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

Chafino etal. eLife 2023;12:e84648. DOI: https://doi.org/10.7554/eLife.84648 1 of 20 Antagonistic role of the BTBzinc finger transcription factors Chinmo and BroadComplex in the juvenile/pupal transition and in growthcontrol Sílvia Chafino1,2,3, Panagiotis Giannios1,3, Jordi Casanova1,3, David Martín2*, Xavier FranchMarro2* 1Institut de Biologia Molecular de Barcelona (CSIC), Barcelona, Spain; 2Institute of Evolutionary Biology (IBE, CSICUniversitat Pompeu Fabra), Barcelona, Spain; 3Barcelona Institute of Science and Technology, Institute for Research in Biomedicine, IRB Barcelona, Barcelona, Spain 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, BrC, 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 BrCindependent 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 wellknown role of chinmo as a prooncogene, BrC 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, BrC 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 Broadindependent 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. RESEARCH ARTICLE *For correspondence: [email protected] (DM); xavier[email protected] (XFM) Competing interest: The authors declare that no competing interests exist. Funding: See page 16 Received: 02 November 2022 Preprinted: 18 November 2022 Accepted: 27 April 2023 Published: 28 April 2023 Reviewing Editor: Vilaiwan M Fernandes, University College London, United Kingdom Copyright Chafino etal. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Research article Developmental Biology | Evolutionary Biology Chafino etal. eLife 2023;12:e84648. DOI: https://doi.org/10.7554/eLife.84648 2 of 20 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 stagespecific differences is mediated by the action of two major developmental hormones, the steroid 20hydroxyecdysone 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 stageidentity factors that compose the metamorphic gene network: the C2H2 zinc finger type factor Krüppelhomolog 1 (Krh1), the helixturnhelix Ecdysone inducible protein 93F (E93), and Broadcomplex (BrC; also known as broad), a member of the bricabractramtrackbroad family (Martín etal., 2021). The deployment of the pupalspecific genetic program is controlled by the expression of BrC at the larvalpupal transition (Truman, 2019; Zhou and Riddiford, 2002). Upon the formation of the pupa, hormone signalling triggers the expression of the helixturnhelix factor E93, whose product represses BrC 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 BrC and E93 are the stagespecifying 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 Krh1, which is present during the larval period and represses BrC and E93 expression during this period (Huang etal., 2011; Ureña etal., 2016). However, although Krh1 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 Krh1 in Drosophila does not prevent normal larval development nor a timely transition to the pupa eLife digest Egg, larva, pupa, adult: the life of many insects is structured around these four welldefined 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 stagespecifying gene. For instance, the activation of the BrC gene triggers the transition from larva to pupa, while E93 initiates the transformation of the pupa into an adult. However, which stagespecifying 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 BrC, an important characteristic since stagespecifying genes usually switch on sequentially by regulating each other. A closer look suggested that, in imaginal cells, chinmo promotes development by inhibiting BrC; in larval cells, however, chinmo not only has a Brcrepressing role but it is also necessary for larval cells to grow. Additional experiments exploring the role of the stagespecifying genes in tumor formation showed that chinmo promotes cells proliferation while BrC and E93 had tumorsuppressing 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 BrC contribute to the emergence of human cancers. Research article Developmental Biology | Evolutionary Biology Chafino etal. eLife 2023;12:e84648. DOI: https://doi.org/10.7554/eLife.84648 3 of 20 (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 BrC 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, BrC, 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 BrC 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 BrCindependent 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 BrC 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 wellcharacterised role of chinmo as a prooncogene, the BrC 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 BTBzinc 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 (NarbonneReveau 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 ActGal4 driver. chinmodepleted 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 chinmodepleted larvae precociously expressed pupal cuticle genes while blocked larvalspecific 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. 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 (fkhGal4), 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 Research article Developmental Biology | Evolutionary Biology Chafino etal. eLife 2023;12:e84648. DOI: https://doi.org/10.7554/eLife.84648 4 of 20 glue 1–3 (ng) and Salivary gland secretion (Sgs), were virtually undetectable in chinmodepleted 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 trhGal4 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. 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 (esgGal4). 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 Figure 1. Chinmo is expressed during early larval stages and is essential for proper larval development. (A)chinmo mRNA levels measured by quantitative realtime reverse transcriptase polymerase chain reaction (qRTPCR) 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 (ActGal4), 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 larvalspecific (Crp47Eg, Lcp65Ag3, and CG30457) and pupalspecific genes (Edg78E) in UASchinmoRNAi L1 larvae measured by qRTPCR. 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). The online version of this article includes the following source data for figure 1: Source data 1. Numerical data for Figure1A and E. Research article Developmental Biology | Evolutionary Biology Chafino etal. eLife 2023;12:e84648. DOI: https://doi.org/10.7554/eLife.84648 5 of 20 the esgGal4 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 chinmodepleted 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 (eyGal4) 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 2. Chinmo is required for proper growth and function of the salivary glands during larval development. (A)DAPI staining of salivary glands from control (fkhGal4) and UASchinmoRNAi 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 UASchinmoRNAi and control larvae at L3W. Error bars indicate the SEM (n = 5–8). (E)Relative expression of ng13 and Salivary glands secretion genes (Sgs) in UASchinmoRNAi L3W animals measured by quantitative realtime reverse transcriptase polymerase chain reaction (qRTPCR). 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). The online version of this article includes the following source data and figure supplement(s) for figure 2: Source data 1. Numerical data for Figure2B–E. Figure supplement 1. The role of Chinmo in the larval tracheal system. Figure supplement 1—source data 1. Numerical data for Figure2—figure supplement 1DF. Research article Developmental Biology | Evolutionary Biology Chafino etal. eLife 2023;12:e84648. DOI: https://doi.org/10.7554/eLife.84648 6 of 20 Figure 3. Chinmo is necessary for wing development during the larval period. Expression of Ct and Wg in wing discs of control (esgGal4) and UASchinmoRNAi L3W larvae. Wing discs were labelled to visualise the esg domain (GFP in green) and nuclei (DAPI). Ct and Wg were not detected in UASchinmoRNAi. Scale bars represent 50µm. Figure 3 continued on next page Research article Developmental Biology | Evolutionary Biology Chafino etal. eLife 2023;12:e84648. DOI: https://doi.org/10.7554/eLife.84648 7 of 20 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 BrC and the adult specifier E93. To this end, we measured the expression of BrC and E93 in chinmodepleted salivary glands and wing discs. Contrary to recently published data (Truman and Riddiford, 2022), both tissues showed a significant and premature increase of BrC protein levels as early as in L1 larvae, while no increase in E93 protein levels was detected in any tissue (Figure4). In view of these results, we speculated whether the impairment of larval development observed in chinmodepleted animals could be the result of precocious presence of the wrong stageidentity factor, in this case, BrC. To address this notion, we precociously expressed BrCZ1, the main BrC isoform expressed during imaginal larval development (NarbonneReveau and Maurange, 2019), in salivary glands and wing discs. As previously described, ectopic expression of BrCZ1 blocked Chinmo activation (NarbonneReveau and Maurange, 2019). As a consequence, precocious upregulation of BrC 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 BrC during the juvenile stages. To confirm this hypothesis, we simultaneously depleted chinmo and BrC in salivary glands and wing discs. Remarkably, The online version of this article includes the following figure supplement(s) for figure 3: Figure supplement 1. Chinmo is required for wing development during the larval period. Figure supplement 2. BrCdependent requirement of chinmo in the eye disc. Figure 3 continued Figure 4. Chinmo represses BrC in salivary glands and wing discs during early larval development. (A–B)Expression of Chinmo, BrC, and E93 in salivary glands of L1 control (fkhGal4) (A),and UASchinmoRNAi (B).(C–D)Expression of Chinmo, BrC, and E93 in wing discs of early L2 control (esgGal4) (C)and UASchinmoRNAi (D).The esg domain is marked with GFP and all cell nucleus with DAPI. In the absence of chinmo only BrC shows early upregulation in both tissues. Scale bars represent 25µm. The online version of this article includes the following figure supplement(s) for figure 4: Figure supplement 1. Overexpression of BrCZ1 phenocopies chinmo loss of function in SGs and wing discs. Research article Developmental Biology | Evolutionary Biology Chafino etal. eLife 2023;12:e84648. DOI: https://doi.org/10.7554/eLife.84648 8 of 20 whereas salivary glands showed the same growth impairment observed upon chinmo depletion (Figure5AF, Figure5—figure supplement 1), depletion of BrC largely rescued the abnormalities in the wing discs caused by depletion of chinmo: the double knockout 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 BrC 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 chinmodepleted tracheal cells (Figure 2—figure supplement 1). Taken together, our results Figure 5. Different requirement of chinmo for the larval growth of salivary glands and wing discs. (A)DAPIstained salivary glands from control (fkhGal4) and UASBrCRNAi; UASchinmoRNAi L3W larvae. In the absence of chinmo and BrC, 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 UASBrCRNAi; UASchinmoRNAi L3W larvae. (E–F)Relative expression of (E)ng13 and (F)Salivary glands secretion genes in control and UASBrCRNAi; UASchinmoRNAi L3W larvae measured by quantitative realtime reverse transcriptase polymerase chain reaction (qRTPCR). 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, BrC, and Wg in wing discs of UASBrCRNAi; UASchinmoRNAi L3W larvae. Wing discs labelled to visualise the esg domain (GFP in green). In the absence of chinmo and BrC, wing discs grow normally and express Wg correctly. Scale bars represent 50µm. The online version of this article includes the following source data and figure supplement(s) for figure 5: Source data 1. Numerical data for Figure5B–F. Figure supplement 1. Effectiveness of chinmo and BrC RNAis in the salivary glands. Figure supplement 2. Expression of Chinmo and BrC in the developing wing disc. (A–C) Control wing discs, esgGal4; UASGFP, were labelled to visualise the esg domain in green and in red (A) BrC expression in L3W larvae, (B) Chinmo expression in earlymid L3 larvae and (C) the morphogenetic marker Wg in L3W larvae. Scale bar represents 50 µm in all panels. Research article Developmental Biology | Evolutionary Biology Chafino etal. eLife 2023;12:e84648. DOI: https://doi.org/10.7554/eLife.84648 9 of 20 suggest that a major regulatory function of chinmo during early larval development in imaginal cells is channelled through the repression of BrC, while in larval tissues chinmo appears to exert specific growthrelated functions that are independent to BrC repression. Thus, in the imaginal cells chinmo appears to ensure the expression of juvenile genes by repressing BrC, a wellknown inhibitor of larval gene expression (Zhou and Riddiford, 2002). In this regard, it is tempting to speculate that BrC 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 BrCdependent Sgs genes fail to be activated in absence of chinmo, when BrC 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 larvapupaadult 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 BrC 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). 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 larvapupal 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 ciGal4 driver impaired its larvapupal transition as abolished BrC expression and induced apoptosis at late L3 as revealed by the high expression of the effector caspase Dcp1 (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 BrC. An alternative way to keep high levels of chinmo at late L3 is by depleting BrC, a wellknown repressor of chinmo from mid L3 (NarbonneReveau and Maurange, 2019). Therefore, we knocked down BrC 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 Dcp1 staining and impairment of ct expression (Figure6E and F). Importantly, simultaneous depletion of chinmo and BrC 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 BrC. 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 E93dependent development of the adult. To this end, we used the thermosensitive 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 E93depleted wings (Ureña etal., 2016; Ureña etal., 2014; Figure7B). Taken together, our results show that chinmo must be downregulated during the Research article Developmental Biology | Evolutionary Biology Chafino etal. eLife 2023;12:e84648. DOI: https://doi.org/10.7554/eLife.84648 16 of 20 BgChinmoF: 5’ CAGC ACCA CTAT GTCC AAGT G’3 BgChinmoR: 5’ CAGG AAAC TGGA GAGG CTTT C’3 BgActin5CF: 5’- AGCT TCCT GATG GTCA GGTG A-3’ BgActin5CR: 5’- TGTC GGCA ATTC CAGG GTAC ATGG T-3’ RNA interference RNA interference (RNAi) in vivo in nymphs was performed as previously described (Cruz etal., 2007; Martín etal., 2006). A dose of 1µl (4–8µg/µl) of the dsRNA solution was injected into the abdomen of newly antepenultimate (N4d0) instar nymphs, and left until analysed. To promote the RNAi effect, the same dose of dsRNAs was reapplied to all treated animals after 3 days (N4d3) from the first injection. Control dsRNA consisted of a noncoding sequence from the pSTBlue1 vector (dsControl). The primers used to generate templates via PCR for transcription of the dsRNA were: BgchinmoF: 5’ CAGC ACCA CTAT GTCC AAGT G’3 BgchinmoR: 5’ GAGT CCTG CATG GCTT CGGA ’3 Imaging acquisition and analysis Images were obtained with the Leica TCS SP5 and the Zeiss LSM880 and LSM780 confocal microscopes. The same imaging acquisition parameters were used for all the comparative analyses. Images were processed with the Imaris Software (Oxford Instruments), Fiji, or Photoshop CS4 (Adobe). For DNA quantification and nuclear size of salivary glands, DNA staining intensity in the salivary glands and tracheal cells was obtained from z stacked images every 0.25μm of DAPIstained L3 larvae. Image analysis was performed using Fiji. For the volumetric calculation of the wing pouch region in Imaris software (Oxford Instruments), the regions of interest were selected based on the RFP fluorescence (induced by nubGal4) in confocal stacks that included the whole disc. The surface function of Imaris was used to segment the wing pouch and the surface volume was calculated by the software. Adult flies, nymphal parts, and adult cockroach images were acquired using AxioImager.Z1 (ApoTome 213 System, Zeiss) microscope, and images were subsequently processed using Photoshop CS4 (Adobe). Statistical analysis Statistical analysis and graphical representations were performed in GraphPad Prism 9 software. All experiments were performed with at least three biological replicates. Twotailed Student’s test and Welch’s ANOVA followed by Dunnett’s T3 post hoc tests were used to determine significant differences. Acknowledgements The ICTS 'NANOBIOSIS', and particularly the Custom Antibody Service (CAbS, IQACCSIC, CIBERBBN), is acknowledged for the assistance and support related to the E93 antibody used in this work. We thank Josefa Cruz for technical support. This project is supported by grants PGC2018098427BI00 and PID2021125661NBI00 to DM and XFM and grant PGC2018094254BI00 and PID2021123392NBI00 to JC funded by MCIN/AEI/10.13039/501100011033 and by grant 2017SGR 1030 to DM and XFM funded by the Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya and through BIST to JC. The research has also benefited from ERDF 'A way of making Europe to JC, DM and XFM. SC is a recipient of a Juan de la Cierva FJC2019041549I contract from the MCIN. Additional information Funding Funder Grant reference number Author Ministerio de Ciencia e Innovación PGC2018-098427-B-I00 David Martín Xavier Franch-Marro Research article Developmental Biology | Evolutionary Biology Chafino etal. eLife 2023;12:e84648. DOI: https://doi.org/10.7554/eLife.84648 17 of 20 Funder Grant reference number Author Ministerio de Ciencia e Innovación PID2021-125661NB-100 David Martín Xavier Franch-Marro Ministerio de Ciencia e Innovación PGC2018-094254-B-100 Jordi Casanova Agència de Gestió d'Ajuts Universitaris i de Recerca 2017-SGR 1030 David Martín Xavier Franch-Marro Ministerio de Ciencia e Innovación PID2021-123392NB-I00 Jordi Casanova The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Author contributions Sílvia Chafino, Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – review and editing; Panagiotis Giannios, Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – review and editing; Jordi Casanova, Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Project administration, Writing – review and editing; David Martín, Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, Visualization, Project administration, Writing – review and editing; Xavier FranchMarro, Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, Visualization, Writing – original draft, Project administration, Writing – review and editing Author ORCIDs Sílvia Chafino http://orcid.org/0000-0001-9679-0622 Panagiotis Giannios http://orcid.org/0000-0002-7881-1431 Jordi Casanova http://orcid.org/0000-0001-6121-8589 David Martín http://orcid.org/0000-0002-9784-647X Xavier FranchMarro http://orcid.org/0000-0002-7465-6729 Decision letter and Author response Decision letter https://doi.org/10.7554/eLife.84648.sa1 Author response https://doi.org/10.7554/eLife.84648.sa2 Additional files Supplementary files • MDAR checklist Data availability All data generated or analysed during this study are included in the manuscript and supporting file. 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