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soul: Biological Overview | References

Soul is a master control gene governing the development of the Drosophila prothoracic gland

The prothoracic gland (PG) is a major insect endocrine organ. It is the principal source of insect steroid hormones, and critical for key developmental events such as the molts, the establishment of critical weight (CW), pupation, and sexual maturation. However, little is known about the developmental processes that regulate PG morphology. This study identified soul, which encodes a PG-specific basic helix-loop-helix (bHLH) transcription factor. Tap, also a bHLH protein, dimerizes with Soul. Both are expressed in the developing PG. Interfering with either soul or tap function caused strikingly similar phenotypes, resulting in small and fragmented PGs, the abolishment of steroid hormone-producing gene expression, larval arrest, and a failure to undergo metamorphosis. Furthermore, both soul and tap showed expression peaks just prior to the CW checkpoint. Disrupting soul- or tap-function before, but not after, the CW checkpoint caused larval arrest, and perturbed highly similar gene cohorts, which were enriched for regulators and components of the steroid hormone biosynthesis pathway. Intriguingly, a chitin-based cuticle gene, Cpr49Ah, and a POU domain transcription factor gene, pdm3, are direct target genes of the Soul/Tap complex, and disruption of either phenocopied key aspects of soul/tap loss-of-function phenotypes. Taken together, these findings demonstrate that the Soul/Tap heterodimer resides at the top of a complex gene hierarchy that drives PG development, CW establishment, and steroid hormone production (liu, 2024).

Holometabolous insects represent the largest and most diverse taxon among all multicellular organisms and comprise species such as butterflies, bees, beetles, and flies. One of the most remarkable aspects of holometabolous insects is their ability to undergo complete metamorphosis, a transformation that radically alters their morphology, behaviors, and survival strategies. Holometabolous development is characterized by four distinct life stages: egg, larva, pupa, and adult. The major developmental transitions such as the molts or pupation are driven by ecdysone, a steroid hormone that is synthesized by the Halloween enzymes in the prothoracic gland (PG). Metamorphosis is the developmental stage during which the larval body plan is remodeled into its adult form and represents a critical phase for insect adaptation and ecological survival. The precise onset of metamorphosis is regulated by intricate networks of hormonal signals and gene cascades, which coordinate the extensive tissue remodeling and organ development necessary for reaching sexual maturity. Understanding the molecular mechanisms that control insect metamorphosis provides an excellent opportunity for studying the complex interplay of i) hormone action and their ability to modulate transcription factor networks, ii) how nutrient status impinges on developmental processes and developmental timing, iii) organogenesis and its regulation, and iv) how environmental conditions modulate these regulatory hierarchies (liu, 2024).

A pivotal moment in the commitment to undergo metamorphosis is the attainment of critical weight (CW), a key developmental milestone first identified in the tobacco hornworm Manduca sexta and subsequently observed in other species such as Drosophila melanogaster. Larvae must acquire sufficient nutrients to surpass the CW threshold, enabling them to initiate metamorphosis later, even under adverse conditions like food scarcity. Several factors influence the achievement of CW. The prothoracicotropic hormone (PTTH), a brain neuropeptide, plays a critical role by stimulating ecdysone production in the PG through the Torso-mediated MAPK pathway. PTTH secretion depends on nutritional status and is necessary for the normal attainment of CW. Under nutritional stress, autophagy is activated in the PG, which limits the entry of sterols that are needed as precursors for ecdysone production. This causes a developmental delay that larvae can exploit to search for a better-suited diet. Additionally, the insulin and TOR signaling pathways are significant regulators of CW attainment. Manipulating insulin signaling in a PG-specific manner alters PG size and disrupts the checkpoint for CW, as well as ecdysone biosynthesis, thereby affecting Drosophila metamorphosis. This suggests a link between nutritional status and CW attainment. Overexpression of FoxO, a transcription factor negatively regulating the insulin and TOR pathways, delays CW attainment. Moreover, TOR signaling, a cellular nutrient sensing pathway, mediates CW exit via Snail (Sna), a transcription factor vital for the endoreplication of the PG genome. However, these studies have primarily focused on the later stages of development, where PG development would normally be complete. This study reports that disrupting either Soul, a PG-specific bHLH transcription factor, or its partner Tap, acts earlier in PG development and thus acts as master control genes controlling the maturation and proper establishment of transcriptional hierarchies in this tissue (liu, 2024).

The ecdysone signaling cascade, which drives developmental transitions such as molts and entrance into metamorphosis, is characterized by a hierarchy of transcription factors, including the Ecdysone Receptor (EcR) and its partner Ultraspiracle Protein (USP), the ecdysone early response gene E74, nuclear receptors DHR3, DHR4, FTZ-F1, and E75, metamorphosis suppressor Chinmo, and the metamorphosis initiation factor Broad-Complex (Br-C). As the principal tissue for ecdysone biosynthesis, the PG exhibits high and specific expression of ecdysone biosynthetic enzyme genes (aka Halloween genes), such as neverland (nvd), spookier (spok), shroud (sro), phantom (phm), shadow (sad), and disembodied (dib). The expression of these enzyme genes is rigorously controlled at the transcriptional level. For instance, transcription factors Séan, Ouija Board (Ouib), and Molting Defective (Mld) cooperatively regulate the transcription of nvd and spok, while the POU domain protein Ventral veins lacking (Vvl) and the nuclear receptor Knirps (Kni) bind to the phm promoter to modulate gene expression. Vvl and Kni also play minor roles in mediating PG size, suggesting that PG growth is under transcriptional control; however, the transcription factor network driving PG growth and development remains largely uncharacterized (liu, 2024).

Utilizing genome-wide microarray analysis, previous work identified 208 genes exhibiting PG-specific expression. In this earlier study, CG33557 (hereafter named soul), which encodes a basic helix-loop-helix (bHLH) transcription factor, was identified. Disrupting soul function in the PG caused developmental arrest, suggesting a role in the ecdysone production pathway. Given that bHLH proteins often act as heterodimers, a PG-specific RNAi screen was conducted targeting each of the 59 bHLH genes in D. melanogaster. This strategy yielded seven candidate lines with severe phenotypes, which included RNAi directed against soul, suggesting that these genes are critical for the regulation of metamorphosis. Expression profiling revealed that soul was the only bHLH gene with high and specific expression in the PG. Physical interaction between Soul and Tap was then demonstrated since tap was one of the seven bHLH candidates. This interaction occurred in vivo in the PG, where the Soul/Tap heterodimer acts at the promoter of the cuticle protein gene Cuticular protein 49Ah (Cpr49Ah) and the POU domain transcription factor pou domain motif 3 (pdm3). Furthermore, Soul/Tap controls PG growth for CW establishment, ultimately regulating ecdysone production and Drosophila metamorphosis. Our study has uncovered a hitherto uncharacterized transcription factor hierarchy that controls PG development and CW establishment, offering insights into the molecular mechanisms governing the transition from a juvenile individual to a sexually reproductive adult (liu, 2024).

Overactivation of insulin signaling in the PG leads to enlarged PG, causing premature metamorphosis in Drosophila, ultimately resulting in smaller individuals. Conversely, specific inhibition of insulin signaling in the PG can lead to smaller PG, thereby delaying metamorphosis and resulting in larger insects, which can be rescued by adding 20E to the food. 20E can also suppress insect growth by antagonizing insulin signaling. Besides insulin signaling, other regulatory factors that govern PG activity and metamorphosis have been identified, such as the epidermal growth factor receptor (EGFR) pathway. Silencing of key genes in the EGFR signaling in the PG significantly inhibits PG growth, causing the fruit fly to arrest at the L3 stage and fail to complete metamorphosis; adding 20E to the food can rescue the larval arrest. Both the insulin and EGFR pathways appear to preferentially act directly on the ecdysone biosynthetic process itself. This view is primarily based on the significant rescue effects mediated by 20E supplementation. This study showed that silencing soul or tap strongly inhibited PG growth and cause tissue fragmentation. Even when GFP was used to mark PG cells, it remained difficult to ascertain the tissue outline. Such severe developmental defects have not been previously reported for the PG. When media was supplemented with 20E, it was posible to reverse the excessive larval growth, consistent with the idea that tap- and soul-RNAi lead to a decrease in ecdysone synthesis. However, feeding 20E could not rescue the larval arrest caused by the loss of soul or tap function, suggesting that other crucial (unidentified) processes were disrupted in these animals. Furthermore, when Tap and Soul depletion was controlled in a spatial and temporal manner, this affected CW establishment. In summary, Soul and Tap play a crucial role in PG development, and interfering with their function has more dramatic effects compared to the disruption of known signaling pathways such as insulin and EGFR on PG development. However, whether Soul and Tap interact with insulin and EGFR signaling to regulate PG development remains to be established in future studies (liu, 2024).

There are no prior reports for Drosophila Soul, except for its brief mentioning of RNAi phenotypes when the gene (CG33557) was identified in a previous study. The mammalian orthologs of Soul are Scleraxis (Scx) and transcription factor 15 (Tcf15), and both have critical roles in chondrogenesis, normal tendon differentiation and formation, somitogenesis, and paraxial mesoderm development. Drosophila Tap and its mammalian ortholog Neurogenin are both required for neurogenesis. Therefore, Soul, Tap, and its mammalian counterparts appear to regulate a range of developmental processes including organ development. The physical interaction between Soul and Tap presented in this study has not yet been described for the mammalian orthologs. Given that this interaction seems to be spatially restricted, it seems plausible that Scx/Tcf15 and Neurogenin may interact only in specific tissues or at specific times during development. The PG is neuronally controlled and is thus equivalent to the hypothalamic-pituitary-gonadal (HPG) axis in mammals. The HPG axis controls puberty, which is not unlike insect metamorphosis, which also ensures sexual maturation. Interestingly, the mammalian orthologs of Soul and Tap are mainly expressed in the brain, the pituitary and thyroid glands, and gonads, consistent with the idea that the Soul/Tap is an ancient regulator of endocrine gland development. According to FlyBase, Soul and Tap are found in various insect taxa, including Diptera, Coleoptera, Hemiptera, and Lepidoptera. Future studies in other insects will clarify whether the roles of Soul and Tap are conserved across insect groups (liu, 2024).

We conducted RNA-seq to identify the transcriptional consequences of soul- and tap-RNAi, which should be enriched for direct targets of this transcription factor heterodimer. When this gene cohort was analyzed via PG-specific RNAi, it was demonstrated that loss of pdm3 and Cpr49Ah function phenocopied many aspects of soul and tap depletion, suggesting a role for these genes in regulating PG growth, CW establishment, and metamorphosis. Importantly, coexpressing pdm3 and Cpr49Ah in the PG rescued the developmental defects and metamorphic block caused by the loss of soul or tap function. Using a luciferase reporter system and EMSA, it was demonstrated that Soul and Tap bind directly to the upstream regions of pdm3 and Cpr49Ah, followed by transcriptional upregulation. Taken together, it is proposed that Soul and Tap form a heterodimer that binds to the promoters of pdm3 and Cpr49Ah, thus controlling PG development in a transcription-dependent manner (liu, 2024).

Cpr49Ah encodes a protein with homology to chitin-based larval cuticle proteins, suggesting it acts as a structural protein in the extracellular space. This is consistent with the idea that this protein is required for tissue integrity of the PG, explaining the severe morphological defects of the PG in PG > soul-RNAi and PG > tap-RNAi larvae. A recent study demonstrated that the chitin-binding protein Obstructor-A (Obst-A) is expressed in PG cells and acts as part of the extracellular matrix, crucial for maintaining the normal morphology and function of the ring gland, thus supporting ecdysone production. Interestingly, Obst-A expression was significantly reduced in late L3 larvae following the knockdown of soul (8.5-fold down, P <0.01) and tap (12-fold down, P < 0.02). Based on these findings, it is proposed that Soul and Tap modulate PG morphology by influencing the properties of the extracellular matrix via the transcriptional regulation of chitin-related genes. pdm3 encodes a POU domain transcription factor that has important roles in regulating olfactory receptor gene expression and axon targeting in olfactory receptor neurons, as well as establishing neuronal diversity and synaptic connectivity. Prior to this study, the only link between pdm3 and ecdysone was the finding that the pdm3 transcript was highly enriched in the PG, but no studies have examined its roles in PG development and metamorphosis. This study found that pdm3 is a direct target of the Soul/Tap heterodimer, and is critical for the larval-pupal transition, likely due to its requirement for the proper expression of the Halloween genes and other PG-specific transcription factors. Compared to the PG fragmentation phenotypes caused by the loss of soul, tap, or Cpr49Ah, RNAi directed against pdm3 had a relatively minor impact on PG morphology as it did not significantly affect the structural integrity of the PG. In summary, Cpr49Ah appears to establish or maintain the structural integrity of the PG tissue, whereas the role of pdm3 appears to be more tightly linked to the regulatory hierarchy controlling ecdysone biosynthesis (liu, 2024).

Based on the RNA-seq results in this study, Soul/Tap appears to also control the expression of ouib, a transcription factor that controls-in cooperation with Mld-the expression of spok, a Halloween gene. As such, it is possible that Soul and Tap regulate spok transcription indirectly via Ouib, since previous studies have found that Ouib specifically regulates spok expression. In addition, Soul and Tap also regulate the expression of multiple transcription factors to varying degrees, including sna, vvl, kni, mld, and sean. Sna is a member of the C2H2 zinc finger superfamily of transcription factor. Sna affects PG size and CW exit by regulating DNA endoreplication within PG cells, thus impinging on ecdysone synthesis. POU domain protein Vvl and nuclear receptor protein Kni can directly bind to the promoters of ecdysteroidogenic genes such as phm to regulate transcription. Both Mld and Sean, also zinc finger proteins, cooperatively regulate nvd transcription. Silencing pdm3 resulted in widespread downregulation of Halloween gene transcription, and caused reduced expression of key transcription factors including sna, ouib, sean, and vvl around 1 to 2 h prior to CW attainment, suggesting that Soul/Tap is a critical component of a transcription factor network controlling PG growth and metamorphosis (liu, 2024).

In Drosophila, a small 20E peak occurs prior to L3-8 h, which likely contributes to CW establishment in the pre-CW phase. This study demonstrated that the expression of soul and tap peaks at L3-4 h, and knockdown of soul or tap in the pre-CW phase rather than post-CW phase blocked PG growth and arrested larval development. Importantly, at L3-4 h, this study found that the expression of the Halloween genes phm, sad, and spok was substantially down-regulated after soul or tap depletion, raising the possibility that Soul and Tap contribute to CW attainment via the production of the small 4-h ecdysone peak during the pre-CW phase. The underlying mechanism of the early L3 ecdysone peak remains unclear, but it is possible that it is tied to the expression or regulation of Cpr49Ah and pdm3 (liu, 2024).

Transcript levels of both soul and pdm3 are highly enriched (>10-fold) in the ring gland (4 h after the L2/L3 molt) compared to whole body samples, suggesting that the Soul/Tap-Pdm3 axis is part of a PG-specific transcription factor hierarchy driving PG development. Given the distinct PG phenotypes after Soul/Tap depletion, it seems plausible that this hierarchy likely operates independently of the insulin pathway, which results in PG overgrowth when impaired. Sna encodes another transcription factor with PG-specific expression, however, its function appears to be involved in CW exit rather than CW establishment (13). Sna has two expression peaks, one at the L2 stage, and the second peak occurring in L3 around 8 to 12 h after the molt, the latter coinciding with the exit of the CW checkpoint. These two sna expression peaks correlate with the occurrence of DNA endoreplication in PG cells, and knockdown of sna in the pre-CW phase blocked CW exit and consequently, prevented the larval-to-pupal transition. Expression peaks of soul, tap, and pdm3 occur at L3-4-h, which is prior to the expression peak of sna, suggesting Sna works downstream of Soul/Tap-Pdm3 signaling. This idea is supported by the observation that pdm3-RNAi caused a ~4-fold reduction of sna expression at L3-8-h. Taken together, it is proposed that Soul/Tap act upstream in a transcriptional cascade comprising Pdm3, Cpr49Ah, and Sna to regulate PG development (liu, 2024).

If the PG’s sole role was producing ecdysteroids, one would expect that the majority of RNAi lines targeting gene function specifically in the PG should be rescuable by dietary supplementation with 20E. However, it is more often the exception than the rule that ecdysone feeding can rescue the lethality caused by PG-specific RNAi. Rescue attempts typically succeed when RNAi targets enzymes involved in ecdysone biosynthesis, yet they often fail when targeting other genes within the PG. For instance, RNAi against EGFR leads to smaller PG and larval arrest. While feeding 20E in this case allows animals to form pupae, it does not suffice to reach adulthood. Similarly, this study found that RNAi targeting soul and tap inhibits PG growth and the larval-to-pupal transition, resulting in larger larvae. Despite restoring larval size with 20E, pupation remains blocked. These observations suggest that the PG has other crucial functions besides producing ecdysone, suggesting the existence of other secreted hormones. The ring gland is enriched for at least nine transcripts that encode peptide hormones, including spatzle 5 and anachronism, suggesting that the PG is a complex hub of endocrine activity. Further investigation into these secretory proteins and their regulatory effects on metamorphosis is needed to fully understand the regulatory roles of the PG. Moreover, 7-dehydrocholesterol (7DC), produced in the PG as a precursor for ecdysone biosynthesis, may have functions beyond its role in ecdysone production. This hypothesis is supported by the observation that while 7DC can rescue loss of neverland function, 20E fails to do so. This is surprising since the ecdysone synthesis pathway is thought to begin with the production of 7DC from a suitable dietary sterol, and if this were a linear pathway, both 7DC and 20E should rescue. To elucidate the additional functions of the PG beyond ecdysone production, future research could examine the effects of PG-specific RNAi targeting secretory protein genes, followed by phenotypic, molecular, and metabolomic analysis. This integrated approach may shed light on the multifaceted roles of the PG in orchestrating metamorphosis (liu, 2024).

In summary, this study reveals that the bHLH transcription factors Soul and Tap act in concert to regulate PG development and CW establishment, thereby orchestrating ecdysone production and metamorphosis in Drosophila. Through directly targeting key genes such as Cpr49Ah and pdm3, Soul and Tap control PG growth and CW attainment in a spatiotemporal-specific manner. This regulatory process involves the activation of transcriptional cascades, potentially implicating multiple transcription factors including Pdm3, Ouib, Sna, Sean, and Vvl. Importantly, the findings suggest that Soul/Tap-mediated PG growth may influence metamorphosis beyond ecdysone production. Overall, this study contributes to our understanding of CW establishment and insect metamorphosis at the molecular level, shedding light on the complex transcriptional regulatory network governing PG development and metamorphosis (liu, 2024).

Search PubMed for articles about Drosophila Soul

Liu, W., Yan, M., King-Jones, K. (2024). Soul is a master control gene governing the development of the Drosophila prothoracic gland. Proc Natl Acad Sci U S A, 121(40):e2405469121 PubMed ID: 39312662

date revised: 5 April 2026

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