Tumor phenotype correlates with the tissue specificity and strength of the driver:
In Drosophila, the collagen-Gal4 (Cg-Gal4) driver induces the strong expression UAS tagged genes in the fatbody as well as in the hematopoietic system 58. The different UAS Hox genes lines, Dfd, Ubx, abd-A and Abd-B, when brought under the Cg-Gal4 driver, induced melanised pseudo-tumors in larvae. This phenotype manifested in 26% of Cg-Gal4>UAS Dfd larvae, 60% of Cg>Ubx larvae, 82% of Cg-Gal4>UAS abd-A larvae and 4% of Cg-Gal4>UAS-Abd-B larvae (Figure 1, 2A, 2B and Supplementary Table S3).
We then used the Hemese-Gal4 (He-Gal4) driver, which expresses throughout the lymph gland as well as in circulating hemocytes, and the HemolectinD3-Gal4 (HmlD3-Gal4) driver, which expresses in the cortical region of the lymph gland as well as in mature circulating hemocytes. While melanised pseudo tumours were observed in these genotypes, they appeared smaller and the penetrance of the phenotype was very low, manifesting in 3% of He-Gal4>UAS Dfd, 6% in HmlD3-Gal4>UAS Dfd, 9% in He-Gal4>UAS Ubx, 2% in HmlD3-Gal4>UAS Ubx, 8% in He-Gal4>UAS abd-A, 4% in HmlD3-Gal4>UAS abd-A, 3% in He-Gal4>UAS Abd-B and 2% in HmlD3-Gal4>UAS Abd-B (Figure 1, 2A, 2B and Supplementary Table S3). He-Gal4 induces expression throughout the lymph gland and in sessile cell pockets which reside underneath the larval cuticle. Thus, it expresses in all areas involved in hematopoiesis 59. Over-expression of Hox genes with the He-Gal4 driver always showed a higher penetrance of the phenotype when compared to HmlD3-Gal4. Lamellocytes are responsible for the encapsulation mechanism in combating an immune challenge, and they do not express Hemolectin. The low penetrance of the phenotype in HmlD3-Gal4 could be due to a lack of expression in lamellocytes 60. Also, Hemolectin does not express in the medullary zone of the lymph gland, where cell proliferation and differentiation takes place 61. It shows the phenotype is associated with active proliferation and differentiation of hemocytes of developing larvae. To test that the phenotype was not due to expression of the Hox genes in the fatbody (as Cg-Gal4 expresses in both blood cells as well as the fatbody) we over-expressed these genes using the fatbody specific driver Lsp2-Gal4. Lsp2-Gal4 functions in L3 larval fat bodies 62. No melanised spots were observed in such larvae, indicating that the pseudo-tumor phenotype is not induced by the misexpression of of Hox gene in the fatbody. To test whether the relative strength of the Gal4s, we overexpressed mcd8-GFP under Cg-Gal4, Hml-Gal4, He-Gal4 and Lsp2-Gal4. Hml-Gal4 was significantly weaker than He-Gal4 and cg-Gal4. He-Gal4 and cg-Gal4 appear to drive expression at similar levels. However, as we compared whole larvae of He-GFP expressing larvae to regions devoid of the fatboy in cg-Gal4 larvae, this similarity may be artefactual (Figure 7A, Supplementary table 11A). Lsp2-Gal4 and cg-Gal4 drove GFP at similar levels in the fatbody (Figure 7B, Supplementary table 11B). of the respective drivers in hemocytes (Figure 1, 2A, 2B and Supplementary Table S3). Taken together, this implied that the melanised pseudo-tumour phenotype we observe is of hemocyte origin.
Tumor phenotype is co-related with lethality at the pupal stage:
We also noticed a significant level of pupal lethality when Hox genes were misexpressed in these conditions. Pupal lethality with the Cg-Gal4 driver was highest when it drives UAS-abd-A (99 %). Cg-Gal4>UAS Dfd (53%) and Cg>Ubx (24%) also show an increased lethality at pupal stage. It was negligible in Cg-Gal4>UAS-Abd-B (2%). We observed lethality when the same genes were over expressed in the fatbody with Lsp2-Gal4. However, Lsp2-Gal4 driven Hox expression induced lethality was lower compared to Cg-Gal4 driven Hox expression induced lethality. But it must be noted that it was greater than that induced by the blood specific drivers used by us. Pupal lethality with Lsp2-Gal4 driver was observed 9% in Lsp2-Gal4>UAS Dfd, 26% in Lsp2-Gal4>UAS Ubx and 31% in Lsp2-Gal4>UAS abd-A. It has previously been shown that aberrant blood cells can induce pupal lethality 63. However, while we did observe some pupal lethality when the Hox genes were expressed under He and Hml, the lethality was most prominent in when the Cg-Gal4 or Lsp2-Gal4 driver was used (Figure 2B, Supplementary Table S4) which supports the earlier report suggesting that Hox genes are repressors of autophagy in the fatbody 29. Thus, while we do observe insignificant lethality with blood specific drivers since the expression of Hox genes in the fatbody does indeed induce lethality, the greater lethality when Cg-Gal4 is used may be due to the concomitant expression induced in the fatbody as well as blood cells.
Hox genes over-expression induces hemocyte proliferation and differentiation:
Change in number cells and types of cells become important considering the phenotype observed upon misexpression of Hox genes. We quantified the number of blood cells in our overexpression lines using a modified version of established methods 56,57. When expressed by blood specific driver, Dfd, Ubx and abd-A led to a significant increase in the number of circulating hemocytes (Figure 3A and 3B, Supplementary Table S5-8). Interestingly, while the penetrance of melanised spots was lower, blood specific drivers showed a larger number of blood cells (Figure 3B). Under the control of, Lsp2, the fatbody exclusive driver, however, Ubx and abd-A gave a significant increase in hemocyte number, despite them not manifesting melanised spots. Our results show that melanised spots (or pseudo-tumors), which have been reported as the hallmarks of a “leukemia-like” phenotype in Drosophila, may not reflect an actual increase in hemocytes. Additionally, many studies have used the strong driver Cg-Gal4, which drives expression in fatbody as well as the blood cells. As our results show that perturbations in the fatbody may indeed lead to an increase in circulating hemocytes. It may also be that the number of circulating cells when we expressed the hox genes under cg-Gal4 may be due to circulating cells being trapped in the melanised peseudo tumors.
Previous studies have shown that cells of the LG do not enter into circulation until the onset of metamorphosis. However, Hml and Cg express in the cortical region of the LG, and He expresses throughout. Thus, the question arose as to whether the increase in cell number was due to an increase in cell proliferation at the LG or were circulating cells proliferating in a cell-autonomous manner. Hence, we checked for the presence of the mitotic cell marker PH3. We observed cells positive for PH3, when Hox genes were expressed in the blood cells, and not when expressed exclusively in the fatbody (Figure 3A, Supplementary Figure 1A-D). Unlike previous reports, we did not find proliferative cells in our control experiments 64. This may be due to a loss of cells in our preparations or more robust immunostaining on our part. Thus, while we cannot rule out the possibility that LG cells contribute to this increase, at least a fraction of the increase takes place due to the cell autonomous division of Hox overexpressing cells. As cells of the LG could potentially prematurely be released into circulation on account of the Hox gene over expression, we checked for the integrity of the LG by overexpressing UAS-Dfd, UAS Ubx and UAS-abd-A in an Hml-Gal4, UAS-GFP background. LGs remained intact 96hrs post egglay (Figure 6)
While imaging the blood cells, we noticed that there were larger, flattened cells in circulation, reminiscent of lamellocytes. To test whether they were bonafide lamellocytes, we stained the hemocytes for the lamellocyte marker myospheroid (Figure 4A, Supplementary Figure 1A-D). Control larvae infrequently showed the presence of lamellocytes. In our overexpression lines, however, we noticed that a significant number of cells were lamellocytes mys+. Some plasmatocytes also stained positive for mys. None of the plasmatocytes in the control flies or those overexpressing Abd-B were positive for mys. Previous reports have sugessteded that circulating plasmatocytes may differentiate into lamellocytes 65,66. Thus, it may be that these circulating mys+ plasmatocyte like cells are differentiating into lamellocytes. However, Lsp2-Gal4>UAS Ubx also had a significant number of lamellocytes. This is in keeping with reports that signals from the fat body can drive lamellocyte differentiation 67,68. Thus, we speculate that these cells, upon Hox overexpression, are pushed toward the lamellocyte fate (Figure 4A, 4B).
Effect of PcG and trxG genes
PcG members are known to function primarily through two distinct complexes, PRC1 (consisting of Pc, Psc, Su(z)2 and Sce) and PRC2 (consisting of E(z), Su(z)12, Esc and Caf 1-55) 69. Members of the PcG and trxG have been shown to have a role in hematological malignancies in different clinicopathological data in leukemic patients and mice models 70,71. To determine their role in melanized pseudo-tumor formation in flies, we over-expressed abd-A using Cg-Gal4 in the background of different PcG and trxG mutants. We selected Psc1, Pc1, Su(z)2, Su(z)12, E(z) and esc2 from the PcG and brm2, Trl from the trxG. Melanotic pseudo-tumor phenotype was used in our study to assay the effect of the mutants as it is convenient and robust. All experiments were performed in biological triplicates. The PcG mutants Pc1, Su(z)123, and trxG member brm2 showed an increase in melanotic body formation (Figure 5A, 5B and Supplementary Table S9), and enhanced the phenotype upto 100 per cent. Pc1 and Su(z)123 not only enhanced the penetrance (percentage phenotype showing larvae) but showed an increase in severity (scored as number and size of the black spots) compared to abd-A over-expressed in absence of mutants (Figure 5A). Pc and Su(z)12, both are the core proteins of PRC1 complex and play a role in negative regulation of their target genes. Our results indicate these proteins might regulate melanotic body formation. Surprisingly, E(z) does not show any significant effect on penetrance. On the other hand, esc2 (PRC2 member) and Psc1 (PRC1 member) showed a significant decrease in penetrance 15% and 17% respectively (Figure 5B, Supplementary Table S9). The severity of the phenotype is also reduced in both the mutant background. These results indicate that genes involved in melanotic pseudo-tumor causing phenotype might be the target of the Esc and Psc proteins. Although it has been shown that Esc-E(z) complex is a thousand times effective to E(z) alone 72,73, our results suggest that Esc regulates its targets independent of E(z) activity or, for that matter, any other member of the PRC2 complex in the observed phenotype. Similarly, Psc mutation rescued the phenotype. We tested whether bringing our overexpression in the PcG and trxG backgrounds affected the number of PH3 positive nuclei. We did not observe a significant change (Figure 5D, Supplementary table 12). As the average number of PH3+ nuclei in cg-Gal4>UAS abd-A larvae was 0.08% of the average of total hemocytes, it may be that the total number of dividing nuclei are too few to significantly differ.
Effect of PcG mutants on the melanized pseudo-tumor related pupal lethality:
To test the effect of mutations on pupal lethality, L3F larvae from each combination, which manifested melanised pseudo-tumours, were transferred to fresh vials and allowed to pupate and eclose. Larvae from overexpressed abd-A (driven by Cg-Gal4) with melanotic body showed up to 99% lethality at the pupal stage. Further, we checked pupal lethality in mutant background. Since all mutants are maintained over balancers (Table S2), we selected overexpressed progenies without balancer to confirm mutant in the same progeny and transferred them in new food vials. Pupal lethality in Su(z)123, Pc1 and Su(z)21.a1 was always 100% while we could get a few survivors from Cg-Gal4>UAS abd-A (Figure 3C, Table S10). A decline in lethality was seen in Psc1,esc2 brm2 and TrlR85. The survivors from Psc1 and esc2 were quite healthy as compared to the survivors of Cg-Gal4>UAS abd-A. This reduction in lethality indicates that Esc and Psc proteins are strongly suppressing the melanotic pseudo-tumour phenotype and its consequences on development. Although brm2 showed an increase in penetrance it decreases pupal lethality 89% compare to abd-A alone. TrlR85 showed a decrease in pupal lethality (79%).