InteractiveFly: GeneBrief

Ptx1: Biological Overview | References

BIOLOGICAL OVERVIEW

Enteroendocrine cells (EEs) in the intestinal epithelium have important endocrine functions, yet this cell lineage exhibits great local and regional variations that have hampered detailed characterization of EE subtypes. Through single-cell RNA-sequencing analysis, combined with a collection of peptide hormone and receptor knockin strains, this study provides a comprehensive analysis of cellular diversity, spatial distribution, and transcription factor (TF) code of EEs in adult Drosophila midgut. Ten major EE subtypes were identified that totally produced approximately 14 different classes of hormone peptides. Each EE on average co-produces approximately 2-5 different classes of hormone peptides. Functional screen with subtype-enriched TFs suggests a combinatorial TF code that controls EE cell diversity; class-specific TFs Mirr and Ptx1 respectively define two major classes of EEs, and regional TFs such as Esg, Drm, Exex, and Fer1 further define regional EE identity. These single-cell data should greatly facilitate Drosophila modeling of EE differentiation and function (Guo, 2019).

Apart from the function in food digestion and absorption, the gastrointestinal tract is also considered as the largest endocrine organ due to the resident enteroendocrine cells (EEs). In mice and humans, EEs are scattered throughout the intestinal epithelium and take up only 1% of total intestinal cells, yet they produce more than 20 types of hormones that regulate a diverse of physiological processes, such as appetite, metabolism, and gut motility. There are at least 12 major subtypes of EEs based on hormones that they produce, and due to their great regional and local cellular diversity, the complete characterization of EE specification and diversification still remains as a challenge (Guo, 2019).

The adult Drosophila midgut has become an attractive model system for the understanding of EE cell diversity and their regulatory mechanisms. The EEs are scattered along the epithelium of the entire midgut, including anterior midgut (regions R1 and R2), middle midgut (the gastric region, R3) and posterior midgut (regions R4 and R5). They have important roles in regulating local stem cell division and lipid metabolism, as well as feeding and mating behaviors. Approximately 10 peptide hormone genes are found to be expressed in EEs, yielding more than 20 different peptide hormones. Studies using RNA in situ hybridization, antibody staining, and gene reporter tools have provided a glimpse of regional EE diversity in terms of peptide hormones that they produce. However, due to limited availability of antibodies against all these hormones and a limit in the number of hormones that can be simultaneously analyzed, the detailed characterization of EE subtypes and peptide profiles is still lacking (Guo, 2019).

As in mammals, EEs in the fly midgut are derived from multipotent intestinal stem cells (ISCs). The initial fate determination between absorptive enterocyte versus secretory EEs is controlled by Notch signaling and appears to be regulated by the antagonistic activities of E(spl)-C genes and achaete-scute complex genes. This is also analogous to the antagonistic activities between Hes1 (orthologous to E(spl)) and Math1 (paralogous to AS-C) in mammalian ISCs that control the initial cell fate decision. The committed EE progenitor cell usually divides one more time to yield a pair of EEs. Interestingly, the two EEs within each pair produce distinct hormone peptides as a result of differentially acquired Notch activity, suggesting that, at least in the posterior midgut, differential Notch signaling defines two major subtypes of EEs. The specification and commitment of EE fate requires the homeodomain transcription factor (TF) Prospero (Pros), and the maturation of peptide hormones in EEs requires a Neuro D family bHLH TF Dimmed (Dimm). Besides these general TFs that promote EE specification and function, little is known regarding the TFs that participate in EE subtype specification and regional EE identity (Guo, 2019).

Single-cell RNA-sequencing (scRNA-seq) has emerged as an efficient tool for revealing cell heterozygosity in different tissues and organisms. By using scRNA-seq and a collection of recently generated peptide and receptor knockin lines, this study provides a comprehensive analysis of EE cell diversity, peptide profiling, and regional distribution along the entire length of the fly midgut at single-cell resolution. In addition, TF enrichment analysis followed by genetic screen allowed thew identification of class and region EE regulators. These results suggest a TF code composed of class-specific and region-specific TFs generates EE cell diversity (Guo, 2019).

Using single-cell transcriptomics in combination with a collection of reporter lines, this study has provided a comprehensive characterization of the EE population in the entire midgut of adult Drosophila. In addition to the two major classes of EEs that respectively produce TK and AstC peptide hormones, a third class of EEs was identified that reside only in the anterior midgut (R2) and produce sNPF and CCHa2. Ten EE subtypes were identified that generally show region-specific distributions. In addition, functional screens with subtype-specific TFs have revealed class- and region-specific TFs that regulate subtype specification. These single-cell data should serve as an important resource for further understanding the differentiation, regulation, and function of EEs using Drosophila midgut as a genetic model system (Guo, 2019).

The single-cell data reveal 14 classes of peptide hormone genes that are expressed in EEs, compared to the previously known 10 classes. The midgut expression patterns of all these peptide hormones, including several peptide hormones whose gut expression patterns have not been clearly defined, such as Gbp5, ITP, and Nplp2 as well as sNPF, are also determined. As EEs perform their endocrine function by secreting various peptide hormones, the types of peptide hormones that they produced are usually used to classify EE subtypes in mammals. Indeed, the exclusive expression pattern of Tk and AstC is sufficient to distinguish between class I EEs and class II EEs. However, although different EE subtypes show distinct peptide hormone expression profiles, the types of peptide hormone expressed and the EE subtypes are not strictly correlated. In fact, the peptide hormone co-expression patterns are highly variable among individual EEs, even for EEs that belong to the same cluster or subtype. For example, for the II-m (C4) subtype, although they commonly produce Tk and NPF, their expression for Mip, Nplp2, and CCAP is highly variable. The external stimuli, such as stress and microbiota, may have an impact on the expression status of these variable peptide hormone genes. Alternatively, EEs could be plastic and change their peptide hormone expression profiles with age. Recent studies demonstrate that the mammalian EEs are plastic and can switch their hormone profiles as they differentiate and migrate upward along the crypt-villus axis (Guo, 2019).

One major limit associated with the scRNA-seq technology is that the spatial information of the cells is lost during tissue dissociation. In a way to overcome this limit, this study has developed a RSGE algorithm based on the region- and cell-type-specific transcriptome database from flygut-seq. As confirmed, for the various peptide hormone markers, including GAL4 knockin lines and antibodies, this algorithm has allowed generation of a reliable distribution map a for all the EE subtype clusters along the length of the midgut. The determination of the spatial distribution of EE subtypes should greatly facilitate the understanding of their regulation and function. For instance, DH31 and ITP expressing EEs are found be located in the posterior-most region of the midgut, and their location is clearly consistent with their known function: DH31 is known to regulate fluid secretion in Malpighian tubules, and ITP is known to regulate ion transport in hindgut. As regional difference for a common cell type is likely a general phenomenon in diverse tissues of many organisms, the algorithm in this study could provide an example of possible approaches for acquiring the lost spatial information of cells when conducting this type of single-cell analysis (Guo, 2019).

By analyzing the TF code for the EE subtypes followed by functional screen, this study has identified a number of TFs that participate in the specification of EE subtypes, including the class-I- and class-II-specific TFs Mirr and Ptx1 for the two major classes of EEs and region-specific TFs such as Esg, Drm, Fer1, and Sug that define regional EE identity. Previous studies in the posterior midgut have revealed that class I and II EEs are specified by differential Notch signaling. In this study, cell-type specific manipulating of Notch activity allows the conclusion that Notch must function transiently at the progenitor stage, between the two immediate daughters of an EEP, to define the two classes of EEs. As Mirr and Ptx1 are expressed only in differentiated EEs, the sequential activity of Notch and Mirr/Ptx1 indicates that these two TFs act downstream of Notch to specify class I versus class II EE type. The regional diversity of EEs is then further specified by region-specific TFs and possibly impacted by other environmental factors. It is proposed that EE cellular diversity is generated by a combination of class-specific and region-specific TFs, with class-specific TFs regulated by Notch signaling and region-specific TFs determined by anterior-posterior body planning during early development. The local EE diversity could also be regulated by environmental changes and age-related cell plasticity, possibilities that remain to be explored in the future (Guo, 2019).

Collectively, these single-cell data have provided a comprehensive characterization of EE cell diversity and their peptide hormone expression profiles. The TF code analysis also provides insights into EE diversity mechanisms. This data should greatly facilitate functional annotations of EE subtypes and gut peptide hormones under diverse physiological and pathological conditions, such as mating, starvation, bacterial infection, and so on. The Perrimon lab recently conducted single-cell transcriptomes for all types of midgut cells using the inDrop method. As EEs only represent a small fraction of total cells analyzed, their analysis primarily focused on progenitor cells and enterocytes (Hung, 2020). Therefore, the data and their scRNA-seq data should serve as complementary resources for understanding Drosophila gut cells. An online searchable database has been established to facilitate the use of these single-cell data (Guo, 2019).

A cell atlas of the adult Drosophila midgut

Studies of the adult Drosophila midgut have led to many insights in understanding of cell-type diversity, stem cell regeneration, tissue homeostasis, and cell fate decision. Advances in single-cell RNA sequencing provide opportunities to identify new cell types and molecular features. This study used single-cell RNA sequencing to characterize the transcriptome of midgut epithelial cells and identified 22 distinct clusters representing intestinal stem cells, enteroblasts, enteroendocrine cells (EEs), and enterocytes. This unbiased approach recovered most of the known intestinal stem cells/enteroblast and EE markers, highlighting the high quality of the dataset, and led to insights on intestinal stem cell biology, cell type-specific organelle features, the roles of new transcription factors in progenitors and regional variation along the gut, 5 additional EE gut hormones, EE hormonal expression diversity, and paracrine function of EEs. To facilitate mining of this rich dataset, a web-based resource is provided for visualization of gene expression in single cells. Altogether, this study provides a comprehensive resource for addressing functions of genes in the midgut epithelium (Hung, 2020).

Like its mammalian counterpart, the adult Drosophila midgut is a complex tissue composed of various cell types performing diverse functions, such as digestion, absorption of nutrients, and hormone production. Enterocytes (ECs) secrete digestive enzymes, and absorb and transport nutrients, whereas enteroendocrine cells (EEs) secrete gut hormones that regulate gut mobility and function in response to external stimuli and bacteria. The fly midgut is a highly regenerative organ that has been used extensively in recent years as a model system to characterize the role of signaling pathways that coordinate stem cell proliferation and differentiation in the context of homeostasis and regeneration. For example, EGFR, JAK/STAT, and Hippo signaling control intestinal stem cell (ISC) growth and proliferation, while Notch signaling regulates ISC differentiation. To maintain homeostasis, ISC proliferates and gives rise to a transient progenitor, the enteroblast (EB), defined by the expression of Su(H)GBE-lacZ, a Notch pathway activity reporter. In addition, both ISCs and EBs express the SNAIL family transcription factor escargot (esg). Polyploid ECs, characterized by the expression of Myosin31DF (Myo1A) and nubbin (also called pdm1), differentiate from EBs. In contrast, EEs, marked by the expression of prospero (pros), are derived from ISCs through distinct progenitors, called pre-EEs, that express Piezo, a cation channel that senses mechanical tension. In addition, the midgut is surrounded by visceral muscles, which control midgut movements and secrete niche signals, such as Wingless (Wg), the EGFR ligand Vein (Vn), and the JAK-STAT ligand Unpaired1 (Upd1) to control ISC activities (Hung, 2020).

Similar to the compartmentalized mammalian digestive tract, the fly midgut can be divided into regions with distinct morphological, histological, and genetic properties. For example, the middle region of the midgut, which contains a group of specialized copper cells, is acidic and resembles the mammalian stomach. In addition, EEs produce at least 10 different gut hormone peptides that are produced in specific regions: Allatostatins (AstA, AstB/Mip, AstC), Tachykinin (Tk), neuropeptide F (NPF), DH31, CCHa1, CCHa2, Orcokinin B, and Bursicon (Burs). AstA-producing EEs are located in the posterior region of the gut, whereas EEs in the anterior, middle, and first half of the posterior midgut produce AstC. Moreover, individual EEs are able to produce 2 combinations of different hormones. In particular, some NPF-producing EEs also produce Tk. The diversity and regional differences in EEs hinder the ability to comprehensively characterize subtypes of EEs using bulk RNA sequencing (RNA-seq) (Hung, 2020).

To further characterize gene expression and cell types in the adult fly midgut, single-cell RNA sequencing (scRNA-seq) was used, as it provides an unbiased approach to survey cell-type diversity, function, and define relationships between cell types. This study reveals molecular markers for each cell type, cell type-specific organelle features, regional differences among ECs, a transitional state of premature ECs, transcriptome differences between ISCs and EBs, 5 additional gut hormones, diverse hormone expression of EEs, paracrine function of EEs, a subset of EEs, and cell-type similarity between the fly and the mammalian gut. This study demonstrates how the dataset can be used to characterize new genes involved in gut cell lineage and in particular, it was demonstrated that the transcription factor klumpfuss suppresses EE formation. Finally, a web-based visualization resource was built that allows users to browse scRNA-seq data, query the expression of any genes of interest in different cell types, and compare the expression of any 2 genes in individual cells. Altogether, this study provides a valuable resource for future studies of the Drosophila midgut (Hung, 2020).

This study surveyed the cell types of the adult intestinal epithelium using scRNA-seq and identified all known cell types, 1 cell type (esg+ pros+) in the middle region of the midgut, differentiating ECs, and 5 unknown cell types (unk1, unk2, EC-like 1 to 3). This study recovered most previously known ISC/EBs and EEs markers, demonstrating the robustness of the scRNA-seq approach. Interestingly, gene expression analysis revealed that ISCs are enriched for free ribosomes and possess mitochondria with fewer cristae. Transcription factors expressed differently along the guts and cytoskeletal proteins and transcription factors preferentially expressed in the ISC/EB population were identified. In particular, this study validated that klu is specifically expressed in EBs, and knockdown of klu in ISC/EBs (with esg-Gal4) results in an increase of EEs, suggesting that klu inhibits EE differentiation. When the scRNA-seq study was performed using inDrop, a clear separation was not seen between cells that expressed Dl and cells expressing Notch downstream targets, E(spl)m3-HLH, E(spl)malpha-BFM, E(spl)mbeta-HLH, and the EB marker, klu. Thus, it was not certain whether this could be resolved using the 10x Genomics technology. Interestingly, using data from 10x Genomics this study was able to detect one subset of cells in the ISC/EB cluster that expresses Dl+ klu- (ISC) and another subset expressing Dl- klu+ (EB). Therefore, using data from 10x Genomics alone allows demarcation of the ISCs and EBs (Hung, 2020).

This study started with a small number of cells in the first sample for inDrop and 10x Genomics technologies, which recovered 344 cells and 256 cells, respectively. This allowed testing and comparison of the 2 technologies. Next, the number of cells was increased for each replicate (7,282 for inDrop and 2,723 for 10x Genomics). The 2 replicates allowed evaluation of the consistency of cell-type discovery between the 2 platforms. Indeed, all of the major cell types (cardia, ISC/EB, EE, dEC, aEC, mEC, pEC, LFC, copper, and iron cells) were detected using both approaches (Hung, 2020).

Cell morphology and digestive functions are different along the length of the Drosophila midgut. For example, ECs in the middle midgut secrete acid and absorb metal ions, whereas ECs in the posterior midgut contain lipid droplets and uptake lipid nutrients. These characteristics reflect regionalized gene-expression differences as previously shown by bulk RNA-seq analyses. Differentially expressed transcription factors were sought that could underlie regionalized gene expression and identified a number of potential candidates. In particular, vnd, odd, caup, and tup are preferentially expressed in the anterior region. Only one discrepancy (odd) was detected between these results and previously published bulk RNA-seq data (expressed in the posterior region from the Flygut-seq). Further studies will be required to resolve these differences. lab, Ptx1, CREG, apt, and dve are preferentially expressed in the middle midgut, consistent with the previous observation that the homeobox genes lab, Ptx1, and dve have been shown to be expressed in the adult middle midgut. Finally, bab2, ham, cad, Ets21C, Hnf4, and hth are preferentially expressed in the posterior midgut; the homeobox gene cad and Ets21C have been previously reported to be expressed in the posterior midgut. The expression pattern of these regionalized transcription factors from the Flygut-seq are listed in an accompanying dataset to help compare these findings. Recently, scRNA-seq of the mouse embryo identified a group of 20 transcription factors that are expressed spatially along the anterior-posterior axis of the gut tube. Interestingly, 6 out of 20 transcription factors expressed in the mouse gut have fly orthologs that are also expressed differently along the anterior-posterior axis of the fly midgut. For example, mouse Irx3 and fly caup are expressed in the anterior region, mouse Hoxb1 and fly lab are expressed in the anterior-middle region, and mouse Cdx2 and fly cad are expressed in the posterior region (Hung, 2020).

The regional expression of the transcription factors described above may also underlie the regionalization of EE populations. For example, cad, which is expressed in posterior ECs, is also highly expressed in AstA-EEs that are localized in the posterior midgut. This study also identified another transcription factor expressed in posterior EEs, Poxn, that is homologous to mouse Pax8, which is expressed regionally in the mouse gut tube. Whether Poxn is expressed in posterior EEs has not yet been experimentally tested. Similarly, stem cell morphology and proliferation activity also differ along the anterior-posterior axis of the gut. However, although previous cell-specific RNA-seq studies revealed regional differences in stem cell transcriptomes, this scRNA-seq analysis was not able to identify subgroups or regional ISC/EB clusters, despite the fact that some stem cells express some regional markers, such as lab or Ptx1. It is possible that the regional differences in ISC transcriptomes are less prominent than the regional differences in EC transcriptomes (Hung, 2020).

Regarding EEs, candidate markers and 5 additional gut hormones were identified: sNPF, ITP, Nplp2, CCAP, and CNMa. In addition, it was found that individual EEs are able to express up to 5 different hormones, in contrast to the traditional view that these cells only produce 2 hormones. Interestingly, a recent mammalian study showed that EEs express different hormones and that they can switch their hormonal repertoire depending on their tissue location. The most frequent combinations of gut hormones were AstA/AstC/Orcokinin/CCHa1/CCHa2 for AstA-EEs, AstC/Orcokinin for AstC-EEs, and NPF/Tk/Nplp2/Orcokinin for NPF-EEs. In addition, it was found that EEs may also act in a paracrine manner because NPF-EEs expressed AstC-R2, which can receive signals from AstC-EEs. Finally, it was shown that a subset of EEs expressing NPF and Tk in the middle of the midgut also expressed the esg progenitor marker (Hung, 2020).

This study provides a rich resource to further characterize the molecular signature of each cell type and gene functions in different cell types in homeostatic conditions. Further scRNA-seq of the fly gut will allow a number of questions to be addressed. These include changes in cell states, cell-type composition, and transcriptomes in the context of regeneration, aging, infection, axenic condition, different diet, various mutant backgrounds, and disease models, such as the Yorkie-induced intestine tumor model. In addition to the higher ISC proliferation activity, the female midgut is larger and longer than the male midgut. Hence, it is highly warranted to use scRNA-seq to delineate the gut at physiological and functional levels based on sex differences. Furthermore, during aging, changes in ISC proliferation, regeneration capacity, innate immune and inflammatory response, and tissue integrity occurs, which can be analyzed using scRNA-seq. Taking these data together, it is felt that future scRNA-seq will provide a fundamental understanding of the changes in cell states and interplay among cell types and disease (Hung, 2020).

Regional cell-specific transcriptome mapping reveals regulatory complexity in the adult Drosophila midgut

Deciphering contributions of specific cell types to organ function is experimentally challenging. The Drosophila midgut is a dynamic organ with five morphologically and functionally distinct regions (R1-R5), each composed of multipotent intestinal stem cells (ISCs), progenitor enteroblasts (EBs), enteroendocrine cells (EEs), enterocytes (ECs), and visceral muscle (VM). To characterize cellular specialization and regional function in this organ, RNA-sequencing transcriptomes were generated of all five cell types isolated by FACS from each of the five regions, R1-R5. In doing so, transcriptional diversities were identified among cell types, and regional differences within each cell type were documented that define further specialization. Cell-specific and regional Gal4 drivers were validated; roles for transporter Smvt and transcription factors GATAe, Sna, and Ptx1 in global and regional ISC regulation were demonstrated, and the transcriptional response of midgut cells upon infection was studied. The resulting transcriptome database will foster studies of regionalization, homeostasis, immunity, and cell-cell interactions (Dutta, 2015).

In this analysis, genes highly expressed in ISCs were uncovered and enriched cis-regulatory motifs upstream of these "ISC-high" genes were identified. This approach identified GATAe, sna, Fos (Kayak), and Ptx1 as central regulators of ISC behavior and has paved the way for future studies on more factors that could influence ISC functionality. sna was confirmed as a regional ISC-high gene that regulates stem cell differentiation in the midgut similar to its paralog, escargot (esg). In contrast, GATAe functions in ISC maintenance, much like its mammalian homolog, GATA6, which is involved in the maintenance and proliferation of stem cells and colorectal cancer. Kay (the fly homolog of Fos) and Da had previously been identified as TFs that control ISC activity and fate. Thus, the results indicate that stem cell activity is controlled not by a single factor but by a combinatorial network of autonomously acting TFs such as GATAe, kayak, and sna and previously studied da, esg, and sc, which in concert regulate stemness in the midgut. This dataset could be a useful resource for identifying mammalian homologs with similar functions in stem and cancer cells and in understanding TF regulatory networks in mammals (Dutta, 2015).

The primary source of biotin for Drosophila is dietary yeast and enteric bacteria. Interestingly, this study shows that the biotin transporter Smvt is essential for ISC maintenance and homeostasis, signifying the importance of nutrients derived from symbiotic organisms in regulating intestinal homeostasis and ISC function (Dutta, 2015).

An evolutionarily conserved feature of the gastrointestinal tract is the division of function between specialized regions. This study reveals that all cell types of the Drosophila midgut have profound regional variation in their gene expression. Nevertheless, differences between intestinal cell types were even more pronounced than regional variations within a specific cell type. For all cell types, it was consistently found the cells of regions R1 and R3 to be vastly different from those of the other regions. This suggests that the specialization of all cells in a region is coordinated (Dutta, 2015).

In the gut epithelium proper, differentiated cells showed the most regionalization, with EEs being the most variant between regions. In agreement with recent studies documenting enteroendocrine cell diversity in the midgut, the results indicate that there are multiple subtypes of the hormone secreting EEs that likely have regionalized functions. EBs and ECs also showed clear regional specificities, including altered expression of genes involved in metabolism and digestion. Although lipases, glycoside hydrolases, and glucose transporters were highly expressed in the anterior midgut, serine endopeptidases and amino acid transporters were primarily expressed in the posterior midgut cells, consistent with the premise that digestion is highly compartmentalized (Dutta, 2015).

Remarkably, multipotent ISCs also displayed transcriptional variation along the length of the gut. Different ISC populations differ in the levels of effectors and targets in key signaling pathways, such as EGFR/Ras/MAPK, Wnt, and JAK/STAT, also showing distinct qualitative differences. Of note, ISCs of the acidic R3 express vacuolar H+ ATPases, indicating an adaptation of ISCs to their local environment. The tests showed that the R3-specific TFs Ptx1 and lab are important for maintaining these regional ISC regional properties. Additional regionalized TFs like exex and Drm were identified, and some of these are likely to control other regional properties. While regionalized gene expression almost certainly determines regional differences in cell morphology and function, enteric environment factors (e.g., microbiota and nutrition distributions) within the gut might also influence regional characteristics (Dutta, 2015).

The transcriptome of visceral muscles varied strongly by region, and studies have suggested that regional identities in the gut are maintained by gradients of morphogens. Accordingly, the morphogens Wg, Wnt 4, Wnt 6, WntD, Dpp, and Vn were expressed in gradients in VM cells, suggesting roles for these components of the stem cell niche in defining a region's transcriptional signature. However, it was noticed that spatial expression of lowly expressed ligands like Dpp and Upd3 varied from previous reports, and thus, this dataset should be used with discretion in such cases. Solely based on gene expression data, it cannot be determined whether the ISCs or niche cells such as VMs are primary in establishing and maintaining regionalization. It will be interesting to test whether differences in the niche are driven by gut-extrinsic factors or whether ISCs engineer their own niche through self-reinforcing feedback, for instance, by epigenetic programming of daughter cells. It is hoped that these data will guide future experiments that test the cross-talk between stem cells and the niche in midgut regionalization (Dutta, 2015).

This study uncovered an unexpected role for EEs as potential players in the immune response to pathogens by inducing AMP expression along with ECs and EBs. Interestingly, the different cell types produce different combinations of AMPs, suggesting a specialization by cell type in the immune response, as also found in the mammalian digestive tract. A model is proposed wherein infection either directly activates EEs to express AMPs or the damaged ECs signal ISCs to proliferate and EEs to produce AMPs (see Cell-Type-Specific Responses upon Infection with P. entomophila). Further studies will be required to clearly define the response of EEs to infection (Dutta, 2015).

In conclusion, this study has systematically characterized transcription by genomic analysis of regions and cell types in the Drosophila midgut. Gut regionalization is a critical factor for human health, as diseases of intestinal origin are often regionalized. It is hoped that the provided dataset (http://flygutseq.buchonlab.com/) will help to pave the way for future studies that elucidate the complex interplay among midgut cells, regions, and microbes that will promote understanding of gut physiology and homeostasis (Dutta, 2015).

A core transcriptional network for early mesoderm development

To delineate the combinatorial relationships between Twist and other TFs, an initial transcriptional network was generated for early mesoderm development. The temporal binding map for Twist was integrated with in vivo binding data for Mef2, Dorsal, and Tinman. A previous study of Mef2-bound enhancers offers the largest collection of regulatory regions bound at this stage of development to date. As it is difficult to visualize all 494 Twist target genes, focus was placed on TFs whose CRMs are cobound by two or more regulators during these stages of development. Therefore, all links in this network represent direct connections to the same CRM at the same stages of development (Sandmann, 2007).

The resulting core network of 51 TFs is already relatively complex, with nine genes [nau, E(spl), eve, bap, Ubx, lbe, odd, hth, and Ptx1] being targeted by three out of the four examined regulators. The topology of the network provides several insights into how Twist functions to regulate multiple aspects of early mesoderm development. Extensive combinatorial binding and feed-forward regulation are abundant features. Dorsal activates twist, which in turn coregulates the majority of known direct Dorsal targets. This network motif is even more prominent within the mesoderm: Twist regulates the expression of Mef2 and tinman, and cobinds with these TFs to many of their targets' enhancers. In fact, Twist co-occupies 42% of all Mef2-bound enhancers during early mesoderm development. Depending on the logical inputs from the two upstream regulators (transcriptional repression or activation), feed-forward loops can aid in cellular decision making by filtering out noisy regulatory inputs or control the timing of a transcriptional response. For example, early gene expression in the mesoderm (e.g., activation of tin) depends on Twist alone, while transcription of other genes initiated at a later stage may require the input from both Twist and Tinman proteins (Sandmann, 2007).

Embryonic expression and characterization of a Ptx1 homolog in Drosophila

This study describes the molecular characterization of the paired-type homeobox gene D-Ptx1 of Drosophila, a close homolog of the mouse pituitary homeobox gene Ptx1 and the unc-30 gene of C. elegans, characterized by a lysine residue at position 9 of the third alpha-helix of the homeodomain. D-Ptx1 is expressed at various restricted locations throughout embryogenesis. Initial expression of D-Ptx1 in the posterior-most region of the blastoderm embryo is controlled by fork head activity in response to the activated Ras/Raf signaling pathway. During later stages of embryonic development. D-Ptx1 transcripts and protein accumulate in the posterior portion of the midgut, in the developing Malpighian tubules, in a subset of ventral somatic muscles, and in neural cells. Phenotypic analysis of gain-of-function and lack-of-function mutant embryos show that the D-Ptx1 gene is not involved in morphologically apparent differentiation processes. It is concluded that D-Ptx1 is more likely to control physiological cell functions than pattern formation during Drosophila embryogenesis (Vorbruggen, 1997).

Two highly homologous proteins specifically interact with the LIM domains of P-Lim/Lhx3 and several other LIM homeodomain factors. Transcripts encoding these factors can be detected as early as mouse E8.5, with maximal expression observed in regions of the embryo in which the LIM homeodomain factors P-Lim/Lhx3, Isl-1, and LH-2 are selectively expressed. These proteins can potentiate transactivation by P-Lim/Lhx-3 and are required for a synergistic activation of the glycoprotein hormone alpha-subunit promoter by P-Lim/Lhx3 and a pituitary Otx class homeodomain transcription factor (P-OTX/Ptx1), with which they also specifically associate. The two new genes are referred to as CLIM-1 and CLIM-2 (cofactor of LIM homeodomain proteins). The CLIM proteins are required for a transcriptional synergy between P-Lim/Lhx3 and P-OTX/Ptx1. The fact that CLIM-encoded mRNAs show a widely overlapping expression pattern with Otx1 and Otx2 in the developing mouse brain suggests that the CLIM protein family may play critical roles in the functional relationships of LIM homeoproteins and additional Otx factors (Bach, 1997).

The Bicoid-related homeoprotein Ptx1 defines the most anterior domain of the embryo and differentiates posterior from anterior lateral mesoderm

Ptx1 is a member of the small bicoid family of homeobox-containing genes; it was isolated as a tissue-restricted transcription factor of the pro-opiomelanocortin gene. The homeodomain of Ptx1 contains a lysine at position 9 of the recognition helix (position 60 of the homeodomain). This residue is strategically placed in the major groove of DNA and it is a major determinant of DNA-binding specificity recognizing the CC doublet of the target site. This lysine residue defines the bicoid subfamily of homeoboxes, including Otx1 and 2 and Goosecoid. Ptx1 expression during mouse and chick embryogenesis was determined by in situ hybridization in order to delineate its putative role in development. In the head, Ptx1 expression is first detected in the ectoderm-derived stomodeal epithelium at E8.0. Initially, expression is only present in the stomodeum and in a few cells of the rostroventral foregut endoderm. A day later, Ptx1 mRNA is detected in the epithelium and in a streak of mesenchyme of the first branchial arch, but not in other arches. Ptx1 expression is maintained in all derivatives of these structures, including the epithelia of the tongue, palate, teeth and olfactory system, and in Rathke's pouch. Expression of Ptx1 in craniofacial structures is strikingly complementary to the pattern of goosecoid expression. Gsc labelling in the mandibular component is confined to a central stripe of mesenchyme whereas Ptx1 labelling is observed more laterally (Lanctot, 1997).

Similarly, the epithelium of the first arch, a site of strong Ptx1 expression, is not labelled by the Gsc probe. Ptx1 is expressed early (E6.8) in posterior and extraembryonic mesoderm, and in structures that derive from these. The restriction of expression to the posterior lateral plate is later evidenced by exclusive labelling of the hindlimb but not forelimb mesenchyme. In the anterior domain of expression, the stomodeum is shown by fate mapping to derive from the anterior neural ridge (ANR) which represents the most anterior domain of the embryo. The concordance between these fate maps and the stomodeal pattern of Ptx1 expression supports the hypothesis that Ptx1 defines a stomodeal ectomere that lies anterior to the neuromeres that have been suggested to constitute units of a segmented plan directing head formation. Drosophila Gsc is expressed in the stomodeal invagination, while vertebrate Gsc is not. Based on these gene expression patterns, it is thought that the vertebrate stomodeum is an evolutionary innovation, assuring the ventral placement of the mouth (Lanctot, 1997).

Parallel genetic origin of foot feathering in birds

Understanding the genetic basis of similar phenotypes shared between lineages is a long-lasting research interest. Even though animal evolution offers many examples of parallelism, for many phenotypes little is known about the underlying genes and mutations. This study used a combination of whole-genome sequencing, expression analyses, and comparative genomics to study the parallel genetic origin of ptilopody (Pti) in chicken. Ptilopody (or foot feathering) is a polygenic trait that can be observed in domesticated and wild avian species and is characterized by the partial or complete development of feathers on the ankle and feet. In domesticated birds, ptilopody is easily selected to fixation, though extensive variation in the type and level of feather development is often observed. By means of a genome-wide association analysis, this study identified two genomic regions associated with ptilopody. At one of the loci, a 17 kb deletion affecting PITX1 expression, a gene known to encode a transcription regulator of hindlimb identity and development, was identified. Similarly to pigeon, at the second loci ectopic expression was observed of TBX5, a gene involved in forelimb identity and a key determinant of foot feather development. It was also observed that the trait evolved only once as foot feathered birds share the same haplotype upstream TBX5. These findings indicate that in chicken and pigeon ptilopody is determined by the same set of genes that affect similar molecular pathways. This study confirms that ptilopody has evolved through parallel evolution in chicken and pigeon (Bortoluzzi, 2020).

Bone-specific overexpression of PITX1 induces senile osteoporosis in mice through deficient self-renewal of mesenchymal progenitors and Wnt pathway inhibition

The cellular and molecular mechanisms underlying senile osteoporosis remain poorly understood. In this study, transgenic mCol1alpha1-Pitx1 mice overexpressing paired-like homeodomain 1 (PITX1), a homeobox transcription factor, rapidly develop a severe type-II osteoporotic phenotype with significant reduction in bone mass and biomechanical strength similar to that seen in humans and reminiscent of the phenotype previously observed in Sca-1 (Ly6a)-null mice. PITX1 plays a critical role in hind limb formation during fetal development, while loss of expression is associated with primary knee/hip osteoarthritis in aging humans. Through in vivo and in vitro analyses, this study demonstrated that Pitx1 directly regulates the self-renewal of mesenchymal progenitors and indirectly regulates osteoclast differentiation through the upregulation of Wnt signaling inhibitors DKK1, SOST, and GSK3-beta. This is confirmed by elevated levels of plasma DKK1 and the accumulation of phospho-beta-catenin in transgenic mice osteoblasts. Furthermore, overexpressed Pitx1 in mice osteoblasts results in severe repression of Sca-1 (Ly6a) that was previously associated with senile osteoporosis. This study is the first to demonstrate the novel roles of PITX1 in senile osteoporosis where PITX1 regulates the self-renewal of mesenchymal stem cells or progenitor cells through Sca-1 (Ly6a) repression and, in addition, inhibits the Wnt signaling pathway (Karam, 2019).

De Novo PITX1 expression controls bi-stable transcriptional circuits to govern self-renewal and differentiation in squamous cell carcinoma

Basal tumor propagating cells (TPCs) control squamous cell carcinoma (SCC) growth by self-renewing and differentiating into supra-basal SCC cells, which lack proliferative potential. While transcription factors such as SOX2 and KLF4 can drive these behaviors, their molecular roles and regulatory interactions with each other have remained elusive. This study shows that PITX1 is specifically expressed in TPCs, where it co-localizes with SOX2 and TRP63 and determines cell fate in mouse and human SCC. Combining gene targeting with chromatin immunoprecipitation sequencing (ChIP-seq) and transcriptomic analyses reveals that PITX1 cooperates with SOX2 and TRP63 to sustain an SCC-specific transcriptional feed-forward circuit that maintains TPC-renewal, while inhibiting KLF4 expression and preventing KLF4-dependent differentiation. Conversely, KLF4 represses PITX1, SOX2, and TRP63 expression to prevent TPC expansion. This bi-stable, multi-input network reveals a molecular framework that explains self-renewal, aberrant differentiation, and SCC growth in mice and humans, providing clues for developing differentiation-inducing therapeutic strategies (Sastre-Perona, 2019).

PITX1 promotes chondrogenesis and myogenesis in mouse hindlimbs through conserved regulatory targets

The PITX1 transcription factor is expressed during hindlimb development, where it plays a critical role in directing hindlimb growth and the specification of hindlimb morphology. While it is known that PITX1 regulates hindlimb formation, in part, through activation of the Tbx4 gene, other transcriptional targets remain to be elucidated. This study used a combination of ChIP-seq and RNA-seq to investigate enhancer regions and target genes that are directly regulated by PITX1 in embryonic mouse hindlimbs. In addition, PITX1 binding sites were analyzed in hindlimbs of Anolis lizards to identify ancient PITX1 regulatory targets. This study found that PITX1-bound regions in both mouse and Anolis hindlimbs are strongly associated with genes implicated in limb and skeletal system development. Gene expression analyses reveal a large number of misexpressed genes in the hindlimbs of Pitx1(-/-) mouse embryos. By intersecting misexpressed genes with genes that have neighboring mouse PITX1 binding sites, 440 candidate targets of PITX1 were identified. Of these candidates, 68 exhibit ultra-conserved PITX1 binding events that are shared between mouse and Anolis hindlimbs. Among the ancient targets of PITX1 are important regulators of cartilage and skeletal muscle development, including Sox9 and Six1. These data suggest that PITX1 promotes chondrogenesis and myogenesis in the hindlimb by direct regulation of several key members of the cartilage and muscle transcriptional networks (Wang, 2018).

Molecular shifts in limb identity underlie development of feathered feet in two domestic avian species

Birds display remarkable diversity in the distribution and morphology of scales and feathers on their feet, yet the genetic and developmental mechanisms governing this diversity remain unknown. Domestic pigeons have striking variation in foot feathering within a single species, providing a tractable model to investigate the molecular basis of skin appendage differences. This study found that feathered feet in pigeons result from a partial transformation from hindlimb to forelimb identity mediated by cis-regulatory changes in the genes encoding the hindlimb-specific transcription factor Pitx1 and forelimb-specific transcription factor Tbx5. This study also found that ectopic expression of Tbx5 is associated with foot feathers in chickens, suggesting similar molecular pathways underlie phenotypic convergence between these two species. These results show how changes in expression of regional patterning genes can generate localized changes in organ fate and morphology, and provide viable molecular mechanisms for diversity in hindlimb scale and feather distribution (Domyan, 2016).

Adaptive evolution of pelvic reduction in sticklebacks by recurrent deletion of a Pitx1 enhancer

The molecular mechanisms underlying major phenotypic changes that have evolved repeatedly in nature are generally unknown. Pelvic loss in different natural populations of threespine stickleback fish has occurred through regulatory mutations deleting a tissue-specific enhancer of the Pituitary homeobox transcription factor 1 (Pitx1) gene. The high prevalence of deletion mutations at Pitx1 may be influenced by inherent structural features of the locus. Although Pitx1 null mutations are lethal in laboratory animals, Pitx1 regulatory mutations show molecular signatures of positive selection in pelvic-reduced populations. These studies illustrate how major expression and morphological changes can arise from single mutational leaps in natural populations, producing new adaptive alleles via recurrent regulatory alterations in a key developmental control gene (Chan, 2010).

Mesenchymal patterning by Hoxa2 requires blocking Fgf-dependent activation of Ptx1

Hox genes are known key regulators of embryonic segmental identity, but little is known about the mechanisms of their action. To address this issue, how Hoxa2 specifies segmental identity in the second branchial arch was analyzed. Using a subtraction approach, it was found that Ptx1 is upregulated in the second arch mesenchyme of Hoxa2 mutants. This upregulation has functional significance because, in Hoxa2^-/-;Ptx1^-/- embryos, the Hoxa2^-/- phenotype is partially reversed. Hoxa2 interferes with the Ptx1 activating process, which is dependent on Fgf signals from the epithelium. Consistently, Lhx6, another target of Fgf8 signaling, is also upregulated in the Hoxa2^-/- second arch mesenchyme. These findings have important implications for the understanding of developmental processes in the branchial area and suggest a novel mechanism for mesenchymal patterning by Hox genes that acts to define the competence of mesenchymal cells to respond to skeletogenic signals (Bobola, 2003).

Pituitary homeobox 1 activates the rat FSHbeta (rFSHbeta) gene through both direct and indirect interactions with the rFSHbeta gene promoter

Molecular mechanisms underlying gonadotrope-specific and hormonal regulation of FSHbeta gene expression remain largely unknown. The role of pituitary homeobox 1 (Ptx1), a transcription factor important for regulation of many pituitary-specific genes, was studied in the regulation of rat FSHbeta (rFSHbeta) gene transcription. Ptx1 was shown to activates the rFSHbeta gene promoter both basally and in synergy with GnRH. The effect of Ptx1 was localized to -140/-50, a region also important for basal activity of the promoter. Two putative Ptx1 binding sites (P1 and P2) homologous to consensus Ptx1 binding elements were identified in this region. Specific binding of Ptx1 was demonstrated to the P2 but not to the P1 site. Furthermore, functional studies indicate that the P2 but not the P1 site mediates activation of the promoter by Ptx1. Residual activation of the promoter by Ptx1 was observed independent of the P2 site. However, no additional Ptx1 binding sites were identified in this region, indicating that the residual activation observed is likely independent of direct Ptx1 binding to the promoter. These results identify a functional Ptx1 binding site in the rFSHbeta gene promoter and suggest the presence of an additional activating pathway that is independent of direct binding of Ptx1 to the promoter (Zakaria, 2002).

The pan-pituitary activator of transcription, Ptx1 (pituitary homeobox 1), acts in synergy with SF-1 and Pit1 and is an upstream regulator of the Lim-homeodomain gene Lim3/Lhx3

The Ptx1 (pituitary homeobox 1) homeobox transcription factor is a transcription factor of the pituitary POMC gene. In corticotrope cells that express POMC, cell-specific transcription is conferred in part by the synergistic action of Ptx1 with the basic helix-loop-helix factor NeuroD1. Since Ptx1 expression precedes pituitary development and differentiation, its expression and function was examined in other pituitary lineages. Ptx1 is expressed in most pituitary-derived cell lines as is the related Ptx2 (Rieger) gene. However, Ptx1 appears to be the only Ptx protein in corticotropes and the predominant one in gonadotrope cells. Most pituitary hormone-coding gene promoters are activated by Ptx1. Thus, Ptx1 appears to be a general regulator of pituitary-specific transcription. In addition, Ptx1 action is synergized by cell-restricted transcription factors to confer promoter-specific expression. Indeed, in the somatolactotrope lineage, synergism between Ptx1 and Pit1 is observed on the PRL promoter, and strong synergism between Ptx1 and SF-1 is observed in gonadotrope cells on the betaLH promoter but not on the alphaGSU (glycoprotein hormone alpha-subunit gene) and betaFSH promoters. Synergism between these two classes of factors is reminiscent of the interaction between the products of the Drosophila genes ftz (fushi tarazu) and ftz-F1. Antisense RNA experiments performed in alphaT3-1 cells that express the alphaGSU gene show that expression of endogenous alphaGSU is highly dependent on Ptx1, whereas many other genes are not affected. Interestingly, the only other gene found to be highly dependent on Ptx1 for expression is the gene for the Lim3/Lhx3 transcription factor. Thus, these experiments place Ptx1 upstream of Lim3/Lhx3 in a cascade of regulators that appear to work in a combinatorial code to direct pituitary-, lineage-, and promoter-specific transcription (Tremblay, 1998).

Search PubMed for articles about Drosophila Ptx1

Bach, I., et al. (1997). A family of LIM domain-associated cofactors confer transcriptional synergism between LIM and Otx homeodomain proteins. Genes Dev. 11(11): 1370-1380. PubMed Citation: 9192866

Bobola, N., et al. (2003). Mesenchymal patterning by Hoxa2 requires blocking Fgf-dependent activation of Ptx1. Development 130: 3403-3414. 12810588

Bortoluzzi, C., Megen, S. H., Bosse, M., Derks, M. F. L., Dibbits, B., Laport, K., Weigend, S., Groenen, M. A. M. and Crooijmans, R. (2020). Parallel genetic origin of foot feathering in birds. Mol Biol Evol. PubMed ID: 32344429

Chan, Y. F., Marks, M. E., Jones, F. C., Villarreal, G., Jr., Shapiro, M. D., Brady, S. D., Southwick, A. M., Absher, D. M., Grimwood, J., Schmutz, J., Myers, R. M., Petrov, D., Jonsson, B., Schluter, D., Bell, M. A. and Kingsley, D. M. (2010). Adaptive evolution of pelvic reduction in sticklebacks by recurrent deletion of a Pitx1 enhancer. Science 327(5963): 302-305. PubMed ID: 20007865

Domyan, E. T., Kronenberg, Z., Infante, C. R., Vickrey, A. I., Stringham, S. A., Bruders, R., Guernsey, M. W., Park, S., Payne, J., Beckstead, R. B., Kardon, G., Menke, D. B., Yandell, M. and Shapiro, M. D. (2016). Molecular shifts in limb identity underlie development of feathered feet in two domestic avian species. Elife 5: e12115. PubMed ID: 26977633

Dutta, D., Dobson, A. J., Houtz, P. L., Glasser, C., Revah, J., Korzelius, J., Patel, P. H., Edgar, B. A. and Buchon, N. (2015). Regional cell-specific transcriptome mapping reveals regulatory complexity in the adult Drosophila midgut. Cell Rep 12: 346-358. PubMed ID: 26146076

Guo, X., Yin, C., Yang, F., Zhang, Y., Huang, H., Wang, J., Deng, B., Cai, T., Rao, Y. and Xi, R. (2019). The cellular diversity and transcription factor code of Drosophila enteroendocrine cells. Cell Rep 29(12): 4172-4185. PubMed ID: 31851941

Hung, R. J., Hu, Y., Kirchner, R., Liu, Y., Xu, C., Comjean, A., Tattikota, S. G., Li, F., Song, W., Ho Sui, S. and Perrimon, N. (2020). A cell atlas of the adult Drosophila midgut. Proc Natl Acad Sci U S A 117(3): 1514-1523. PubMed ID: 31915294

Karam, N., Lavoie, J. F., St-Jacques, B., Bouhanik, S., Franco, A., Ladoul, N. and Moreau, A. (2019). Bone-specific overexpression of PITX1 induces senile osteoporosis in mice through deficient self-renewal of mesenchymal progenitors and Wnt pathway inhibition. Sci Rep 9(1): 3544. PubMed ID: 30837642

Lanctot, C., Lamolet, B. and Drouin, J. (1997). The bicoid-related homeoprotein Ptx1 defines the most anterior domain of the embryo and differentiates posterior from anterior lateral mesoderm. Development 124(14): 2807-2817. PubMed ID: 9226452

Sandmann, T., et al. (2007). A core transcriptional network for early mesoderm development in Drosophila melanogaster. Genes Dev. 21: 436-449. PubMed ID: 17322403

Sastre-Perona, A., Hoang-Phou, S., Leitner, M. C., Okuniewska, M., Meehan, S. and Schober, M. (2019). De novo PITX1 expression controls bi-stable transcriptional circuits to govern self-renewal and differentiation in squamous cell carcinoma. Cell Stem Cell 24(3): 390-404 e398. PubMed ID: 30713093

Tremblay, J. J., Lanctot, C. and Drouin, J. (1998). The pan-pituitary activator of transcription, Ptx1 (pituitary homeobox 1), acts in synergy with SF-1 and Pit1 and is an upstream regulator of the Lim-homeodomain gene Lim3/Lhx3. Mol Endocrinol 12(3): 428-441. PubMed ID: 9514159

Vorbruggen, G., Constien, R., Zilian, O., Wimmer, E. A., Dowe, G., Taubert, H., Noll, M. and Jackle, H. (1997). Embryonic expression and characterization of a Ptx1 homolog in Drosophila. Mech Dev 68(1-2): 139-147. PubMed ID: 9431811

Wang, J. S., Infante, C. R., Park, S. and Menke, D. B. (2018). PITX1 promotes chondrogenesis and myogenesis in mouse hindlimbs through conserved regulatory targets. Dev Biol 434(1): 186-195. PubMed ID: 29273440

Zakaria, M. M., Jeong, K. H., Lacza, C. and Kaiser, U. B. (2002). Pituitary homeobox 1 activates the rat FSHbeta (rFSHbeta) gene through both direct and indirect interactions with the rFSHbeta gene promoter. Mol Endocrinol 16(8): 1840-1852. PubMed ID: 12145338

date revised: 13 December 2023

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