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

  1. Sílvia Chafino
  2. Panagiotis Giannios
  3. Jordi Casanova
  4. David Martín
  5. Xavier Franch-Marro

  • Curated by eLife

    eLife logo

    eLife assessment

    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 (exemplified in the wing disc) and in a Broad-independent manner in other larval tissues such as the salivary gland. Finally, they 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.

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

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.

Article activity feed

  1. Version published to 10.7554/elife.84648 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    This study demonstrates that Chinmo promotes larval development as part of the metamorphic gene network (MGN), in part by regulating Br-C expression in some tissues (exemplified in the wing disc) and in a Br-C independent manner in other tissues such as the salivary gland. I have included below the following comments on the submitted version of this manuscript:

    1. The authors have shown experimentally that Chinmo regulates Br-C expression in the wing disc but not the larval salivary gland. Based on this, they posit that Chinmo promotes larval development in a Br-C-dependent manner in imaginal tissues and a Br-C-independent manner in other larval tissues. This generalization of Chinmo's role in development would be more compelling if the relationship between Chinmo and Br-C were explored in other examples of imaginal/larval tissues.

    We agree with the referee that confirmation of our observations in other tissues might help to generalize Chinmo’s role. To this aim, we have analyzed the role of chinmo in an additional larval, the larval tracheal system, and imaginal tissue, the eye disc. Consistent with the results reported in the manuscript, we found that the mode of action of Chinmo is conserved, as depletion of Br-C in the eye disc is able to rescue the lack of chinmo, whereas in the tracheal system it is not. We included this new information in the main text and in new SFigures 1 and 3.

    1. Chinmo, Br-C, and E93 have all been shown to be EcR-regulated in larval tissues, including the brain and wing disc (as in Zhou et al. 2006, Dev Cell; Narbonne-Reveau and Maurange 2019, PLOS Biology; Uyeharu et al. 2017, ). It would be interesting (and I believe relevant to this study) to know whether the roles of these factors in their respective developmental stages are EcR-dependent and whether their regulation by EcR (or lack thereof) depends on whether the tissue is larval or imaginal.

    Although the relevance of EcR on the regulation of the genes that conform the metamorphic gene network has been already established, a different response of EcR-mediated signalling of these genes in larval and imaginal tissues is still not properly addressed. Finding this possible different output of the EcR signalling would be very interesting. However, we think that this is out of scope of this report as the main aim of this study was to determine the main role of the temporal genes during development and their repressive interactions.

    1. In the chinmo qPCR analysis shown in Fig1A, whether animals were sex-matched or controlled was not indicated. Since Chinmo has a published role in regulating sexual identity (Ma et al. 2014, Dev Cell; Grmai et al. 2018, PLOS Genetics), and since growth/body size is known to be a sexually dimorphic trait (Rideout et al. 2015, PLOS Genetics), it seems important to establish whether the requirement of Chinmo for larval development and/or growth. I recommend either 1) controlling for sex by repeating qPCRs in Fig 1A in either males or females, or 2) reporting male/female chinmo levels at each stage side-by-side.

    As the referee pointed out, chinmo has been related to sexual identity raising the possibility of a different effect of chinmo in growth of males and females during development. However, several observations discard this option. First of all, the role of chinmo in sexual identity has been only reported in adult testis and specifically in cyst stem cells. In fact, specific mutations of chinmo that only affects the expression of chinmo in testis, do not affect testis formation but its maturation, suggesting a role of chinmo in sex determination specifically in the testis cyst stem cells (Ma et al. 2014, Dev Cell; Grmai et al. 2018, PLOS Genetics). Second, it has been described a sex dependent growth rate during larval development (Rideout et al. 2015, PLOS Genetics; Sawala A. and Gould AP, PLoS Biol, 2017). However, the main difference in growth rate between males and females is found in L3 larvae (Sawala A. and Gould AP, PLoS Biol, 2017), when the expression of chinmo strongly declines in both males and females, indicating that chinmo impact on sex dimorphism during larval development might be at least, limited.

    Thus, considering that, based on our results, chinmo exerts its main role in larval tissue growth during L1 and L2 stages and that body growth is practically identical in male and female during these stages (Sawala A. and Gould AP, PLoS Biol, 2017), we can assume that chinmo might not contribute to sexual body size dimorphism.

    Nevertheless, we would like to clarify that we have performed the measurements of chinmo expression always in females, when sex identification was possible, namely in L3 larvae. L1 and L2 larvae qPCRs were not sex-discriminated as sex identification was not possible in our conditions.

    1. In Fig2E, the authors show that salivary gland secretion (sgs) genes are repressed in salivary glands lacking chinmo. Sgs genes are expressed during late larval stages as the animal prepares to pupate. Thus, based on the proposed model where Chinmo promotes larval development and represses the larval-to-pupal transition, one might expect that larval salivary glands lacking chinmo would express higher than normal levels of sgs genes. This expectation directly opposes the observed result - it would be helpful to speculate on this in the interpretation of results.

    This is an interesting observation. As Sgs genes are regulated by Br-C (Duan et al. Cell Reports 2020), precocious expression of this transcription factor in chinmo depleted animals might result in an early activation of those genes. Interestingly, we were not able to detect any Sgs genes expression in chinmo depleted salivary glands. We think that this is due to the fact that in absence of chinmo, this organ does not properly develop and mature, and therefore it is unable to express Sgs genes. Proof of that is that the double knockdown of Br-C and chinmo shows the same dramatically low levels of those genes. Altogether, these results strongly suggest that SGs lacking chinmo expression are unable to grow and synthesise Sgs proteins, even in the premature presence of Br-C. We discussed this point in the main text of the edited Ms. Please also see the response to referee 2.

    Reviewer #2 (Public Review):

    The evolution and control of the three-part life history of holometabolous insects have been controversial issues for over a century. While the functioning of broad as a master gene controlling the pupal stage and of E93 as a master gene for the adult stage has been known for about a decade or more, chinmo has only recently been proposed as being the master gene responsible for maintaining the larval stage (Truman & Riddiford, 2022). While the former paper focused on the embryonic and early larval function of Chinmo, this paper explores its metamorphic effects and defines the roles of Broad and E93 in the phenotypes produced by manipulations of Chinmo expression.

    Overall, the paper is well presented but in places, readers would be helped if the authors were more explicit about the logic and details of their manipulations. There are a couple of conceptual issues that the authors should address.

    The role of Broad in larval tissues:

    One intriguing issue relates to the relationship of Chinmo to Broad and E93 in larval versus imaginal tissues prior to metamorphosis. The knock-down of chinmo in imaginal discs results in severe suppression of growth and the lack of metamorphic patterning genes such as cut and wingless. Normal growth and patterning are reestablished though, if broad is also knocked-down, supporting the notion that the effects of the lack of Chinmo are mediated through the premature expression of Broad.

    In the salivary glands, by contrast, chinmo knock-down suppresses growth, and this growth suppression is not reversed by simultaneous broad knockdown. They properly conclude that the role of Chinmo in supporting the growth of larval tissues does not involve Broad, but their data on the expression of salivary gland proteins suggest that Broad still plays some role in Chinmo function in salivary glands. Fig. 5E shows the levels of various salivary glue proteins in the glands of Chinmo knock-down larvae. The levels are reduced, as expected by the lack of salivary gland growth, but a significant finding is that they are there at all! The Costantino et al. (2008) paper shows that these genes are only induced in the mid-L3. Ecdysone, acting through Broad isoforms, is necessary for their appearance and these SGS genes can be induced in the L1 and L2 stages by ectopic expression of some Broad isoforms. Their low levels in Fig 5, would be due to the small size of the gland, but the gland's premature expression of Broad likely causes their induction. In larval cells, then, Chinmo may feed into two parallel pathways, one that does not involve broad and regulates growth and the other, utilizing Broad, regulating premetamorphic changes.

    It would be useful to look at early larval salivary gland proteins such as ng-1 to -3 that are expressed in salivary glands before the critical weight. Also, it would be interesting if the appearance of the SGS proteins after chinmo knock-down (Fig 5E) is abolished by simultaneous knock-down of broad.

    This is an interesting observation. We think that the main problem has derived from the way we presented the data. Our results showed that depletion of chinmo in the SGs dramatically impairs the induction of Sgs gene expression, even with the premature presence of Br-C, which has been shown to be responsible for Sgs expression (Duan et al. Cell Reports 2020). The confusion might come from the way we presented the level of expression of those genes. In fact, the levels of Sgs in both chinmoRNAi and chinmoRNAi/Br-CRNAi SGs were virtually undetectable, suggesting that chinmo in the SG is not only required for Br-C repression but also for proper development of the gland. We believe that based on the fact that the very low levels of expression of Sgs genes in chinmo depleted SGs are still detected in the double knockdown chinmoRNAi/Br-CRNAi. Dramatically reduced expression of the early larval SGs ng1-3 genes in chinmoRNAi and double knockdown chinmoRNAi/Br-CRNAi supports this statement. Altogether these results suggest that Br-C is necessary but not sufficient for the expression of those specific SGs genes. We have changed the plots in Figure 2 and 3 to clarify this point and added the levels of expression of ng1-3.

    Role of Chinmo and Broad in Hemimetabolous insects:

    In the conclusion of their comparative studies on the cockroach (line 342), the authors state that Broad exerts no role in the development of hemimetabolous insects. However, this conclusion is not consistent with the literature. The first study of broad knockdown in a hemimetabolous insect was in the milkweed bug Oncopeltus fasciatus by Erezyilmaz et al. (2006). Surprisingly to Erezyilmaz et al., broad knock-down in early-stage nymphs did not cause premature metamorphosis. However, Broad expression was essential for tissues of the wing pads and dorsal thorax to undergo morphogenetic growth (rather than simple isomorphic growth), and for stage-specific changes in coloration through the nymphal series (but not for the nymph to adult color change). A similar function for Broad on wing growth during the later nymphal stages was later shown in Blattella (Fernandez-Nicolas et al., 2022; Huang et al., 2013). The wing- and genital pads represent "imaginal" tissues in the nymph and the need for Broad in these tissues are the same as seen in imaginal discs as the latter shift from isomorphic growth to morphogenesis at the critical weight checkpoint in the L3. This would suggest that important roles for Broad and E93 are already established in the hemimetabolous insects with E93 controlling the shift from immature (nymphal) to adult phenotypes and Broad controlling the premetamorphic growth of imaginal tissues in early-stage nymphs. Chinmo might then be needed to keep both in check.

    We are sorry for not having dealt with these observations in the submitted manuscript. We have taken them into consideration in the new version to discuss about the role of Br-C in the transition from hemimetabolous to holometabolous.

  3. eLife

    eLife assessment

    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 (exemplified in the wing disc) and in a Broad-independent manner in other larval tissues such as the salivary gland. Finally, they 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.

  4. eLife

    Reviewer #1 (Public Review):

    This study demonstrates that Chinmo promotes larval development as part of the metamorphic gene network (MGN), in part by regulating Br-C expression in some tissues (exemplified in the wing disc) and in a Br-C independent manner in other tissues such as the salivary gland. I have included below the following comments on the submitted version of this manuscript:

    1. The authors have shown experimentally that Chinmo regulates Br-C expression in the wing disc but not the larval salivary gland. Based on this, they posit that Chinmo promotes larval development in a Br-C-dependent manner in imaginal tissues and a Br-C-independent manner in other larval tissues. This generalization of Chinmo's role in development would be more compelling if the relationship between Chinmo and Br-C were explored in other examples of imaginal/larval tissues.

    2. Chinmo, Br-C, and E93 have all been shown to be EcR-regulated in larval tissues, including the brain and wing disc (as in Zhou et al. 2006, Dev Cell; Narbonne-Reveau and Maurange 2019, PLOS Biology; Uyeharu et al. 2017, ). It would be interesting (and I believe relevant to this study) to know whether the roles of these factors in their respective developmental stages are EcR-dependent and whether their regulation by EcR (or lack thereof) depends on whether the tissue is larval or imaginal.

    3. In the chinmo qPCR analysis shown in Fig1A, whether animals were sex-matched or controlled was not indicated. Since Chinmo has a published role in regulating sexual identity (Ma et al. 2014, Dev Cell; Grmai et al. 2018, PLOS Genetics), and since growth/body size is known to be a sexually dimorphic trait (Rideout et al. 2015, PLOS Genetics), it seems important to establish whether the requirement of Chinmo for larval development and/or growth. I recommend either 1) controlling for sex by repeating qPCRs in Fig 1A in either males or females, or 2) reporting male/female chinmo levels at each stage side-by-side.

    4. In Fig2E, the authors show that salivary gland secretion (sgs) genes are repressed in salivary glands lacking chinmo. Sgs genes are expressed during late larval stages as the animal prepares to pupate. Thus, based on the proposed model where Chinmo promotes larval development and represses the larval-to-pupal transition, one might expect that larval salivary glands lacking chinmo would express higher than normal levels of sgs genes. This expectation directly opposes the observed result - it would be helpful to speculate on this in the interpretation of results.

  5. eLife

    Reviewer #2 (Public Review):

    The evolution and control of the three-part life history of holometabolous insects have been controversial issues for over a century. While the functioning of broad as a master gene controlling the pupal stage and of E93 as a master gene for the adult stage has been known for about a decade or more, chinmo has only recently been proposed as being the master gene responsible for maintaining the larval stage (Truman & Riddiford, 2022). While the former paper focused on the embryonic and early larval function of Chinmo, this paper explores its metamorphic effects and defines the roles of Broad and E93 in the phenotypes produced by manipulations of Chinmo expression.

    Overall, the paper is well presented but in places, readers would be helped if the authors were more explicit about the logic and details of their manipulations. There are a couple of conceptual issues that the authors should address.

    The role of Broad in larval tissues:
    One intriguing issue relates to the relationship of Chinmo to Broad and E93 in larval versus imaginal tissues prior to metamorphosis. The knock-down of chinmo in imaginal discs results in severe suppression of growth and the lack of metamorphic patterning genes such as cut and wingless. Normal growth and patterning are reestablished though, if broad is also knocked-down, supporting the notion that the effects of the lack of Chinmo are mediated through the premature expression of Broad.
    In the salivary glands, by contrast, chinmo knock-down suppresses growth, and this growth suppression is not reversed by simultaneous broad knockdown. They properly conclude that the role of Chinmo in supporting the growth of larval tissues does not involve Broad, but their data on the expression of salivary gland proteins suggest that Broad still plays some role in Chinmo function in salivary glands. Fig. 5E shows the levels of various salivary glue proteins in the glands of Chinmo knock-down larvae. The levels are reduced, as expected by the lack of salivary gland growth, but a significant finding is that they are there at all! The Costantino et al. (2008) paper shows that these genes are only induced in the mid-L3. Ecdysone, acting through Broad isoforms, is necessary for their appearance and these SGS genes can be induced in the L1 and L2 stages by ectopic expression of some Broad isoforms. Their low levels in Fig 5, would be due to the small size of the gland, but the gland's premature expression of Broad likely causes their induction. In larval cells, then, Chinmo may feed into two parallel pathways, one that does not involve broad and regulates growth and the other, utilizing Broad, regulating premetamorphic changes.
    It would be useful to look at early larval salivary gland proteins such as ng-1 to -3 that are expressed in salivary glands before the critical weight. Also, it would be interesting if the appearance of the SGS proteins after chinmo knock-down (Fig 5E) is abolished by simultaneous knock-down of broad.

    Role of Chinmo and Broad in Hemimetabolous insects:
    In the conclusion of their comparative studies on the cockroach (line 342), the authors state that Broad exerts no role in the development of hemimetabolous insects. However, this conclusion is not consistent with the literature. The first study of broad knockdown in a hemimetabolous insect was in the milkweed bug Oncopeltus fasciatus by Erezyilmaz et al. (2006). Surprisingly to Erezyilmaz et al., broad knock-down in early-stage nymphs did not cause premature metamorphosis. However, Broad expression was essential for tissues of the wing pads and dorsal thorax to undergo morphogenetic growth (rather than simple isomorphic growth), and for stage-specific changes in coloration through the nymphal series (but not for the nymph to adult color change). A similar function for Broad on wing growth during the later nymphal stages was later shown in Blattella (Fernandez-Nicolas et al., 2022; Huang et al., 2013). The wing- and genital pads represent "imaginal" tissues in the nymph and the need for Broad in these tissues are the same as seen in imaginal discs as the latter shift from isomorphic growth to morphogenesis at the critical weight checkpoint in the L3.
    This would suggest that important roles for Broad and E93 are already established in the hemimetabolous insects with E93 controlling the shift from immature (nymphal) to adult phenotypes and Broad controlling the premetamorphic growth of imaginal tissues in early-stage nymphs. Chinmo might then be needed to keep both in check.

  6. Version published to 10.1101/2022.11.17.516883v1 on bioRxiv