Editor's evaluation: Antagonistic role of the BTB-zinc finger transcription factors Chinmo and Broad-Complex in the juvenile/pupal transition and in growth control

Abstract

Full text Figures and data Side by side Abstract Editor's evaluation eLife digest Introduction Results and discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract During development, the growing organism transits through a series of temporally regulated morphological stages to generate the adult form. In humans, for example, development progresses from childhood through to puberty and then to adulthood, when sexual maturity is attained. Similarly, in holometabolous insects, immature juveniles transit to the adult form through an intermediate pupal stage when larval tissues are eliminated and the imaginal progenitor cells form the adult structures. The identity of the larval, pupal, and adult stages depends on the sequential expression of the transcription factors chinmo, Br-C, and E93. However, how these transcription factors determine temporal identity in developing tissues is poorly understood. Here, we report on the role of the larval specifier chinmo in larval and adult progenitor cells during fly development. Interestingly, chinmo promotes growth in larval and imaginal tissues in a Br-C-independent and -dependent manner, respectively. In addition, we found that the absence of chinmo during metamorphosis is critical for proper adult differentiation. Importantly, we also provide evidence that, in contrast to the well-known role of chinmo as a pro-oncogene, Br-C and E93 act as tumour suppressors. Finally, we reveal that the function of chinmo as a juvenile specifier is conserved in hemimetabolous insects as its homolog has a similar role in Blatella germanica. Taken together, our results suggest that the sequential expression of the transcription factors Chinmo, Br-C and E93 during larva, pupa an adult respectively, coordinate the formation of the different organs that constitute the adult organism. Editor's evaluation This important study demonstrates that the transcription factor Chinmo is a master regulator that maintains larval growth and development as part of the metamorphic gene network in Drosophila. Chinmo does so in part by regulating Broad expression in imaginal tissues (e.g. eye and wing discs) and in a Broad-independent manner in other larval tissues such as the salivary glands and larval trachea. Finally, the authors demonstrate that the role of Chinmo in promoting larval development is conserved between holometabolous insects and hemimetabolous insects, which lack a pupal stage. The data were collected and analyzed using solid and validated methodology and will be of interest to a broad audience including those interested in development and evolution. https://doi.org/10.7554/eLife.84648.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Egg, larva, pupa, adult: the life of many insects is structured around these four well-defined stages of development. After hatching, the larva grows until it reaches a certain size; when the right conditions are met, it then becomes a pupa and metamorphoses into an adult. Most larval cells die during metamorphosis; only a group known as imaginal cells survives, dividing and maturing to create pupal and adult tissues. Each of these developmental steps are linked to a particular genetic program deployed in response to a single stage-specifying gene. For instance, the activation of the Br-C gene triggers the transition from larva to pupa, while E93 initiates the transformation of the pupa into an adult. However, which stage-specifying gene controls larval identity remains unclear. Recent studies suggest that in fruit flies, a gene known as chinmo could be playing this role. In response, Chafino et al. explored how chinmo shapes the development of fruit fly larvae. The experiments showed that chinmo is activated in the juvenile stage, and that it is required for the larvae to grow properly and for larval and imaginal tissues to form. Conversely, it must be switched off for the insect to become a pupa and then an adult. Further work suggested that the role of chinmo as a larval specifier could have emerged early in insect evolution. Moreover, Chafino et al. revealed that chinmo could repress Br-C, an important characteristic since stage-specifying genes usually switch on sequentially by regulating each other. A closer look suggested that, in imaginal cells, chinmo promotes development by inhibiting Br-C; in larval cells, however, chinmo not only has a Brc-repressing role but it is also necessary for larval cells to grow. Additional experiments exploring the role of the stage-specifying genes in tumor formation showed that chinmo promotes cells proliferation while Br-C and E93 had tumor-suppressing properties. Overall, the work by Chafino et al. sheds new light on the genetic control of insect development, while also potentially providing a new perspective on how genes related to chinmo and Br-C contribute to the emergence of human cancers. Introduction Animal development passes through various stages characterised by distinct morphological and molecular changes. In humans, for instance, development continues from birth through to childhood and puberty to give rise to the adult form. As in many animals, in holometabolous insects such as Drosophila melanogaster, the developmental stages are sharply defined: embryogenesis gives rise to the larva, a juvenile stage, which, upon different rounds of growth and moulting, brings about a new stage structure, the pupa, when most of the larval cells die and the adult progenitor cells (imaginal cells) develop to generate the adult organism. The regulation of stage-specific differences is mediated by the action of two major developmental hormones, the steroid 20-hydroxyecdysone and the terpenoid juvenile hormone (Hiruma and Kaneko, 2013; Jindra et al., 2013; Truman, 2019; Truman and Riddiford, 2007; Truman and Riddiford, 2002; Yamanaka et al., 2013). Both hormones exert this precise developmental control by regulating the expression of three critical genes that encode for the stage-identity factors that compose the metamorphic gene network: the C2H2 zinc finger type factor Krüppel-homolog 1 (Kr-h1), the helix-turn-helix Ecdysone inducible protein 93F (E93), and Broad-complex (Br-C; also known as broad), a member of the bric-a-brac-tramtrack-broad family (Martín et al., 2021). The deployment of the pupal-specific genetic program is controlled by the expression of Br-C at the larval-pupal transition (Truman, 2019; Zhou and Riddiford, 2002). Upon the formation of the pupa, hormone signalling triggers the expression of the helix-turn-helix factor E93, whose product represses Br-C expression and directs the formation of the final differentiated adult structures (Chafino et al., 2019; Martín et al., 2021; Ureña et al., 2014). While it is firmly established that Br-C and E93 are the stage-specifying genes for the pupal and adult states, the nature of the larval specifying gene has been elusive. To date, larval identity has been attributed to Kr-h1, which is present during the larval period and represses Br-C and E93 expression during this period (Huang et al., 2011; Ureña et al., 2016). However, although Kr-h1 is undoubtedly critical for maintaining the larval state, evidence has shown that this factor cannot be considered the larval specifier per se. For example, depletion of Kr-h1 in Drosophila does not prevent normal larval development nor a timely transition to the pupa (Beck et al., 2004; Pecasse et al., 2000). In this regard, the product of chronologically inappropriate morphogenesis (chinmo) gene, another member of the BTB family of transcription factors, has been recently proposed to be responsible for larval identity in Drosophila (Truman and Riddiford, 2022). First isolated based on its requirement for the temporal identity of mushroom body neurons (Zhu et al., 2006), the identification of Chinmo as a more general larval specifier has provided invaluable insights into the molecular mechanisms underlying the control of juvenile identity. Yet, little is known about how this factor exerts its function along with Br-C and E93. Moreover, given that holometabolous insects are comprised of both larval tissues and pools of adult progenitor cells (known as imaginal cells), a central issue in the understanding of how larval identity is controlled is how larval and imaginal cells respond differentially to the same set of temporal transcription factors. Furthermore, in the sequential activation of chinmo, Br-C, and E93, the extent of the activity directly attributable to each transcription factor or to their mutual repression is still unclear. Here, we confirm the role of chinmo as larval specifier in larval and imaginal cells and establish its regulatory interactions with the other temporal specifiers. We also examine how the temporal sequence of Chinmo and Br-C differently affects with the genetic program that establishes larval vs. imaginal identity. Thus, we found that Chinmo controls larval development of larval and imaginal tissues in a Br-C-independent and -dependent manner, respectively. According to these data, and in the context of the metamorphic gene network, we also show that chinmo absence is critical for the transition from larva to pupa and then to adult, as it acts as a repressor of both Br-C and E93. In addition, we report that the chinmo homologue has a similar role in the cockroach Blattella germanica, thereby indicating that its function as a juvenile specifier precedes the hemimetabolous/holometabolous split. Finally, we show that in contrast to the well-characterised role of chinmo as a pro-oncogene, the Br-C pupal and E93 adult specifiers act mainly as tumour suppressor genes. These characteristics are maintained beyond insects and may account for the different role of some human BTB-zinc finger transcription factors in tumourigenesis. Results and discussion chinmo is expressed throughout larval stages and is required in larval and imaginal tissues Examination of chinmo expression revealed that it is expressed during embryogenesis and early larval development and that it is strongly downregulated from L3 (Figure 1A). Immunostaining analysis in imaginal and larval tissues confirmed the presence of Chinmo in L1 and L2 stages and its disappearance in late L3 (Figure 1B and C), an expression profile that is in agreement with previous studies (Narbonne-Reveau and Maurange, 2019; Truman and Riddiford, 2022). We next addressed its functional requirement by knocking down this factor with an RNAi transgene controlled by the ubiquitous Act-Gal4 driver. chinmo-depleted animals showed developmental arrest at the end of the first instar larval stage presenting a tanned cuticle clearly reminiscent of the tanned larval cuticle of the puparium (Figure 1D). Consistent with the phenotype, we found that arrested chinmo-depleted larvae precociously expressed pupal cuticle genes while blocked larval-specific genes activation (Figure 1E). These results confirm that chinmo is required for normal progression of the organism during the larval period, as proposed by Truman and Riddiford, 2022. Figure 1 Download asset Open asset Chinmo is expressed during early larval stages and is essential for proper larval development. (A) chinmo mRNA levels measured by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) from embryo to the wandering stage of L3 (L3W). Transcript abundance values were normalised against the Rpl32 transcript. Fold changes were relative to the expression of embryo, arbitrarily set to 1. Error bars indicate the SEM (n = 3). (B–C) Chinmo protein levels in the wing disc (B) and salivary glands (C) of larval L1, L2, and L3W (females) stages. (D) Compared with the control (Act-Gal4), overexpression of UAS chinmoRNAi in the whole body induced developmental arrest at the L1 stage. Scale bars represent 50 µm (B and C) and 0.5 mm (D). (E) Relative expression of larval-specific (Crp47Eg, Lcp65Ag3, and CG30457) and pupal-specific genes (Edg78E) in UAS-chinmoRNAi L1 larvae measured by qRT-PCR. Transcript abundance values were normalised against the Rpl32 transcript. Fold changes were relative to the expression in control larvae, arbitrarily set to 1 (dashed black line). Error bars indicate the SEM (n = 3). Statistical significance was calculated using t test (***p≤0.001; **p≤0.005). Figure 1—source data 1 Numerical data for Figure 1A and E. https://cdn.elifesciences.org/articles/84648/elife-84648-fig1-data1-v2.xlsx Download elife-84648-fig1-data1-v2.xlsx Since Drosophila larva consists of a combination of larval and imaginal tissues, we then analysed the contribution of chinmo to the development of these two types of tissues. Regarding the former, chinmo was selectively depleted in the salivary glands using the forkhead (fkh) driver (fkh-Gal4), which is active in this tissue from embryogenesis onwards. The salivary glands are a secretory organ that develops from embryonic epithelial placodes (Abrams et al., 2003; Bradley et al., 2001; Edgar et al., 2014; Zielke et al., 2013). This tissue is responsible for producing glycosylated mucin for the lubrication of food during the larval period (Costantino et al., 2008; Farkaš et al., 2014; Riddiford, 1993; Syed et al., 2008) and for synthesising glue proteins for the attachment of the pupa to a solid surface at the onset of metamorphosis (Andres et al., 1993; Costantino et al., 2008; Kaieda et al., 2017). As it is shown in Figure 2A, although depletion of chinmo in the salivary glands did not affect the formation of this organ, it caused a dramatic decrease in normal larval development, as revealed by the strong reduction in size and DNA content of the gland cells (Figure 2B–D). Consistently, the expression levels of both early and late specific salivary gland protein encoding genes, such as new glue 1–3 (ng) and Salivary gland secretion (Sgs), were virtually undetectable in chinmo-depleted salivary glands compared to control (Figure 2E). Remarkably, to further study the requirement of chinmo for larval tissue growth, we analyzed the role of this factor in the larval tracheal system. Although depletion of chinmo specifically in the tracheal cells, using a trh-Gal4 driver, resulted in many arrested L2 larvae with a necrotic tracheal system, escapers that reached L3 presented reduction in nuclear size and DNA content of tracheal cells as well as reduced length of the organ (Figure 2—figure supplement 1), thus confirming that Chinmo is required for proper growth of larval tissues. Figure 2 with 1 supplement see all Download asset Open asset Chinmo is required for proper growth and function of the salivary glands during larval development. (A) DAPI staining of salivary glands from control (fkh-Gal4) and UAS-chinmoRNAi larvae at L3W. Scale bar represents 50 µm. (B–D) Comparison of the relative size of salivary glands (n = 10 for each genotype) (B), DAPI intensity (n = 50 for each genotype) (C), and nucleic size of salivary glands (n = 50 for each genotype) (D) between UAS-chinmoRNAi and control larvae at L3W. Error bars indicate the SEM (n = 5–8). (E) Relative expression of ng1-3 and Salivary glands secretion genes (Sgs) in UAS-chinmoRNAi L3W animals measured by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). Transcript abundance values were normalised against the Rpl32 transcript. Error bars indicate the SEM (n = 5–8). Statistical significance was calculated using t test (***p≤0.001). Figure 2—source data 1 Numerical data for Figure 2B–E. https://cdn.elifesciences.org/articles/84648/elife-84648-fig2-data1-v2.xlsx Download elife-84648-fig2-data1-v2.xlsx Regarding the role of chinmo in imaginal tissues, we knocked down this factor in the pouch region of wing imaginal discs from the embryonic period onwards using the escargot (esg) driver (esg-Gal4). As before, depletion of chinmo in the esg domain did not alter the specification of the disc, but strongly impeded its larval development. Thus, in late L3 wing discs only the notum, which does not express the esg-Gal4 driver, was observed while the wing pouch, revealed by positive GFP signal, was strongly reduced and did not show the expression of patterning genes such as wingless (wg) and cut (ct) (Figure 3A). In line with these results, although most of the chinmo-depleted animals arrested development as pharate adults, escapers that were able to eclose (15%) had no wings (Figure 3—figure supplement 1). Similarly, depletion of chinmo in the eye disc using a specific driver (ey-Gal4) induced similar effects abolishing the developing tissue and the formation of the adult eye (Figure 3—figure supplement 2). Taken together, these data show that Chinmo is required during the larval period to control the development and function of larval and imaginal tissues. Figure 3 with 2 supplements see all Download asset Open asset Chinmo is necessary for wing development during the larval period. Expression of Ct and Wg in wing discs of control (esg-Gal4) and UAS-chinmoRNAi L3W larvae. Wing discs were labelled to visualise the esg domain (GFP in green) and nuclei (DAPI). Ct and Wg were not detected in UAS-chinmoRNAi. Scale bars represent 50µm. Distinct roles of Chinmo in larval and progenitor cells A critical feature of the metamorphic gene network factors is that their sequential expression is achieved through a series of regulatory interactions between them. Therefore, we next sought to characterise the regulatory interactions of Chinmo with the pupal specifier Br-C and the adult specifier E93. To this end, we measured the expression of Br-C and E93 in chinmo-depleted salivary glands and wing discs. Contrary to recently published data (Truman and Riddiford, 2022), both tissues showed a significant and premature increase of Br-C protein levels as early as in L1 larvae, while no increase in E93 protein levels was detected in any tissue (Figure 4). Figure 4 with 1 supplement see all Download asset Open asset Chinmo represses Br-C in salivary glands and wing discs during early larval development. (A–B) Expression of Chinmo, Br-C, and E93 in salivary glands of L1 control (fkh-Gal4) (A), and UAS-chinmoRNAi (B). (C–D) Expression of Chinmo, Br-C, and E93 in wing discs of early L2 control (esg-Gal4) (C) and UAS-chinmoRNAi (D). The esg domain is marked with GFP and all cell nucleus with DAPI. In the absence of chinmo only Br-C shows early upregulation in both tissues. Scale bars represent 25 µm. In view of these results, we speculated whether the impairment of larval development observed in chinmo-depleted animals could be the result of precocious presence of the wrong stage-identity factor, in this case, Br-C. To address this notion, we precociously expressed Br-CZ1, the main Br-C isoform expressed during imaginal larval development (Narbonne-Reveau and Maurange, 2019), in salivary glands and wing discs. As previously described, ectopic expression of Br-CZ1 blocked Chinmo activation (Narbonne-Reveau and Maurange, 2019). As a consequence, precocious upregulation of Br-C blocked development in both tissues, phenocopying the loss of function of chinmo (Figure 4—figure supplement 1). This result suggests that a fundamental function of Chinmo is to suppress the expression of the pupal specifier Br-C during the juvenile stages. To confirm this hypothesis, we simultaneously depleted chinmo and Br-C in salivary glands and wing discs. Remarkably, whereas salivary glands showed the same growth impairment observed upon chinmo depletion (Figure 5A-F, Figure 5—figure supplement 1), depletion of Br-C largely rescued the abnormalities in the wing discs caused by depletion of chinmo: the double knock-out wing discs developed in a regular manner to reach normal size by the end of L3 and showed proper expression of patterning genes such as wg (Figure 5G, Figure 5—figure supplement 2). The difference between larval and imaginal tissues was also observed in the analysis of the tracheal system and the eye imaginal disc. Whereas depletion of Br-C in the eye disc rescued the phenotype induced by the absence of chinmo (Figure 3—figure supplement 1), the larval trachea failed to restore the growth defects observed in chinmo-depleted tracheal cells (Figure 2—figure supplement 1). Taken together, our results suggest that a major regulatory function of chinmo during early larval development in imaginal cells is channelled through the repression of Br-C, while in larval tissues chinmo appears to exert specific growth-related functions that are independent to Br-C repression. Thus, in the imaginal cells chinmo appears to ensure the expression of juvenile genes by repressing Br-C, a well-known inhibitor of larval gene expression (Zhou and Riddiford, 2002). In this regard, it is tempting to speculate that Br-C might repress the early expression of critical components of signaling pathways such as Wg and EGFR, involved in wing fate specification in early larval development (Ng et al., 1996; Wang et al., 2000; Zecca and Struhl, 2002). In contrast, in larval tissues chinmo seems to exert an active role promoting growth and maturation. The fact that the Br-C-dependent Sgs genes fail to be activated in absence of chinmo, when Br-C is prematurely expressed, supports this idea (Figure 2E). This different response could be explained by the nature of the larval and imaginal tissues. While larval tissues are mainly devoted to growth during the larval period and then fated to die during the metamorphic transition, the developmental identity of the imaginal cells is modified along the larva-pupa-adult temporal axis to give rise to the adult structures. This difference could also account for the distinct roles of the other members of the metamorphic gene network in larval and imaginal tissues. Thus, while Br-C is necessary for the degeneration of the larval salivary glands during the onset of the pupal period (Jiang et al., 2000), it is critical for the correct eversion of the imaginal wing disc and for the temporary G2 arrest that synchronises the cell cycle in the wing epithelium during early pupa wing elongation (Guo et al., 2016). Likewise, E93 is necessary to activate autophagy for elimination of the larval mushroom body neuroblasts in late pupae (Pahl et al., 2019), whereas it controls the terminal adult differentiation of the imaginal wing during the same period (Ureña et al., 2016; Uyehara et al., 2017). Figure 5 with 2 supplements see all Download asset Open asset Different requirement of chinmo for the larval growth of salivary glands and wing discs. (A) DAPI-stained salivary glands from control (fkh-Gal4) and UAS-Br-CRNAi; UAS-chinmoRNAi L3W larvae. In the absence of chinmo and Br-C, salivary glands did not grow. (B–D) Comparison of the relative size of salivary glands (n = 10 for each genotype) (B), DAPI intensity (n = 50 for each genotype) (C), and nucleic size of salivary glands (n = 30 for each genotype) (D) of control and UAS-Br-CRNAi; UAS-chinmoRNAi L3W larvae. (E–F) Relative expression of (E) ng1-3 and (F) Salivary glands secretion genes in control and UAS-Br-CRNAi; UAS-chinmoRNAi L3W larvae measured by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). Transcript abundance values were normalised against the Rpl32 transcript. Error bars in B and C indicate the SEM (n = 5–8). Statistical significance was calculated using t test ( ****p≤0.001). (G) Expression of Chinmo, Br-C, and Wg in wing discs of UAS-Br-CRNAi; UAS-chinmoRNAi L3W larvae. Wing discs labelled to visualise the esg domain (GFP in green). In the absence of chinmo and Br-C, wing discs grow normally and express Wg correctly. Scale bars represent 50 µm. Figure 5—source data 1 Numerical data for Figure 5B–F. https://cdn.elifesciences.org/articles/84648/elife-84648-fig5-data1-v2.xlsx Download elife-84648-fig5-data1-v2.xlsx Downregulation of chinmo is required during metamorphosis The functional and expression data reported above show that Chinmo acts as a larval specifier in Drosophila. From this, we could infer that its absence by the end of larval development is required first for the transition to the prepupa, and then to allow terminal adult differentiation during the pupal period. If this were the case, maintenance of high levels of chinmo during late L3 would interfere with the larva-pupal transition. To test this possibility, we maintained high levels of chinmo in late L3 wing discs using the Gal4/Gal80ts system. Consistent with this hypothesis, overexpression of chinmo from early L3 in the anterior compartment of the disc using the cubitus interruptus ci-Gal4 driver impaired its larva-pupal transition as abolished Br-C expression and induced apoptosis at late L3 as revealed by the high expression of the effector caspase Dcp-1 (Figure 6A). As a result, the size of the anterior compartment was dramatically reduced, and the expression of patterning genes such as ct was halted (Figure 6B). Impairment of ct expression was not just a consequence of cell death, as ct expression was neither detected in wing discs overexpressing both chinmo and the p35 inhibitor of effector caspases (Hay et al., 1994; Figure 6C and D), but instead to a distinct response to the sustained expression of chinmo or to the consequent depletion of Br-C. Figure 6 Download asset Open asset chinmo depletion during late L3 is required for proper larva to pupa transition. (A–H) Images of wing imaginal discs from L3W larvae. The indicated constructs were expressed under the control of the ci-Gal4 driver. Overexpression or depletion of the transgenes was activated in early L3 larvae and analyzed at the L3W stage. An UAS-GFP construct was used to mark the anterior region of the disc where the transgenes were induced or repressed (green). (A) Overexpression of chinmo repressed Br-C, induced Dcp-1, and (B) abolished Ct. (C) Overexpression of chinmo together with p35 repressed Br-C and blocked Dcp-1, but fails to restore normal expression of Ct (D). (E) Depletion of Br-C induced Chinmo and Dcp-1 and (F) repressed Ct. (G) In double depletion of Br-C and chinmo (H), Dcp-1 was not detected. Scale bars represent 50 µm. An alternative way to keep high levels of chinmo at late L3 is by depleting Br-C, a well-known repressor of chinmo from mid L3 (Narbonne-Reveau and Maurange, 2019). Therefore, we knocked down Br-C in the anterior compartment of the wing disc and confirmed that Chinmo levels remained high in this compartment by late L3. Also, in this case we observed a strong Dcp-1 staining and impairment of ct expression (Figure 6E and F). Importantly, simultaneous depletion of chinmo and Br-C from early L3 did not lead to an increase in apoptosis (Figure 6G) nor altered the expression of patterning genes (Figure 5F), which indicates that tissue death at the end of the larval period is due to sustained expression of chinmo rather than the absence of Br-C. Altogether, these results confirm that the transition from larva to pupa must take place in the absence of the larval specifier Chinmo. Next, we analyzed whether lack of chinmo is also important during the pupal period to allow the E93-dependent development of the adult. To this end, we used the thermo-sensitive system to overexpress chinmo in the anterior part of the wing specifically during the pupal stage. To that aim, larvae were maintained at 18°C until 12 hr after pupa formation (APF) and then shifted to 29°C to allow the Gal4 to function. The resulting ectopic expression of chinmo led to a marked decrease in E93 protein levels (Figure 7A). As a result, the anterior compartment of the wing was strongly undifferentiated, a phenotype reminiscent of that observed in E93-depleted wings (Ureña et al., 2016; Ureña et al., 2014; Figure 7B). Taken together, our results show that chinmo must be downregulated during the initiation and throughout the metamorphic transition to allow the sequential expression of the pupal specifier Br-C and the adult specifier E93. Figure 7 Download asset Open asset Presence of Chinmo during pupal development blocks adult differentiation. (A) Overexpression of chinmo in the anterior part of the pupal wing at 72 hr after pupa formation (APF) using ci-Gal4 driver represses E93 expression and produced alterations in phalloidin (Pha) pattern. (B) Cuticle preparation of a pupal wing at 96 hr APF expressing chinmo under the control of the ci-Gal4 driver. Bottom panels are magnifications from upper images. The scale bars represent 50 μm (top panels) and 100 μm (bottom panels). Antagonistic effects of chinmo and Br-C/E93 in tumour growth Chinmo and Br-C belong to the extended family of BTB-zinc finger transcription factors, which are not restricted to insects. In humans, many such factors have been implicated in cancer, where they have opposing effects, from oncogenic to tumour suppressor functions (Siggs and Beutler, 2012). However, while overexpression of Drosophila chinmo has been found to cooperate with Ras or Notch to trigger massive tumour overgrowth (Doggett et al., 2015), changes in Drosophila Br-C expression have not been associated with any effect on tumourigenesis. Since the results described here, and those from other labs (Narbonne-Reveau and Maurange, 2019), indicate that chinmo and Br-C have antagonistic effects in terms of proliferation vs. differentiation, we addressed whether these opposite features might also be associated with pro-oncogenic or tumour suppressor properties, respectively. To test this notion, we resorted to the well-defined tumourigenesis model in Drosophila generated by the depletion of cell polarity genes