Results – The characterisation of Mats loss-of-function and transgenic flies

Lai et al (2005) observed that mats loss-of-function causes embryonic lethality, with increased cell proliferation and tissue overgrowth in mosaic clones. More importantly, they showed that expression of human MOB1A(wt) in Mats mutant clones compensated for the mats loss-of- function in larval eye discs (Lai, Wei et al. 2005). Since MOB1A expression can rescue mats loss-of-function phenotype in mosaic clones, we speculated that MOB1A expression may compensate for the embryonic lethality caused by mats loss-of-function. To test this hypothesis, we generated transgenic flies expressing myc-tagged MOB1A(wt) under the control of the ubiquitin

63E promoter (ubi>myc-hMOB1A(wt). Furthermore, to understand which

PPIs between MOB1A and Wts, Trc, Hpo are required to prevent embryonic lethality, we also generated transgenic flies expressing our MOB1A variants in the same locus using the ΦC31 integrase system. However, before we could test whether the embryonic lethality caused by mats loss of function can be rescued by MOB1A(wt) expression, we confirmed that mats trans- heteroallelic combinations result in the homozygous embryonic lethality in our setting. The crosses outlined in Figure 3.20.

Figure 3.20. Confirmation of the homozygous lethality caused by mats loss-of- function.

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The aim of this cross was to confirm that the lethality is caused by mats loss-of-function, independently of additional mutations in in the available mats mutants stocks. Female virgin w;; FRT82B, matsroo/ TM6B flies were crossed with male w;; FRT82B, matse235/ TM6B flies as outlined. After this crossing, only heterozygous flies developed to adulthood, while homozygous flies failed to develop

To confirm that the homozygous lethality driven by mats results from mats loss-of-function, flies carrying two independent mats mutant alleles (matsroo and matse235) were crossed (Figure 3.20). The matse235 mutant allele was generated by P-element mobilisation (Lai, Wei et al. 2005), which is the process of imprecise transposon excision leading to deletion of surrounding genomic sequences. mats235 results in deletion of most of the mats coding sequence. The matsroo mutant allele is an insertion of a 428 base pair Roo transposon at the beginning of the mats third exon (Lai, Wei et al. 2005), leading to premature termination of the Mats protein. Both alleles are though to be null for the mats locus. As expected, these alleles failed to complement each other, confirming the lethality of the mats mutations (Figure 3.20).

After confirming that mats loss-of-function results in homozygous embryonic lethality (see section 3.5.3 above), we sought to test whether

MOB1A(wt) can compensate for the embryonic lethality driven by mats

loss-of-function. To do so, the matsroo transgene was recombined into the proximity of the chromosomal arm where our MOB1A(wt) transgene was integrated (see section 2.2.5.3). MOB1A(wt) and MOB1A variants were inserted on third chromosome at cytological location 89E11 using the ΦC31 integrase-mediated transgenesis system, which utilises the recombination between an attP docking site and the bacterial attachment attB site (Figure 3.21a). For the recombination of the MatsRoo transgene, Virgin w;; ubi>myc-hMOB1A, endogenous Mats/ FRT82B, Matsroo flies were crossed with male +/TM6B flies (see section 2.2.5 for further details). Importantly, to ensure that MOB1A(wt) and MOB1A mutants are expressed equally, we performed Western blot analysis of transgenic flies (see Figure 3.21b). Our results presented in Figure 3.21b show that MOB1A(wt) and MOB1A variants are expressed equally. After the required recombination

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and confirmation of equal expression of our MOB1A variants in the different fly lines, we performed the crosses presented in Figure 3.21c.

Figure 3.21. Ubiquitous expression of human MOB1(wt) or MOB1(K104E/K105E) rescues larval lethality of mats deficient flies.

(a) Schematic representation of the generation of MOB1A transgenic flies. Plasmids which

can ubiquitously express N-terminally myc-tagged wild-type (wt) or mutant human MOB1 (ubi>-myc-hMOB1A) were inserted into an identical genome location (attP site at 89E11 on chromosome 3) using ΦC31-mediated recombination. (b) Whole flies of indicated genotypes were analysed by Western blotting. Equal expression of the myc-tagged MOB1A transgenes was observed. DV/KE/KE, D63V/K104E/K105E. (c) Genetic schemes illustrating how we tested which MOB1A transgenes can rescue the larval lethality of Mats deficient flies. wt, wild-type; DV, D63V; KE/KE, K104E/K105E; DV/KE/KE, D63V/K104E/K105E. Adult flies expressing myc-MOB1A(wt) or myc- MOB1A(K104E/K105E) in a mats null (matsroo/matse235) genetic background are viable and fertile, indicating that the interaction of MOB1 with Hippo is dispensable for normal

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fly development. In contrast, adult flies expressing myc-MOB1A(D63V) or myc- MOB1A(D63V/K104E/K105E) in a mats null background were not observed, indicating that the interaction of MOB1 with Wts is essential for normal fly development.

To determine whether MOB1A can rescue the embryonic lethality caused by mats loss-of-function, w;; ubi>myc-MOB1A(wt), matsroo/ TM6B flies were mated with w;; FRT82B, matse235/ TM6B flies as outlined in Figure 3.20. This cross revealed that homozygous embryonic lethality caused by Mats loss-of-function can be rescued by transgenic ubiquitous expression of human MOB1A (wt) (Figure 3.21c, first cross).

We identified and characterized MOB1A variants with respect to binding to Hpo, Wts, and Trc (defined in sections 3.2 and 3.3 above). Therefore, considering further that MOB1A(wt) can rescue the homozygous lethality caused by homozygous mats loss-of-function (Figure 3.21c, first cross), we sought to test which PPIs of MOB1A with the Hippo core kinases are required to prevent embryonic lethality of Mats null flies. To do so, we generated transgenic flies expressing our MOB1A mutants from one fixed chromosomal location (Figure 3.21a) and performed compensation crossings, as shown in Figure 3.21 c. As expected, transgenic flies expressing MOB1A(DV) or MOB1A(DVKEKE) failed to compensate for the lethality driven by mats loss-of-function (Figure 3.21c). In stark contrast, transgenic flies expressing MOB1A(KEKE) were viable and fertile in a mats null background, hence indicating that expression of MOB1A(KEKE) fully compensates for mats loss-of-function like flies expressing wild-type MOB1A (Figure 3.21c). Considering that MOB1A(DV) is deficient in binding to Wts, while MOB1A(KEKE) does not associate with Hpo (see section 3.2 above), these results collectively indicate that the binding of MOB1A to Wts is necessary for normal fly development, while complex formation between MOB1A and Hpo is dispensable.

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3.5.4. Results – The characterisation of transgenic flies expressing