InteractiveFly: GeneBrief

Sox21a: Biological Overview | References

Gene name - Sox21a

Synonyms -

Cytological map position - 70D2-70D2

Function - Transcription factor

Keywords - essential for enterocyte differentiation from enteroblast in the midgut - involved in feedback amplification loop that leads to rapid production from intestinal stem cells of differentiation-defective enteroblasts and subsequent tumorigenesis - Sox100B directly regulates Sox21a to promote differentiation - Sox21a and GATAe form a functional relay to orchestrate EB differentiation - genetic interaction between peroxisomes and the JAK and STAT signaling restores the expression defect of Sox21a in peroxisome defective cells

Symbol - Sox21a

FlyBase ID: FBgn0036411

Genetic map position - chr3L:14,103,481-14,108,118

Classification - SOX-TCF_HMG-box, class I member of the HMG-box superfamily of DNA-binding protein

Cellular location - nuclear

NCBI links: EntrezGene, Nucleotide, Protein

Sox21 orthologs: Biolitmine
Recent literature
Wu, K., Tang, Y., Zhang, Q., Zhuo, Z., Sheng, X., Huang, J., Ye, J., Li, X., Liu, Z. and Chen, H. (2021). Aging-related upregulation of the homeobox gene caudal represses intestinal stem cell differentiation in Drosophila. PLoS Genet 17(7): e1009649. PubMed ID: 34228720
The differentiation efficiency of adult stem cells undergoes a significant decline in aged animals, which is closely related to the decline in organ function and age-associated diseases. However, the underlying mechanisms that ultimately lead to this observed decline of the differentiation efficiency of stem cells remain largely unclear. This study investigated Drosophila midguts and identified an obvious upregulation of caudal (cad), which encodes a homeobox transcription factor. This factor is traditionally known as a central regulator of embryonic anterior-posterior body axis patterning. This study reports that depletion of cad in intestinal stem/progenitor cells promotes quiescent intestinal stem cells (ISCs) to become activate and produce enterocytes in the midgut under normal gut homeostasis conditions. However, overexpression of cad results in the failure of ISC differentiation and intestinal epithelial regeneration after injury. Moreover, this study suggests that cad prevents intestinal stem/progenitor cell differentiation by modulating the Janus kinase/signal transducers and activators of the transcription pathway and Sox21a-GATAe signaling cascade. Importantly, the reduction of cad expression in intestinal stem/progenitor cells restrained age-associated gut hyperplasia in Drosophila. This study identified a function of the homeobox gene cad in the modulation of adult stem cell differentiation and suggested a potential gene target for the treatment of age-related diseases induced by age-related stem cell dysfunction.

Homeostatic renewal of many adult tissues requires balanced self-renewal and differentiation of local stem cells, but the underlying mechanisms are poorly understood. This study identified a novel feedback mechanism in controlling intestinal regeneration and tumorigenesis in Drosophila. Sox21a, a group B Sox protein, is preferentially expressed in the committed progenitor named enteroblast (EB) to promote enterocyte differentiation. In Sox21a mutants, EBs do not divide, but cannot differentiate properly and have increased expression of mitogens, which then act as paracrine signals to promote intestinal stem cell (ISC) proliferation. This leads to a feedback amplification loop for rapid production of differentiation-defective EBs and tumorigenesis. Notably, in normal intestine following damage, Sox21a is temporally downregulated in EBs to allow the activation of the ISC-EB amplification loop for epithelial repair. It is proposed that executing a feedback amplification loop between stem cells and their progeny could be a common mechanism underlying tissue regeneration and tumorigenesis (Chen, 2016).

Adult stem cells have important roles in maintaining tissue and organ homeostasis by their prolonged ability to produce progenitor cells that differentiate into multiple types of mature cells. Production of the progenitor cells from stem cells must be coordinated with cell differentiation and the overall tissue demand, as disruption of this coordination could lead to tissue degeneration, if cell production is not sufficient, or hyperplasia/ tumorigenesis, if the cell production is unrestricted and exceeds the pace of cell differentiation. However, the molecular mechanism that coordinates progenitor cell proliferation with cell differentiation is largely unknown (Chen, 2016).

The adult Drosophila midgut has been established as a simple and useful system for the study of the stem cell behavior during homeostatic tissue renewal and in response to environmental changes. Like mammalian intestine, the Drosophila midgut epithelium is constantly replenished by adult intestinal stem cells (ISCs), although at a relatively slower pace. In addition, signaling pathways that regulate mammalian ISC activity, such as Wnt, JAK/STAT, EGFR/Ras, Hippo, BMP and Notch, also play important roles in regulating Drosophila ISC activity during normal homeostasis and/or stress conditions. The Drosophila ISC, which generates a relatively simple stem cell lineage, can be specifically marked by Delta (Dl), the Notch ligand. After each asymmetric division, an ISC will produce a new ISC and a committed progenitor cell named enteroblast (EB), which will further differentiate into either an enterocyte or an enteroendocrine cell, depending on the levels of Notch activation it received from ISCs. Enterocyte differentiation from EB requires high levels of Notch activation, and JAK/STAT signaling activity is required for both enterocyte and enteroendocrine cell differentiation from EB. Aside from the signaling pathways, many transcription factors have been identified as important regulators of cell differentiation. Enterocyte differentiation from EB requires downregulation of Escargot (Esg) and activation of Pdm1, whereas enteroendocrine cell differentiation from EB requires release of the inhibition by the transcriptional repressor Tramtrack and activation of acheate-scute complex (AS-C) genes and Prospero (Pros), the enteroendocrine cell determination factor. It is largely unclear how these signaling pathways and transcription factors are coordinately regulated for balanced self-renewal of ISCs and differentiation of EBs to maintain intestinal (Chen, 2016).

Sox family transcription factors, which share a DNA binding high-mobility-group domain, are known as important regulators of cell fate decisions during development and in adult tissue homeostasis. In mouse small intestine, Sox2 is expressed in ISCs and progenitor cells and is critical for ISC maintenance and differentiation of Paneth cells. Several Sox family proteins have been identified in Drosophila, but their potential roles in the ISC lineage are unclear. This study characterized the function of a Drosophila Sox gene, Sox21a, in the ISC lineage. Sox21a is expressed in EBs and acts as a tumor suppressor in the midgut epithelium. One important function of Sox21a is to promote enterocyte differentiation by inducing Pdm1 expression. By studying its tumor suppressing function, a novel feedback amplification loop was identified between ISC and EB, which is normally suppressed by Sox21a. Temporal activation of this loop is essential for damage-induced intestinal regeneration, whereas sustained activation of this loop leads to tumorigenesis. Therefore, this study has revealed a novel mechanism that coordinates stem cell activity with progenitor cell differentiation, and connects regeneration with tumorigenesis (Chen, 2016).

Sox family proteins in metazoan are divided into different groups based on their similarity in biochemical properties, and Sox21a belongs to the SoxB2 subgroup whose function is relatively less studied compared to the most related SoxB1 subgroup proteins, such as the pluripotency factor Sox2. By cellular and genetic analysis, this study has characterized the functions of Sox21a, a member of the SoxB2 group in Drosophila; two major roles were revealed. First, Sox21a is essential for EC differentiation from EB. Sox21a protein is mainly expressed in differentiating EBs, a pattern that is consistent with its role in EB differentiation. Loss of Sox21a causes accumulation of undifferentiated cells that fail to express Pdm1, the EC marker. The majority of these mutant cells remain as diploid EBs and some begin to show polyploidy, indicating that mechanisms controlling EC differentiation and cell ploidy can be uncoupled. On the other hand, forced transgene expression of Sox21a in EBs accelerates their differentiation into ECs as evidenced by precocious expression of Pdm1. However, forced expression of Sox21a in ISCs does not induce their differentiation, suggesting that Sox21a is necessary but not sufficient for inducing EC differentiation from ISCs. One possible explanation for this is that EC differentiation requires both Sox21a and Notch activity. Indeed, although Notch activity is known to be both necessary and sufficient for inducing EC differentiation from ISCs, Notch activated EBs that have already been presented along the length of midgut in young flies remain dormant for several days until there is a need for cell replacement. These observations indicate that activation of Notch alone is not sufficient to induce EB differentiation, but only primes EB for EC differentiation. Notch-activated EBs will stay in undifferentiated state until Sox21a is activated, which then promotes differentiation of the primed EBs into ECs (Chen, 2016).

Another surprising role for Sox21a revealed in this study is that it provides a feedback regulation of ISC activity by suppressing mitogenic signals from differentiating EBs. This function is important for controlling the strength of the ISC-EB amplification loop for balanced self-renewal of ISCs and differentiation of EBs. Because Sox21a is also required for EB differentiation, disruption of this function will cause sustained activation of the ISC-EB amplification loop as well as blocked EB differentiation, leading to the formation of EB-like tumors. Importantly, following epithelial damage, Sox21a is quickly downregulated in EBs. This allows temporal activation of the ISC-EB loop for rapid production of progenitor cells prepared for epithelial repair. During recovery, Sox21a is then temporally upregulated in EBs. This not only stops the ISC-EB amplification loop to avoid excessive EB production, but also accelerates cell differentiation for epithelial repair. Therefore, Sox21a does not simply act as a tumor suppressor in intestine. It is dynamically regulated to control the process of epithelial regeneration in response to various environmental changes via regulating the strength of the ISC-EB amplification loop. Because Sox21a expression in intestine is dynamic during normal adult development, it is conceivable that its expression could possibly be influenced by physiological changes, such as food intake and activity of symbiotic bacteria, and fine-turning Sox21a activity could be important for maintaining regular epithelial turnover. How Sox21a expression is regulated is unclear, but signaling pathways that are implicated in regulating intestinal regeneration, such as JAK/STAT or EGFR/Ras pathways are potential candidate regulators, especially JAK/STAT, which is known to be essential for EB differentiation. During the preparation of this manuscript, two groups have reported the function of Sox21a in Drosophila midgut (Meng, 2015; Zhai, 2015). Meng did not observe the tumor suppressive function of Sox21a, possibly because they used a weak mutant allele of Sox21a in their study. Notably, Zhai observed a similar tumor suppressive role for Sox21a as reported here. Interestingly, they suggest that Sox21a is regulated by JAK/STAT signaling, as Sox21a transgene expression is able to rescue the differentiation defects caused by disrupted JAK/STAT signaling. However, the current found that Sox21a was still expressed in JAK/STAT compromised EBs. Therefore, how Sox21a is regulated during normal homeostasis and regeneration remains to be further explored, and it is possible that Sox21a could be controlled by a combination of regulators in a cell type-specific manner, with different mechanisms in ISCs and EBs. Because Sox proteins commonly function together with other cell type-specific co-factors in regulating gene transcription, Sox21a could function with different co-factors in ISCs and EBs. These are interesting questions worthy of further investigation (Chen, 2016).

Cells in a given tumor are usually heterogeneous and based on the ability to initiate tumors, tumor cells can be divided into tumor-initiating cells and non-tumor-initiating cells. In this case, Sox21 mutant EBs can be regarded as the tumor-initiating cells in vivo. Depleting Sox21a specifically in EBs is sufficient to initiate EB-like tumors. Conversely, restoring Sox21a function specifically in EBs is sufficient to prevent tumor development in Sox21a mutant intestine. However, unlike typical tumor-initiating cells, Sox21a mutant EBs are post-mitotic cells. In addition, their ability to initiate tumors depends on the activity of local ISCs. Therefore, this study also reveals a novel example of tumor-initiating cells in vivo that do not divide themselves, but can 'propagate' themselves by utilizing local stem cells (Chen, 2016).

In short, by studying the function of Sox21a in Drosophila ISC lineages, this study identified a novel feedback amplification loop between stem cells and their progeny that mediates epithelial regeneration and tumorigenesis. It has long been suggested that tissue regeneration and tumorigenesis are intimately associated, although the mechanistic connection is still obscure. The Sox21a-Spi mediated- ISC-EB amplification loop revealed in this study may provide a simple example of potential mechanisms that could connect tissue regeneration with tumorigenesis: transient activation of the stem cell- progeny amplification loop promotes regeneration, whereas sustained or irreversible activation of the amplification loop promotes tumorigenesis. It is proposed that this could be a general mechanism underlying tissue regeneration and tumorigenesis in other tissues, including that in mammals and humans (Chen, 2016).

Sox100B regulates progenitor-specific gene expression and cell differentiation in the adult Drosophila intestine

Robust production of terminally differentiated cells from self-renewing resident stem cells is essential to maintain proper tissue architecture and physiological functions, especially in high-turnover tissues. However, the transcriptional networks that precisely regulate cell transition and differentiation are poorly understood in most tissues. This study identified Sox100B, a Drosophila Sox E family transcription factor, as a critical regulator of adult intestinal stem cell differentiation. Sox100B is expressed in stem and progenitor cells and required for differentiation of enteroblast progenitors into absorptive enterocytes. Mechanistically, Sox100B regulates the expression of another critical stem cell differentiation factor, Sox21a. Supporting a direct control of Sox21a by Sox100B, a Sox21a intronic enhancer was identified that is active in all intestinal progenitors and directly regulated by Sox100B. Taken together, these results demonstrate that the activity and regulation of two Sox transcription factors are essential to coordinate stem cell differentiation and proliferation and maintain intestinal tissue homeostasis (Meng, 2020).

The proper maintenance of tissue homeostasis is essential for their normal architecture and physiological functions, especially in high-turnover tissues, such as intestinal epithelium. In most tissues, this is achieved by their resident stem cells, which are capable of self-renewing and differentiating into a variety of cell types within tissues. To answer the fundamental question of how tissue homeostasis is properly maintained, it is critical to identify the genetic networks that control stem cell proliferation and differentiation. Although proliferation has been extensively studied over the decades, mechanisms by which progressive and robust differentiation is achieved in vivo remain less understood in many lineages (Meng, 2020).

The adult Drosophila intestinal epithelium provides a genetically tractable experimental system to examine molecular mechanisms regulating stem cell activities. The adult midgut epithelium is actively maintained by multipotent intestinal stem cells (ISCs), which self-renew to maintain a stable stem cell population and give rise to post-mitotic progenitors committed to one of two distinct cell lineages: diploid secretary enteroendocrine cells (EEs) and polyploid absorptive enterocytes (ECs). In the EC lineage, ISCs turn on the Notch signaling in the daughter cells termed enteroblasts (EBs) that are committed to differentiation into the absorptive fate. EBs then go through several rounds of endo-replication and finally differentiate into Pdm1-positive ECs. To maintain the secretory lineage, ISCs give rise to Prospero-positive pre-EE daughter cells. A number of signaling pathways and transcription factors have been implicated in regulating ISC differentiation, including Delta/Notch, JAK/STAT92E, escargot, Sox21a. However, understanding of the transcriptional network involved in the control of EB differentiation remains incomplete (Meng, 2020).

Sox (Sry-related HMG Box) family transcription factors are important regulators of cell fate specification and cell differentiation during development and in multiple adult stem cell populations. Sox21a, a Drosophila Sox B gene, is specifically expressed in ISCs and EBs and plays important roles in regulating ISC proliferation and EB differentiation into EC, both at homeostasis and under stress conditions (Chen, 2016, Meng, 2015, Zhai, 2015, Zhai, 2017). However, how ISC- and EB-specific Sox21a expression pattern is established remains unknown. This study investigated the expression and function of another Sox family transcription factor, the Sox E factor Sox100B, and found that it is required for ISC differentiation into the EC lineage. Sox100B is shown to be required for both Sox21a protein expression and the activity of a transcriptional enhancer located in the first intron of the Sox21a gene. This identification of Sox100B binding sites in this intronic enhancer strongly supports the notion that Sox21a is a direct Sox100B target gene. The results identify an essential player in the transcriptional network that regulates the complex process of stem cell differentiation in the adult Drosophila intestine (Meng, 2020).

This work shows that the Sox E family transcription factor Sox100B is expressed in ISCs and EBs in the adult Drosophila intestine, consistent with a recent report using a GFP-tagged genomic BAC construct for Sox100B (Doupe, 2018). Recently, another Sox family transcription factor Sox21a has already been shown to be expressed specifically in ISCs and EBs (Chen, 2016, Meng, 2015, Zhai, 2015). These two Sox transcription factors do not simply share an overlapping expression pattern, as demonstrated by this work, but Sox100B is required for proper Sox21a expression in both cell types. These data altogether have led to a proposal of a transcriptional cascade in which Sox21a is a Sox100B target gene. The overlapping expression pattern is not surprising, given the fact that Sox factors are commonly co-expressed in several tissues. For example, Drosophila B Group Sox factors SoxNeuro and Dichaete are co-expressed in part of the neuroectoderm during early development of the CNS. However, this study presents evidence showing a direct transcriptional regulation between different Sox factors in the somatic stem cell lineage in Drosophila (Meng, 2020).

Previous studies have shown that transcription factors, such as Stat92E and AP-1 factor Fos are involved in regulating Sox21a expression at basal and stress conditions (Chen, 2016, Meng, 2015, Zhai, 2015). At basal condition, Stat92E has been implicated in regulating Sox21a expression, and the second intron of Sox21a alone is sufficient to drive gene expression in ISCs and EBs (Zhai, 2015); however, whether this intronic enhancer is directly regulated by Stat92E has not been addressed. This study has identified that the first intron of Sox21a is also sufficient to direct endogenous Sox21a expression pattern. Interestingly, this intronic enhancer is not dependent on Stat92E, suggesting that parallel signal inputs from both Sox21a introns act together to robustly control the ISC/EB-specific Sox21a expression pattern. In support of this model, this study found that the first intronic enhancer is specifically responsive to Sox100B. This model is consistent with the observation that Sox21a expression is strongly reduced but not absent in Sox100B mutant clones, suggesting that parallel inputs mediated by the second intron via other factors, such as Stat92E contribute to Sox21a expression even in the absence of Sox100B. This model also partially accounts for the notion that depleting Sox100B does not inhibit ISC proliferation while depleting Sox21a strongly inhibits ISC proliferation (Meng, 2015), and it is likely that residual Sox21a expression in Sox100B loss-of-function conditions allows ISC proliferation. In response to stress, Sox21a expression is strongly induced which involves multiple stress-sensing signaling pathways and factors, such as JNK, EGFR, AP-1 factor Fos, and Stat92E (Meng, 2015, Zhai, 2015, Zhai, 2017). This study has shown that Sox100B is required for stress-, JNK-, and RasV12-induced Sox21a expression but that Sox100B overexpression in ISCs and EBs is not sufficient to induce Sox21a expression. The current data suggest a model where Sox100B provides cell-type specificity and allows Sox21a expression in intestinal progenitors, ISCs and EBs, while other pathways control Sox21a induction during the differentiation process or in response to stresses. This raises an interesting question of whether Sox100B directly interacts with stress-sensing and differentiation pathways to ensure that Sox21a expression is stress inducible and gradually increases during differentiation. As an example of such a mechanism, mammalian Sox9 has been shown to physically interact with AP-1 factors to co-activate target gene expression in developing chondrocytes. It is anticipated that the identification of Sox100B-interacting cofactors will provide a better view regarding the mechanism(s) by which Sox100B cooperates with stress-sensing signaling and other differentiation pathways to precisely adapt stem cell activities to tissue demands (Meng, 2020).

The last decade has witnessed significant advances in understanding the mechanisms by which ISC activities are regulated in response to infection, tissue injury, and during aging, with a strong focus on ISC proliferation. In contrast, a well-defined progressive differentiation process is still inadequately understood. Sox100B has been recently implicated in regulating acute gut regeneration in response to pathogenic bacteria Pseudomonas entomophila (Lan, 2018); however, the exact role of Sox100B in this process has not been characterized. This study found that Sox100B is functionally required for robust stem cell differentiation, as a strong reduction in the EC lineage and a reduction to a lesser extent in the EE lineage were observed in the Sox100B mutant clones. This study further showed that the expression of a critical differentiation regulator Sox21a is reduced in Sox100B mutants, establishing a transcriptional cascade during the ISC differentiation process. However, in preliminary experiments, Sox21a overexpression alone is not sufficient to rescue the differentiation defects of the Sox100B mutants, suggesting that other Sox100B targets, in addition to Sox21a, are required for proper differentiation. Further identification of Sox100B transcriptional target genes is needed to fully decipher the role of Sox100B during the differentiation process (Meng, 2020).

Interestingly, Sox9, the mammalian counterpart of Drosophila Sox100B, is expressed in ISCs and differentiated Paneth cells at the bottom of the crypts. In Sox9 knockout intestine, Paneth cells are found missing and crypt hyperplasia is widely observed (Bastide, 2007, Mori-Akiyama, 2007). This study also observed a mild but significant increase of ISC proliferation in Sox100B loss-of-function conditions, which could be due to disruption of normal differentiation process, since it has been well documented that blocking differentiation process by genetically manipulating Notch and Sox21a causes strong pro-mitotic feedback regulation on ISC proliferation (Chen, 2016, Patel, 2015, Zhai, 2015). In addition to Sox9, several other Sox family factors are expressed in the mammalian intestine, and their expression pattern and function remain elusive. The transcriptional regulatory pattern between different Sox factors in the intestine could be conserved from flies to mammals, and such possibility needs to be further examined (Meng, 2020).

The Drosophila ortholog of mammalian transcription factor Sox9 regulates intestinal homeostasis and regeneration at an appropriate level

Balanced stem cell self-renewal and differentiation is essential for maintaining tissue homeostasis, but the underlying mechanisms are poorly understood. This study identified the transcription factor SRY-related HMG-box (Sox) 100B, which is orthologous to mammalian Sox8/9/10, as a common target and central mediator of the EGFR/Ras and JAK/STAT signaling pathways that coordinates intestinal stem cell (ISC) proliferation and differentiation during both normal epithelial homeostasis and stress-induced intestinal repair in Drosophila. The two stress-responsive pathways directly regulate Sox100B transcription via two separate enhancers. Interestingly, an appropriate level of Sox100B is critical for its function, as its depletion inhibits ISC proliferation via cell cycle arrest, while its overexpression also inhibits ISC proliferation by directly suppressing EGFR expression and additionally promotes ISC differentiation by activating a differentiation-promoting regulatory circuitry composed of Sox100B, Sox21a, and Pdm1. Thus, this study reveals a Sox family transcription factor that functions as a stress-responsive signaling nexus that ultimately controls tissue homeostasis and regeneration (Jin, 2020).

Homeostatic renewal of many adult tissues requires balanced stem cell proliferation and differentiation, a process that is commonly compromised in cancer and in tissue degenerative diseases. The intestinal epithelium in adult Drosophila midgut provides a genetically tractable system for understanding the underlying mechanisms of tissue homeostasis and regeneration driven by resident stem cells. The intestinal stem cells (ISCs) of the Drosophila midgut normally divide to renew themselves and give rise to two different types of progenitor cells that respectively differentiate into enterocyte cells (ECs) and enteroendocrine cells (EEs). Normally, ISCs divide occasionally and thereby maintain the ongoing renewal of the epithelium, a slow process that takes approximately 2-4 weeks. However, upon damage or infection, ISCs are able to rapidly divide to facilitate accelerated epithelial repair in as fast as two days (Jin, 2020).

Extensive studies have implicated the JAK/STAT and the EGFR/Ras/mitogen-activated protein kinase (MAPK) as the two major signaling pathways that regulate ISC proliferation and differentiation during both normal epithelial homeostasis and stress-induced intestinal repair. The EGFR signaling is considered to play a predominant role in the regulation of ISC proliferation because it is required for the JAK/STAT signaling activation-induced ISC proliferation, whereas the JAK/STAT signaling is not essential for EGFR/Ras signaling activation-induced ISC proliferation. The EGFR signaling is also important for remodeling of the differentiated cells, including the exclusion of damaged/aged ECs and incorporation of new cells. The JAK/STAT pathway is also essential for ISC differentiation. ISCs with compromised JAK/STAT activity generate progenitor cells that are incapable of further differentiation. Despite the importance of the two signaling pathways in controlling intestinal homeostasis, their downstream targets-which integrate pathway activities to coordinate ISC proliferation and differentiation-remain elusive (Jin, 2020).

Sox (SRY-related HMG-box) family transcription factors (TFs) are known to have diverse roles in cell-fate specification and differentiation in multicellular organisms. In mouse-small intestine, Sox9, a SoxE subfamily member, is expressed in ISCs to regulate ISC proliferation and differentiation, but whether it acts as an oncogene or a tumor suppressor is still in debate. In Drosophila midgut, Sox21a, a SoxB2 subfamily member, is specifically expressed in ISCs and transient progenitor cells, and is essential for progenitor cell differentiation into mature cells (Chen, 2016, Zhai, 2015, Zhai, 2017). This study identified Sox100B, the Drosophila ortholog of Sox9, as a common downstream gene target for both the JAK/STAT and the EGFR signaling in regulating ISC proliferation and differentiation. This study also revealed that an appropriate level of Sox100B is critical for its function in regulating ISC proliferation, in that it may allow it to serve as an important mediator for a balanced process of ISC proliferation and differentiation, thereby maintaining intestinal homeostasis (Jin, 2020).

Although it has been well established that in the Drosophila midgut, the stress-responsive JAK/STAT signaling and EGFR/Ras/MAPK signaling are the two major signaling pathways that regulate ISC proliferation and differentiation, the downstream signaling targets that coordinate ISC proliferation and differentiation for intestinal regeneration are still yet to be identified. Sox100B identified in this study may represent such a key target. First, the expression of Sox100B is regulated by both JAK/STAT- and EGFR-signaling pathways. Normally Sox100B is expressed specifically in ISCs and EBs, where JAK/STAT- and EGFR/Ras/MAPK-pathway activities are high, and its expression is highly dependent on the activity of JAK/STAT- and EGFR/Ras/MAPK-signaling activities. Second, similar to the functions of JAK/STAT and EGFR signaling, Sox100B is critically required for both ISC proliferation and differentiation. The sustained EGFR/Ras/MAPK activity in EBs is important for the initiation of DNA endoreplication during the process of EC differentiation, and the sustained JAK/STAT signaling activity in EBs is essential for terminal differentiation toward both EC and EE lineage. Depletion of Sox100B causes ISC quiescence, similar to that caused by the disruption of EGFR signaling, as well as arrest of EB differentiation, similar to that caused by the disruption of JAK/STAT signaling. Third, an appropriate level of Sox100B expression appears to be critical for intestinal homeostasis. This effect by the expression level, as well as its responsiveness to JAK/STAT, EGFR, and potentially other stress-induced signaling activities (not shown), such as Wnt and Hippo signaling, may position Sox100B as a central mediator that coordinates ISC proliferation and differentiation during intestinal homeostasis and regeneration in Drosophila (Jin, 2020).

Sox100B is a Sox family group-E transcription factor, homolog of mammalian Sox8/9/10. In mouse small intestine, Sox9 is expressed in stem cells and progenitor cells at the base of crypts, and loss of Sox9 in the intestinal epithelium causes ISC hyperplasia and failure of Paneth cell differentiation (Bastide, 2007, Mori-Akiyama, 2007). Interestingly, in the stem cell zone, Sox9 is expressed at low levels in ISCs and high levels in the quiescent or reserved stem cells that are also considered as the secretory progenitors. A possible explanation for these observations is that a low level of Sox9 sustains actively dividing ISCs, while an increase of SOX9 converts these proliferating ISCs into quiescent ISCs that will eventually differentiate into Paneth cells. Similarly, Sox9 is also implicated in regulating colorectal cancer cells, but there are conflicting data regarding whether Sox9 functions as an oncogene or a tumor suppressor. These seemingly contradictory results can be reconciled with a proposed model that Sox9 functions at an appropriate level, with a critical dose of Sox9 that exhibits proliferation-promoting activity, while increasing or decreasing this dose both result in proliferation-inhibitory activity. It is worthy to note that the differentiation-promoting function of Sox9 could potentially further complicate the interpretation of the mutant phenotype. It has been shown in Drosophila gut that defects in differentiation can induce a stressed microenvironment that promotes cell proliferation and propels tumor development (Jin, 2020).

The results of this study suggest many aspects of functional conservation of this Sox E subfamily gene in ISCs from Drosophila to mammals. Sox100B regulates both ISC proliferation and differentiation in the Drosophila intestine, and in terms of regulating ISC proliferation, Sox100B also requires an appropriate expression level. This study has demonstrated that this modulation of Sox100B expression is largely due to a negative feedback mechanism, in which increased Sox100B caused by elevated EGFR/Ras/MAPK signaling in turn suppresses the expression of EGFR, thereby leading to damped EGFR-signaling activity. Of note, contradictory data were recently reported on the roles of Sox100B and Sox21a in regulating ISC proliferation: both a proliferation-promoting role (Meng, 2015) and a tumor-suppressive role (Chen, 2016, Zhai, 2015) for Sox21a in ISCs have been reported; as for the role of Sox100B, Lan (2018) showed in an RNAi genetic screen that Sox100B is required for P.e.-induced ISC proliferation, whereas Doupé (2018) showed that depletion of Sox100B by RNAi causes increased ISC proliferation. Consideration of the effects caused by different levels of Sox100B expression that was observed in the present study may help resolve understanding of apparently disparate functions for these genes as central coordinators of both ISC proliferation and differentiation. It is proposed that, normally, a low level of Sox protein expression sustains ISC proliferation. A transient increase of Sox protein may not only promote cell cycle exit but also activate programs for terminal differentiation, thereby leading to a coordinated ISC proliferation and differentiation and, consequently, a coherent process of epithelial renewal (Jin, 2020).

This study demonstrates that Sox100B directly regulates Sox21a to promote differentiation. One important downstream target of Sox100B and Sox21a appears to be Pdm1, a known EC-fate-promoting factor. Interestingly, overexpression of Pdm1 in progenitor cells rapidly shuts down both Sox100B and Sox21a expression, indicating a negative feedback mechanism. Therefore, the induced Sox100B-Sox21a-Pdm1 axis in the differentiating ECs not only promotes cell differentiation, but also acts in a feedback mechanism to turn down EGFR and JAK/STAT signaling activities, thereby allowing ECs to terminally differentiate. This differentiation-promoting axis might also have a role in turning down ISC-specific programs, which are independently regulated by EGFR or JAK/STAT signaling pathways. For example, downregulation of the stem-cell-factor Esg is required for EB differentiation, and ectopic expression of Pdm1 is able to antagonize Esg expression in progenitor cells. These kinds of feedback regulation could be a common strategy used for initiation and finalization of a cell-differentiation program (Jin, 2020).

In summary, this study identified the transcription factor Sox100B as a major effector downstream of JAK/STAT and EGFR pathways that acts at an appropriate level to coordinate ISC proliferation and differentiation during both normal intestinal homeostasis and during damage- and infection-induced intestinal regeneration in Drosophila. With the 'just-right' effect endowed by a feedback mechanism, Sox100B behaves as a homeostatic sensor in the intestinal epithelium that coordinates stem cell proliferation with stem cell differentiation under various environmental conditions. It is proposed that this expressional and functional modulation associated with Sox family transcription factors may be a general mechanism for maintaining tissue homeostasis and regeneration in many organs, including those in mammals, and that deregulation of this mechanism may lead to tissue degeneration or cancer development (Jin, 2020).

Peroxisome elevation induces stem cell differentiation and intestinal epithelial repair

Epithelial-repair-dependent mucosal healing (MH) is associated with a more favorable prognosis for patients with inflammatory bowel disease (IBD). MH is accomplished via repair and regeneration of the intestinal epithelium. However, the mechanism underlying MH is ill defined. This study found a striking upregulation of peroxisomes in the injured crypts of IBD patients. By increasing peroxisome levels in Drosophila midguts, it was found that peroxisome elevation enhanced RAB7-dependent late endosome maturation, which then promoted stem and/or progenitor-cell differentiation via modulation of Janus Kinase (JAK) and Signal Transducer and Activator of Transcription (STAT)-SOX21A signaling. This in turn enhanced ISC-mediated regeneration. Importantly, RAB7 and SOX21 were upregulated in the crypts of IBD patients. Moreover, administration of drugs that increased peroxisome levels reversed the symptoms of dextran sulfate sodium (DSS)-induced colitis in mice. This study demonstrates a peroxisome-mediated epithelial repair mechanism, which opens a therapeutic avenue for the enhancement of MH in IBD patients (Du, 2020).

Inflammatory bowel disease (IBD) encompasses a range of complex, long-lasting, and relapsing-remitting disorders, of which ulcerative colitis (UC) and Crohn's disease (CD) are the two most prevalent manifestations. Currently, the incidence of IBD is increasing globally, and the prevalence has increased to just below 0.5% in the Western population. Although genetic predisposition, inappropriate immune responses, and environmental factors have been reported to be closely associated with IBD, its exact etiology and pathogenesis remain poorly understood (Du, 2020).

A series of recent clinical studies indicated that complete regeneration of the intestinal mucosa (also called 'mucosal healing') is associated with a more favorable prognosis for patients with IBD. In the mammal intestine, pluripotent intestinal stem cells (ISCs), located at the base of the epithelial crypts, are responsible for the repair of the damaged epithelium by differentiating into multiple epithelial progenies. Thus, a better understanding of cellular and molecular mechanisms that underlie ISC-mediated epithelial repair could not only provide insight into the etiology but also the therapeutic targets of IBD (Du, 2020).

To identify the mechanism with which peroxisomes regulate the differentiation of ISC progenies during midgut regeneration, RNA sequencing (RNA-seq) was performed on dissected midguts from the Pex10-null and WT flies. This study showed that Sox21a, a transcription factor essential for ISC-to-EC differentiation, had a significantly lower level in mutant midguts compared with that in WT midguts after injury. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differentially expressed genes, which only appeared in WT midguts but not in Pex10-null midguts during regeneration, showed significant enrichment of genes involved in the endocytosis process. A number of these endocytosis genes have been reported to regulate stem- and/or progenitor-cell differentiation in diverse animal models, such as polo, vap, Pcyt1, and Ubi-p63E. To confirm the results of the RNA-seq analyses, real-time quantitative PCR (qPCR) analyses were performed using sorted ISCs and EBs (esg-GFP+ cells). The results of real-time qPCR analyses of the selected genes, including Sox21a, vap, and rl/ERK showed similar expression patterns to those of RNA-seq analysis during regeneration. Thus, peroxisomes may regulate the ISC-to-EC differentiation by promoting the expression of Sox21a and/or that of specific endocytosis-related genes during regeneration (Du, 2020).

Since the Sox21a mutant has a similar differentiation defect (Zhai, 2015, Zhai, 2017, Chen, 2016) to Pex10 and Pex2 mutants, first, it was verified whether peroxisomes regulate the differentiation of ISCs and EBs through Sox21a-mediated signaling. The endogenous SOX21A reporter line Sox21a-HA was generated using a CRISPR-Cas9 knockin system. As expected, prior to injury, the expression of the SOX21A-HA protein in Pex10-null ISCs and EBs was similarly weak compared with its expression in control ISCs and EBs. However, after injury, while SOX21A expression was dramatically upregulated in control ISCs and EBs, it still retained a relatively low level in Pex10-null ISCs and EBs. Flip-out RNAi clone analysis consistently showed that peroxisomes promoted SOX21A expression after injury (Du, 2020).

More importantly, forced expression of either Sox21a or GATAe (one of the downstreams of SOX21A, which also showed a similar expression change pattern with Sox21a in Pex10-null mutant clone cells partially rescued the differentiation defect of ISC progenies in Pex10-null clones as evidenced by the generation of Pdm1+ cells. Consistently, forced expression of either Sox21a or GATAe in ISCs and EBs also partially restored the defects of esg-GFP+ cell and pH3+ cell accumulation in Pex10-null midguts (Du, 2020).

Since previous studies have shown that the JAK and STAT signaling functions upstream of Sox21a in ISCs and EBs to promote EC formation (Zhai, 2015, 2017), this study tested the genetic interaction between peroxisomes and the JAK and STAT signaling. After injury, while the activity of the JAK and STAT signaling was significantly upregulated in control midguts as indicated by the mRNA expression of the STAT target Socs36E and the 10XSTAT-GFP transcriptional reporter, it still retained a relatively low level in Pex10-null midguts. More importantly, forced expression of a constitutively active version of JAK (hopscotch [hop]) (UAS-hopTUM) significantly restored the expression defect of SOX21A in Pex10-depleted cells and the differentiation defect of ISCs and EBs in Pex10 RNAi clones as evidenced by the generation of Pdm1+ cells. In addition, depletion of PEX10 did not affect the activity of Notch signaling, and overexpression of constitutively active Notch (Notchintra) did not show obvious rescue of the ISC-to-EC differentiation defect of Pex10 mutant flies (Du, 2020).

Similar to the human intestine, the adult midgut in Drosophila uses resident ISCs to replenish damaged and lost epithelial cells after injury. The Drosophila ISCs divide to self-renew and produce non-dividing enteroblasts (EBs) or enteroendocrine mother cells (EMCs) depending on Notch activity. Post-mitotic EBs with high levels of Notch signaling further differentiate into absorptive enterocytes (ECs). The EMCs divide once to produce a pair of secretory enteroendocrine cells (EEs). In response to Drosophila midgut injury, ISCs transiently increase their proliferation rates and initiate the production of differentiated ECs to compensate for the damaged and lost cells. This mechanism lasts for a few days until the damaged midgut recovers its normal morphology. In addition to the activation of numerous signals, intestinal damage results in clear changes of cellular structures including the organelles of stem and/or progenitor cells. Although the signaling requirements for the stem cell function in intestinal regeneration have been reported in detail, the contribution of organelle dynamics in stem cells to intestinal repair remains poorly understood (Du, 2020).

Among different organelles, peroxisomes are remarkably plastic with the capability to change their composition, abundance, and morphology in response to environmental stimuli, which in turn may play a role in intestinal repair. By analyzing samples from human patients, utilizing the Drosophila midgut model, and leveraging the mouse IBD model, this study elucidates a conserved molecular pathway with which peroxisome proliferation induces stem-cell-mediated intestinal repair. Furthermore, a new therapeutic approach for the treatment of IBD has been suggested (Du, 2020).

To meet various cellular requirements, organelles in one cell do not function as isolated or static units but rather form dynamic contacts between each other. The findings of this study show that peroxisomes likely modulate the maturation of late endosomes via modulation of vascular transportation, which had been reported to regulate several types of signaling, such as Notch and JAK and STAT signaling. Further studies should aim to understand the detailed mechanism of how peroxisomes modulate the maturation of late endosomes during tissue regeneration (Du, 2020).

This study showed that administration of peroxisome-proliferating agents, NaPB and fenofibrate, which are two drugs approved by the food and drug administration (FDA), effectively reversed the symptoms of DSS-induced colitis in mice. Since NaPB and fenofibrate have a long history of use for disease treatment in human patients, they can be applied to determine their therapeutic effect of enhancing mucosal healing in patients with IBD. Since the peroxisome enhances intestinal repair by promoting stem cell differentiation, it will likely not cause stem cell over-proliferation and tumor formation. These findings not only have important implications for deciphering the functions of the peroxisome in stem cells, tissue regeneration, and injury-induced human diseases but also suggest the peroxisome as a promising therapeutic target of IBD (Du, 2020).

A genetic framework controlling the differentiation of intestinal stem cells during regeneration in Drosophila

The speed of stem cell differentiation has to be properly coupled with self-renewal, both under basal conditions for tissue maintenance and during regeneration for tissue repair. Using the Drosophila midgut model, this study analyzed at the cellular and molecular levels the differentiation program required for robust regeneration. The intestinal stem cell (ISC) and its differentiating daughter, the enteroblast (EB), were observed to form extended cell-cell contacts in regenerating intestines. The contact between progenitors is stabilized by cell adhesion molecules, and can be dynamically remodeled to elicit optimal juxtacrine Notch signaling to determine the speed of progenitor differentiation. Notably, increasing the adhesion property of progenitors by expressing Connectin is sufficient to induce rapid progenitor differentiation. It was further demonstrated that JAK/STAT signaling, Sox21a and GATAe form a functional relay to orchestrate EB differentiation. Thus, this study provides new insights into the complex and sequential events that are required for rapid differentiation following stem cell division during tissue replenishment (Zhai, 2017).

Key questions in stem cell biology are how the pool of stem cells can be robustly expanded yet also timely contracted through differentiation to generate mature cells according to the need of a tissue, and what are the underlying mechanisms that couple stem cell proliferation and differentiation. Over the last years, the mechanisms underlying intestinal stem cell activation have been extensively studied in both flies and mammals, while the genetic control of progenitor differentiation, especially during regeneration, has only recently begun to be understood (Zhai, 2017).

The transcription factor Sox21a has recently been the focus of studies in fly intestines. Using a Sox21a-sGFP transgene, this study uncovered its dynamic expression pattern in intestinal progenitors. Higher levels of Sox21a were found in ISC during homeostatic conditions but in EB during regeneration, supporting the roles of Sox21a in both ISC maintenance and EB differentiation at different conditions. The highly dynamic expression pattern of Sox21a revealed by this sGFP-tagged transgene per se argues against accumulation and perdurance of GFP fusion protein. Indeed, immunostaining using an antibody against Sox21a also indicated stronger Sox21a expression in ISC in homeostatic condition and global activation of Sox21a in progenitors under DSS-induced regeneration. However, Chen (2016) suggested that Sox21a levels are always higher in EB than in ISC by applying another antibody against Sox21a. The inconsistency between these studies may have arisen from the differences in EB stages examined or the sensitivity of respective detection approaches (Zhai, 2017).

This study has analyzed the cellular processes required for efficient progenitor differentiation during regeneration. Three main findings are reported revealing: i) the importance of extended contact between a stem cell and its differentiating daughter, ii) the existence of specific mechanisms allowing fast differentiation during regeneration, and iii) the characterization of a genetic program instructing the transition from EB to EC. These results together led to a proposal of a molecular framework underlying intestinal regeneration that is discussed below step by step (Zhai, 2017).

By studying the mechanisms of Sox21a-induced differentiation, this study found that ISC establishes extended contact with its differentiating daughter within a progenitor pair. Increased interface contact was not only observed upon Sox21a expression but also during regeneration after bacterial infection and DSS-feeding. Since the presence of extended contact is rare in intestinal progenitors under homeostatic conditions, it is hypothesized that extended contact between progenitors is related to increased epithelial renewal as a mechanism to elicit optimal juxtacrine Notch signaling to accelerate the speed of progenitor differentiation. The observations that down-regulation of the cell adhesion molecules E-Cadherin or Connectin suppresses rapid progenitor differentiation upon regeneration, and that overexpression of Connectin is sufficient to promote differentiation, underline the importance of increased cell-cell contact in rapid differentiation. This study shows that one early role of Sox21a is to promote the formation of this contact zone, possibly through transcriptional regulation of Connectin. Further studies should identify the signals and pathways leading to the change of contact between progenitors to adjust the rate of differentiation (Zhai, 2017).

Intestinal progenitors with extended contact in non-homeostatic midguts have been observed in some studies, but their role and significance have not been analyzed. Previous studies have also shown that progenitor nests are outlined by E-Cadherin/β-Catenin complexes, yet it was not known whether different degrees of progenitor contact are associated with their ISC versus EB fate. Consistent with these results, recent modeling analyses suggested a positive correlation between the contact area of progenitor pairs and the activation of Notch signaling. Thus, it seems that an increase in the contact area between intestinal progenitors is a hallmark of progenitors that are undergoing accelerated differentiation towards ECs. Another study has suggested an inhibitory role of prolonged ISC-EB contact to restrict ISC proliferation. Collectively, these studies and the current findings suggest that the strong contact between ISC and EB promotes on one hand the efficient differentiation of EBs into mature intestinal cells while on the other hand preventing stem cells from over-dividing. Thus, it is hypothesize that alteration in the contact zone provides a mechanism for ensuring both the appropriate speed of differentiation and the timely resolution of stem cell proliferative capacity (Zhai, 2017).

A second finding of this study consists in revealing the existence of specific mechanisms accelerating differentiation for tissue replenishment. In addition to the extended contact discussed above, a difference was observed in the pattern of ISC division between homeostatic and highly regenerative intestines. The modes of ISC division in Drosophila have been the topic of intense discussion, and the general consensus is that it is associated with an asymmetric cell fate outcome, in which one cell remains an ISC and the other engages in differentiation. In line with these previous studies, the results support the notion that asymmetric cell division is the most prevalent mode of ISC division under homeostatic conditions, where the rate of epithelial renewal is low. However, use of ISC- and EB-specific markers shows that upon rapid regeneration an ISC divides into two cells both expressing the ISC marker Dl-GFP but with one cell showing weak Notch activity. Similarly to other Notch-mediated cell-fate decision systems, this study suggests that the two resulting Dl-GFP+ cells from a symmetric division stay in close contact and compete for the stem cell fate. While this study is not the first to postulate the existence of symmetric ISC division, the use of reliable ISC- and EB-specific markers allows better visualization of this process. Applying a dual-color lineage tracing system to unravel the final fate of respective cells in a Dl+-Dl+ pair could reinforce the existence of symmetric stem cell division. This is nevertheless technically challenging to apply here since all the current available lineage-tracing settings require a heat shock to initiate the labeling, which affects intestinal homeostasis (Zhai, 2017).

Importantly, this study shows that the genetic program required for fast intestinal regeneration differs from the one involved in basal intestinal maintenance. This study indicates that GATAe, Dpp signaling, and the cell adhesion molecules E-cadherin and Connectin are not critical for progenitor differentiation when the rate of epithelial renewal is low, whereas their roles become crucial upon active regeneration. It is speculated that many discrepancies in the literature can be reconciled by taking into consideration that some factors are required only for rapid differentiation but not in basal conditions. For instance, the implication of Dpp signaling in differentiation has been disputed, since Zhou (2015) focused on bacterial infection-induced regeneration while two other studies dealt with basal conditions. The current study points to a clear role of Dpp signaling in the differentiation process upon regeneration. Therefore, better defining the genetic program that allows adjusting the speed of differentiation would be of great interest (Zhai, 2017).

Cell fate determination and differentiation involve extensive changes in gene expression and possibly also gradual change of cell morphology. The EB to EC differentiation in the adult Drosophila intestine provides a model of choice to study this process. This transition includes changes in cell shape, an increase in cell size, DNA endoreplication leading to polyploidy and the activation of the set of genes required for EC function. This study has integrated a number of pathways (Notch, JAK/STAT and Dpp/BMP) and transcription factors (Sox21a and GATAe) into a sequential framework. It was further shown that Sox21a contributes to the EB-EC transition downstream of JAK/STAT but upstream of Dpp signaling and GATAe. The recurrent use of several factors, namely JAK/STAT, Sox21a and GATAe at different processes including ISC self-renewal and EB-EC differentiation is likely to be a general feature during cell fate determination, and somehow also complicates the study of differentiation. Future work should analyze how each of the factors interacts with the other in a direct or indirect manner. It would be interesting as well to further study how these factors shape intestinal regionalization as the gut exhibits conspicuous morphological changes along the length of the digestive tract (Zhai, 2017).

Several of the findings described in this study are likely to apply to the differentiation program that takes place in mammals. Since Notch signaling plays major roles in stem cell proliferation and cell fate specification from flies to mammals, it would be interesting to decipher whether in mammals changes in progenitor contact also impact differentiation speed and whether a specific machinery can accelerate progenitor differentiation when tissue replenishment is required (Zhai, 2017).

Accumulation of differentiating intestinal stem cell progenies drives tumorigenesis

Stem cell self-renewal and differentiation are coordinated to maintain tissue homeostasis and prevent cancer. Mutations causing stem cell proliferation are traditionally the focus of cancer studies. However, the contribution of the differentiating stem cell progenies in tumorigenesis is poorly characterized. This paper reports that loss of the SOX transcription factor, Sox21a, blocks the differentiation programme of enteroblast (EB), the intestinal stem cell progeny in the adult Drosophila midgut. This results in EB accumulation and formation of tumours. Sox21a tumour initiation and growth involve stem cell proliferation induced by the Unpaired 2 mitogen released from accumulating EBs generating a feed-forward loop. EBs found in the tumours are heterogeneous and grow towards the intestinal lumen. Sox21a tumours modulate their environment by secreting matrix metalloproteinase and reactive oxygen species. Enterocytes surrounding the tumours are eliminated through delamination allowing tumour progression, a process requiring JNK activation. These data highlight the tumorigenic properties of transit differentiating cells (Zhai, 2015).

The data show that Sox21a, a previously uncharacterized transcription factor of the SOX family, plays a major role in the terminal differentiation of ISC progenies. Although the Drosophila genome encodes eight Sox genes, Sox21a is the only one enriched in the midgut. Sox21a is specifically expressed in progenitor cells along the entire midgut, and acts downstream of the JAK/STAT signalling to permit the transformation of EBs into enterocytes and enteroendocrine cells. Although Sox21a is required for both differentiated cell types, overexpression of Sox21a drives differentiation into enterocytes. It cannot be excluded that high level of Sox21a due to overexpression approach favours enterocyte rather than the enteroendocrine cell fate. Overexpression of Sox21a rescues the differentiation marker Pdm1 that is lost in JAK/STAT-deficient clones, demonstrating that Sox21a contributes significantly to EB differentiation downstream of this pathway. Consistent with this notion, RNA-seq analysis demonstrates that Sox21a regulates a large set of genes including Pdm1, which encodes a transcription factor involved in the terminal differentiation of enterocytes. This study also shows that Sox21a contributes to stem cell division notably in the posterior midgut. This is similar to the JAK/STAT pathway that impacts both stem cell division and differentiation. The observation that Sox21a flies are viable indicates that the role of Sox21a is likely restricted to the adult intestinal homeostasis. Moving on, functional characterization of Sox21a target genes and identification of Sox21a-binding sites are now required to better understand the role of this transcription factor in ISC proliferation and progenitor differentiation (Zhai, 2015).

An unexpected observation of this work was that Sox21a flies developed tumours that increase in size and grow towards the intestinal lumen over time. Sox21a tumours are mainly composed of post-mitotic progenitors, the EBs that are blocked in their differentiation. This study shows that the growth of Sox21a tumours relies on ISC division. The requirement for ISC proliferation to drive Sox21a tumours explains why tumours are not observed in the posterior midgut and are more frequent when flies are infected with bacteria, a condition that stimulates stem cell proliferation. The results indicate that the release of a mitogen, Upd2, by accumulating EBs provides a feed-forward loop stimulating ISCs to divide and differentiate, leading to a further increase in the number of EBs. It is likely that Sox21a tumours are initiated at random sites where EB clustering leads to a local increase of Upd2. Like other cancer model, Sox21a tumours also express matrix metalloproteinase, which probably shapes the tumour local environment to promote tumour progression. Accumulating EBs display a shift in metabolism with an increased expression of ROS-producing factors, such as Duox and a higher number of mitochondria and peroxisomes. This metabolic shift is likely to underlie the increase of ROS at the vicinity of the tumour. It was observed that the progenitors express at high-level ROS detoxification enzymes. Thus, the concomitant high-level synthesis of ROS and detoxifying enzymes by accumulating EBs restricts high ROS levels to the tumour borders. It is likely that ROS production promotes Sox21a tumour growth by eliminating flanking enterocytes in a JNK-dependent manner. Further experiments are required to determine whether JNK activation in flanking enterocytes is induced by ROS or by mechanical constraints from the tumours or by simultaneously both processes. This tumour model introduces a new concept highlighting the active role of differentiating stem cell daughters in tumour formation. This model highlights the tumorigenic properties of transit differentiating cells and is in contrast to the current paradigm that emphasizes exclusively on the role of stem cells. In light of these findings, it is speculated that the plasticity of these differentiating cells underlies their cancerous properties (Zhai, 2015).

Mechanistic studies of several intestinal tumour models have been reported previously in Drosophila. The Sox21a tumour model is unique in its simplicity compared with other models that require complex genetic manipulations. Similar to Notch loss-of-function tumour model, it reveals how a differentiation defect in the stem cell progenies can drive tumorigenesis. Both models are caused by a defect in the stem cell differentiation program, rely on stem cell division, and involve the elimination of flanking enterocytes by delamination through the JNK pathway. There are, however, major differences. First, Notch tumours are caused by a blockage of differentiation at the ISC stage, while Sox21a is required later in the differentiation of EBs. This explains their distinct dynamics of tumour formation. Compared with the acute formation of Notch ISC tumour, the formation of Sox21a EB tumour is a slow and stochastic process. While Notch tumours can be observed only a few days after induction of Notch-deficient cells, >20 days is required to observe grade 3 tumours in Sox21a flies. Second, the growth of Notch tumour relies on the autocrine EGF ligand Spitz and the JAK/STAT ligand Upd2, while the growth of Sox21a tumour requires the paracrine Upd2, and to a much lesser extent Spitz from EBs. Third, Sox21a tumours display a higher level of cellular heterogeneity, which has not been described for Notch tumours. Fourth, Sox21a tumours are not formed in mosaic intestine where Sox21a is mutated clonally, while Notch-deficient stem cells can grow into tumour in clones. Finally, this study uncovers a non-cell autonomous effect of ROS in tumour progression caused by metabolic changes in the tumour cells. The implication of ROS in cancer is an emerging theme of research. Thus, this model is likely to serve as a useful tool to study how ROS could play a signalling role to mediate short-range communication between tumour cells and their neighbours (Zhai, 2015).

Many human tumours are composed of cells with different degrees of differentiation, including differentiating progenitors derived from stem cells. This study highlights the cancerous properties of the differentiating stem cell progenies, which can stimulate stem cells proliferation and display a high cellular plasticity. Promoting the terminal differentiation of cancer cells has long been proposed and studied as a promising therapeutic strategy. With increasing knowledge of genetic control of stem cell differentiation, it would be interesting to explore whether modulating progenitor cell differentiation can combat the progression of cancers (Zhai, 2015).

A Sox transcription factor is a critical regulator of adult stem cell proliferation in the Drosophila intestine

Adult organs and their resident stem cells are constantly facing the challenge of adapting cell proliferation to tissue demand, particularly in response to environmental stresses. Whereas most stress-signaling pathways are conserved between progenitors and differentiated cells, stem cells have the specific ability to respond by increasing their proliferative rate, using largely unknown mechanisms. This study shows that a member of the Sox family of transcription factors in Drosophila, Sox21a, is expressed in intestinal stem cells (ISCs) in the adult gut. Sox21a is essential for the proliferation of these cells during both normal epithelium turnover and repair. Its expression is induced in response to tissue damage, downstream of the Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) pathways, to promote ISC proliferation. Although short-lived, Sox21a mutant flies show no developmental defects, supporting the notion that this factor is a specific regulator of adult stem cell proliferation (Meng, 2015).

This work demonstrates that Sox21a, a member of the Sox2 sub-family of transcription factors, is essential for cell proliferation in the adult Drosophila intestine under homeostatic conditions and in response to stress. Strikingly, although it was found that Sox21a mutant adult flies have dramatic ISCs proliferation defects and are short lived, they do not display any visible developmental phenotype, recapitulating a reported analysis of null Sox21a mutants. Thus, this demonstrates that ISCs use stem-cell-specific mechanisms to control cell proliferation. Further studies will be required to understand how Sox21a interacts with other transcription factors that have been shown to regulate ISC proliferation, such as Myc, Nrf2, Stat92E, and Yorkie. In addition, it is anticipated that the identification of Sox21a transcriptional targets in ISCs will be required to fully decipher the mechanism by which this factor controls ISC cell cycle and/or quiescent state. This also constitutes a unique opportunity to study the adult-specific functions of a Sox factor, apart from their requirement during development (Meng, 2015).

This study shows that JNK and Ras/ERK signaling, as well as the AP-1 transcription factor Fos, are required for Sox21a induction in response to tissue damage. Although the data support a model in which the activity of Fos is regulated by JNK and ERK to control Sox21a expression, further studies will be necessary to investigate the potential mechanisms of such regulation and test whether Fos directly binds to the Sox21a locus and controls its transcription. Whereas the transcriptional response to various stresses or the activation of these pathways has been investigated in developing tissues and other adult organs, Sox21a has not been identified as a target of these pathways. Thus, the current findings suggest that unidentified stem-cell-specific factor(s) cooperate with Fos to control Sox21a expression in ISCs and EBs. Additional work will be necessary to carefully describe the regulation of Sox21a and the possible role of ISC-specific factors, such as esg. It will also be interesting to test whether other signaling pathways, such as JAK/STAT and Hippo/Yorkie, are involved in the regulation of Sox21a expression. The identification of potential common transcriptional targets will help to understand how these signaling pathways crosstalk in ISCs and how different signals are integrated into a coordinated proliferative response (Meng, 2015).

Interestingly, like the activation of stress-signaling pathways, expression of Sox factors is essential for tumor formation in many tissues. Therefore, it will be interesting to test whether, similarly to the regulation described in this study in ISCs, stress pathways, such as JNK and Ras/ERK, directly control the expression of Sox factor(s) in mammals. In this context, the results may provide new insights in the mechanisms that control tissue repair and tumorigenesis in higher organisms, including in humans (Meng, 2015).

Parallel expansions of Sox transcription factor group B predating the diversifications of the arthropods and jawed vertebrates

Group B of the Sox transcription factor family is crucial in embryo development in the insects and vertebrates. Sox group B, unlike the other Sox groups, has an unusually enlarged functional repertoire in insects, but the timing and mechanism of the expansion of this group were unclear. Data for Sox group B was collected and analyzed from 36 species of 12 phyla representing the major metazoan clades, with an emphasis on arthropods, to reconstruct the evolutionary history of SoxB in bilaterians and to date the expansion of Sox group B in insects. It was found that the genome of the bilaterian last common ancestor probably contained one SoxB1 and one SoxB2 gene only and that tandem duplications of SoxB2 occurred before the arthropod diversification but after the arthropod-nematode divergence, resulting in the basal repertoire of Sox group B in diverse arthropod lineages. The arthropod Sox group B repertoire expanded differently from the vertebrate repertoire, which resulted from genome duplications. The parallel increases in the Sox group B repertoires of the arthropods and vertebrates are consistent with the parallel increases in the complexity and diversification of these two important organismal groups (Zhong, 2011).

Previous studies have suggested two incompatible models for the expansion of Sox group B in Drosophila. One of these models places one of the four SoxB members into subgroup B1, and the other three into subgroup B2. Although there is agreement on the assignment of SoxNeuro (SoxB1) into subgroup B1 and Sox21a (SoxB2a) into subgroup B2, the other model maintains that Dichaete (SoxB2b1) and Sox21b (SoxB2b2) are both co-orthologous to both vertebrate Sox1 and Sox2 rather than to the vertebrate SoxB2 members, and that the Protostome-Deuterostome LCA had a three-member complement of Sox group B proteins. The resolution of this dispute lies in the correct orthology assignments of the Drosophila SoxB members with the vertebrate ones, and a valid reconstruction of the ancestral SoxB repertoire at key phylogenetic nodes. A related and interesting question concerns the phylogenetic timing of the expansion of Sox group B in Drosophila. Initially, this expansion was attributed to relatively recent duplications, but later research involving more insect taxa indicated that the four-member SoxB inventory is phylogenetically old, and was at least present in the LCA of the Hymenoptera and Diptera. However, whether this expansion is even older remained an open question at that time (Zhong, 2011).

A comparison of the two models of Sox group B evolution is presented. The first model is for Sox group B evolution proposed by a previous study. In this model, an ancestral SoxB generate original Dichaete and SoxNeuro by an ancient genome duplication, a subsequent tandem duplication generate original Sox21a before the Deuterostome/Protostome split. After the Deuterostome/Protostome split, a further tandem duplication generated Sox21b in insects and an independent genome duplication event increased the copy number of SoxB in vertebrates. The second model shows this study's proposal for Sox group B evolution. In this model, the Protostome-Deuterostome last common ancester had one SoxB1 and one SoxB2 generated by an ancient tandem duplication of an ancestral SoxB. After the Deuterostome/Protostome split, two further tandem duplications gave rise to the additional two copies of SoxB2 in arthropods, and a linkage break between SoxB1 and SoxB2s occurred in the ancestor of Drosophila, resulting in the different chromosome locations of SoxB1 and SoxB2s in Drosophila; independently, the vertebrates increased their copy number of SoxB through the two rounds of genome duplication. Forks on the rectangles indicate pseudogenization leading to gene loss. SoxB2b1, SoxB2b2, and SoxB2a are the preferred synonyms for Dichaete, Sox21a, and Sox21b, respectively. Sry is currently considered to have evolved from allele Sox3 on the Y chromosome, and is therefore not shown in the models. The second model, described in this work, shows Sox neuro as belonging to subgroup SoxB1, homolgous to Sox1 and Sox2 of vertebrates. The SoxB2 subgroup in vertebrates is represented by Sox21 and Sox14 (Zhong, 2011).


Search PubMed for articles about Drosophila Sox21a

Bastide, P., Darido, C., Pannequin, J., Kist, R., Robine, S., Marty-Double, C., Bibeau, F., Scherer, G., Joubert, D., Hollande, F., Blache, P. and Jay, P. (2007). Sox9 regulates cell proliferation and is required for Paneth cell differentiation in the intestinal epithelium. J Cell Biol 178(4): 635-648. PubMed ID: 17698607

Chen, J., Xu, N., Huang, H., Cai, T. and Xi, R. (2016). A feedback amplification loop between stem cells and their progeny promotes tissue regeneration and tumorigenesis. Elife 5. PubMed ID: 27187149

Doupe, D. P., Marshall, O. J., Dayton, H., Brand, A. H. and Perrimon, N. (2018). Drosophila intestinal stem and progenitor cells are major sources and regulators of homeostatic niche signals. Proc Natl Acad Sci U S A 115(48): 12218-12223. PubMed ID: 30404917

Du, G., Xiong, L., Li, X., Zhuo, Z., Zhuang, X., Yu, Z., Wu, L., Xiao, D., Liu, Z., Jie, M., Liu, X., Luo, G., Guo, Z. and Chen, H. (2020). Peroxisome elevation induces stem cell differentiation and intestinal epithelial repair. Dev Cell 53(2): 169-184. PubMed ID: 32243783

Jin, Z., Chen, J., Huang, H., Wang, J., Lv, J., Yu, M., Guo, X., Zhang, Y., Cai, T. and Xi, R. (2020). The Drosophila ortholog of mammalian transcription factor Sox9 regulates intestinal homeostasis and regeneration at an appropriate level. Cell Rep 31(8): 107683. PubMed ID: 32460025

Lan, Q., Cao, M., Kollipara, R. K., Rosa, J. B., Kittler, R. and Jiang, H. (2018). FoxA transcription factor Fork head maintains the intestinal stem/progenitor cell identities in Drosophila. Dev Biol 433(2): 324-343. PubMed ID: 29108672

Meng, F. W. and Biteau, B. (2015). A Sox transcription factor is a critical regulator of adult stem cell proliferation in the Drosophila intestine. Cell Rep 13(5): 906-914. PubMed ID: 26565904

Meng, F. W., Rojas Villa, S. E. and Biteau, B. (2020). Sox100B regulates progenitor-specific gene expression and cell differentiation in the adult Drosophila intestine. Stem Cell Reports 14(2): 226-240. PubMed ID: 32032550

Mori-Akiyama, Y., van den Born, M., van Es, J. H., Hamilton, S. R., Adams, H. P., Zhang, J., Clevers, H. and de Crombrugghe, B. (2007). SOX9 is required for the differentiation of paneth cells in the intestinal epithelium. Gastroenterology 133(2): 539-546. PubMed ID: 17681175

Patel, P. H., Dutta, D. and Edgar, B. A. (2015). Niche appropriation by Drosophila intestinal stem cell tumours. Nat Cell Biol 17(9): 1182-1192. PubMed ID: 26237646

Zhai, Z., Kondo, S., Ha, N., Boquete, J. P., Brunner, M., Ueda, R. and Lemaitre, B. (2015). Accumulation of differentiating intestinal stem cell progenies drives tumorigenesis. Nat Commun 6: 10219. PubMed ID: 26690827

Zhai, Z., Boquete, J. P. and Lemaitre, B. (2017). A genetic framework controlling the differentiation of intestinal stem cells during regeneration in Drosophila. PLoS Genet 13(6): e1006854. PubMed ID: 28662029

Zhong, L., Wang, D., Gan, X., Yang, T. and He, S. (2011). Parallel expansions of Sox transcription factor group B predating the diversifications of the arthropods and jawed vertebrates. PLoS One 6(1): e16570. PubMed Citation: 21305035

Biological Overview

date revised: 22 October 2020

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