the V2 blastomere of an 8- cell stage embryo had no effect on cell division (A). The same injection of 50 pg p27Xic1mRNA caused a repeatable cell cycle effect, cells around the site of injection were larger than normal (B). Injection of 500 pgGadd45γmRNA had no obvious effect on cell division at stage 8.5 despite its well characterised ability to inhibit the cell cycle (C). Co-injection of 50 pgp27Xic1mRNA and 500 pgGadd45γmRNA had a severe effect on cell division, inhibiting the cell cycle and producing much larger cells than occur during normal development. This co-injection suggests Gadd45γis capable of augmenting the cell cycle exit function of p27Xic1(D).

Figure 4.5 Over-expression ofp27Xic1andGadd45γinhibits pronephros anlagen formation.Embryos were injected into the V2 blastomere of an 8-cell stage embryo with 400 pgβgalmRNA to act as a lineage tracer. Embryos were then left to develop to stage 24 where they were fixed, sorted for correct targeting and whole mount in situ hybridised forLim-1, a marker of the early pronephros anlagen. Injection ofβgal mRNA had no effect on pronephros anlagen formation (A). Injection of 50 pgp27Xic1 mRNA, as described in Chapter 3, reducedLim-1expression on the injected side (B). Injection of 500 pgGadd45γmRNA also reduced the size of the pronephros anlagen, with similar severity top27Xic1over-expression (C). Co-injection ofp27Xic1mRNA andGadd45γmRNA reduced the size of the pronephros with a higher frequency than either the single injections of p27Xic1or Gadd45γ(D).

Gadd45γ, and co-injection of p27Xic1 mRNA/ Gadd45γ mRNA produced a more severe reduction in pronephros anlagen formation than the single injections.

At stage 41 p27Xic1 over-expression, as described in Chapter 3, reduced the size of the pronephros detectable with 3G8 and 4A6 antibodies. Here p27Xic1mRNA single injections reduced 3G8/ 4A6 antibody staining in 59% of embryos (n=54, Fig 4.6B). We also observed a marginal, but still statistically significant, reduction of 3G8/ 4A6 antibody staining in 12% of embryos injected withGadd45γmRNA (n=68, Fig 4.6C). Co-injection of p27Xic1 mRNA and Gadd45γ mRNA reduced 3G8/ 4A6 antibody staining in 64% of embryos (n=58, Fig 4.6D). This increase in frequency is not of statistical significance to confirm Gadd45γ has an additive effect on p27Xic1 cell cycle exit function. However comparative analysis of the number of embryos that had completely absent pronephroi after single injections ofp27Xic1mRNA or co- injections ofp27Xic1mRNA/Gadd45γmRNA suggested that the co-injected embryos acquired a more severe pronephic phenotype (Appendix 2). Only one embryo (3% of those scored) had a completely absent pronephros in the single p27Xic1 mRNA injected sample of embryos. Co-injection of p27Xic1 mRNA/ Gadd45γ mRNA completely prevented 11 embryos (30% of those scored) developing a pronephros. In conclusion we are unable to confirm whether Gadd45γ acts synergistically or additively with p27Xic1to promote cell cycle exit.

To directly and quantitatively observe if Gadd45γ is able to promote the cell cycle exit function of p27Xic1 we injected one cell of a two cell embryo with p27Xic1 mRNA,Gadd45γmRNA and a combination ofp27Xic1mRNA/Gadd45γmRNA, left the embryos to develop to approximately stage 17, when they were fixed, stained for

Figure 4.6 Over-expression ofp27Xic1andGadd45γinhibits pronephros development.Embryos were injected into the V2 blastomere of an 8-cell stage embryo with 400 pgβgalmRNA to act as a lineage tracer. Embryos were then left to develop to stage 41 where they were fixed, sorted for correct targeting and whole mount antibody stained using antibodies 3G8, which detects the nephrostomes and proximal tubules, and 4A6, which detects the intermediate and distal tubules. Injection ofβgalmRNA had no effect on pronephrogenesis (A). Injection of 50 pg p27Xic1mRNA, as described in Chapter 3, reduced 3G8/ 4A6 antibody staining on the injected side (B). Injection of 500 pgGadd45γmRNA also reduced the size of the pronephros at stage 41, but with less severity thanp27Xic1over-expression (C). Co- injection ofp27Xic1mRNA andGadd45γmRNA reduced the size of the pronephros with a frequency higher than either the single injections of p27Xic1or Gadd45γ(D).

theβgal lineage label and then whole mount antibody stained for phosphohistone H3 (pH3, a marker of dividing cells). 12-14 embryos of each injection were scored and the difference between the number of positively stained pH3 cells on the injected and un-injected sides was compared. Injection of just the βgalmRNA lineage tracer had a statistically insignificant effect on cell division with an average 13% more pH3 stained cells on the un-injected side (n=12, Fig 4.7A). Injection of p27Xic1 mRNA reduced the number of positively stained pH3 cells on the injected side by 60% on average (n=13, Fig 4.7B). Gadd45γ over-expression reduced the number of pH3 positive cells by 27% on average on the injected side, although this result is surprisingly statistically insignificant (n=14, Fig 4.7C). Co-injection of p27Xic1 mRNA and Gadd45γ mRNA reduced pH3 staining on the injected side by 62% on average (n=14, Fig 4.7D). In conclusion, this result suggests Gadd45γ is unable to promote the cell cycle exit function of p27Xic1, despite our previous observation at stage 8.5that suggested Gadd45γ does augment the cell cycle exit function of p27Xic1.