BIOLOGICAL OVERVIEW

The eye is composed of dorsal and ventral fields of photoreceptor clusters called ommatidia. Ommatidia in the dorsal half of the eye are the mirror image of those in the ventral half. mirror (mrr), a component of the iroquois locus that also includes araucan and caupolican, is expressed in the dorsal half of the eye imaginal disc; as such it is a primary determiner of the border between dorsal and ventral halves of the eye (see Specification of the eye disc primordium and establishment of dorsal/ventral asymmetry) and has a marked effect on eye symmetry (McNeill, 1997).

The boundary where dorsal and ventral fields meet is known as the equator. The equator bisects the eye from anterior to posterior, rarely deviating by more than one ommatidial width as it crosses the eye. Preclusters (the progenitors of ommatidia) rotate in opposite directions as they mature in dorsal and ventral regions. Asymmetries are incorporated into the preclusters as they rotate, resulting in ommatidia of opposing polarity and chirality (handedness) in the adult eye. Recent work has demonstrated that the degree of ommatidial rotation is controlled by several genes, including nemo and roulette, and has suggested that tissue polarity genes such as spinylegs, prickly-spinylegs, frizzled, and dishevelled are necessary for regulating DV polarity in the eye (McNeill, 1997 and references).

The equator might be the source of D/V patterning information. For example, frizzled is a transmembrane protein required to interpret and relay a signal emitted by the equator (Zheng, 1995). A number of genes appear to be expressed either solely at the equator or in a gradient from the equator. Four-jointed, either a cell surface or a secreted protein, is expressed in a graded fashion around the equator; mutants show a variable deformation of the eye surface. Morphological analysis of normal eyes indicates that ommatidial differentiation in each row proceeds from the equator onward. It seems that more mature ommatidia provide D/V polarity information to their less differentiated neighbors (McNeill, 1997 and references).

The iroquois locus was initially defined by iroquois mutation, isolated in a search for mutations that alter the pattern of macrochaetae (Leyns, 1995). Two transcriptions units, araucan and caupolican, were initially detected within the iroquois locus The proteins possess homeodomains similar to those of Drosophila Extradenticle and mammalian Pbx1 (Gómez-Skarmeta, 1996). Subsequently, the gene mirror was identified, closely linked to ara and caup. mrr was identified using a P element insertion element that carried two markers, white and lacZ, whose expression is restricted to cells in the dorsal half of the eye. The expression patterns of P element vectors is determined by the regulatory elements of the gene into which the vector inserts. Thus the expression pattern of white and lacZ potentially marks a promoter directing expression of a gene to the dorsal half of the eye. Mirror also proves to be a homeoprotein, and is the third PBX-class homeoprotein of the IROC complex (McNeill, 1997).

Since strong mirror mutants show an embryonic or early larval lethality, the function of mirror in the eye was examined by generating loss-of-function clones. Loss of mirror function has different effects in different parts of the eye. There are no effects in the ventral half, consistent with the observation that mirror expression is restricted to the dorsal half of the eye. Dorsal clones also have no significant differences in ommatidial polarity or chirality within the clone. These results suggest that mirror is not required for the implementation of dorsal identity. There is, however, a dramatic alteration in patterning at the border of certain clones. Ectopic equators form at the equatorial borders of dorsal anterior clones where mirror-minus tissue abuts mirror-expressing tissue. The wild-type ommatidia just outside the clone adopt ventral polarity and chirality. Ventral patterning extends for one to two ommatidial widths, then the tissue resumes normal dorsal patterning. The formation of ectopic equators where mirror-expressing and -nonexpressing cells meet suggests that the juxtaposition of mirror-expressing and -nonexpressing cells serves to define the normal equator (McNeill, 1997).

An examination of mirror-minus clones that cross the normal equator reveals that where the equator approaches a patch of mirror-minus tissue, the path of the equator is diverted to follow the new border of mirror expression. After taking several dorsal steps paralleling the border of mirror-expressing and -nonexpressing cells, the equator resumes its posterior to anterior path across the eye rather than continuing to follow the edge of the clone. This result suggests that the pattern of mirror expression is not the sole determinant of equator position and that there may be spatial restrictions on the ability of mirror to define the equator (McNeill, 1997).

The mechanism of D/V boundary determination in the wing may provide a model for understanding equator formation in the eye. Dorsal identity in the wing is controlled by the homeodomain protein Apterous, which is expressed solely by dorsal cells. The juxtaposition of Apterous-expressing and -nonexpressing cells defines the D/V border in part by directing dorsal expression of the secreted protein Fringe. Fringe then directs patterning at the D/V border in part by controlling the Notch pathway. It will be interesting to see if Apterous and mirror are found to control some of the same genes (McNeill, 1997).

Mirror also is involved in segmentation. The onset of mirror expression in each segment comes after the establishment of parasegmental signaling, but prior to and during the determination of the segmental border and the refinement of Engrailed and Wingless expression. As the border between Engrailed- and Wingless-expressing cells determines the parasegmental boundary, so might the juxtapositioning of mirror-expressing and -nonexpressing cells be important for defining the segmental border. Consistent with this hypothesis is the obervation that mirror mutant embryos have segmental patterning defects. mirror might therefore function both in the eye and during segmentation to define borders (McNeill, 1997).

The Mirror transcription factor links signalling pathways in Drosophila oogenesis

Many genetic cascades trigger different responses and hence determine different cell fates at specific times and positions in development. At stage 10 of oogenesis, mirror is expressed in anterior-dorsal follicle cells, and this is dependent upon the Gurken signal from the oocyte. The fringe gene is expressed in a complementary pattern in posterior-ventral follicle cells at the same stage. Ectopic expression of mirror represses fringe expression, thus linking the epidermal growth factor receptor (Egfr) signaling pathway to the Fringe signaling pathway via Mirror. The Egfr pathway also triggers the cascade that leads to dorsal-ventral axis determination in the embryo. twist was used as an embryonic marker for ventral cells. Ectopic expression of mirror in the follicle cells during oogenesis ultimately represses twist expression in the embryo, and leads to phenotypes similar to those that occur due to the ectopic expression of the activated form of Egfr. Thus, mirror also controls the Toll signaling pathway, leading to Dorsal nuclear transport. In summary, the Mirror homeodomain protein provides a link that coordinates the Gurken/Egfr signaling pathway (initiated in the oocyte) with the Fringe/Notch/Delta pathway (in follicle cells). This coordination is required for epithelial morphogenesis, and for producing the signal in ventral follicle cells that determines the dorsal/ventral axis of the embryo (Zhao, 2000).

mirror is expressed in a dynamic pattern during oogenesis. This includes expression in the germarium and in centrally-located follicle cells at stage 6 and the anterior-dorsal and centripetal follicle cells at stage 10. This paper concentrates on the regulation and function of mirr expression at stage 10 of oogenesis. At this time, mirr is expressed in the cells in the follicular epithelium that receive the Grk signal. This signal is known to be required in these cells to establish the dorsal-ventral axis of the egg and embryo. In grk-mutant ovaries mirr expression disappears or is occasionally reduced to a small group of follicle cells at the dorsal midline. In fs(1)k10 mutant ovaries, where grk transcripts diffuse from anterior-dorsal towards anterior-ventral within the oocyte at stage 9, all anterior follicle cells receive the Grk signal and an expanded expression of mirr is observed. Thus mirr expression is downstream of the Grk signal, and those cells that have activated Egfr signaling at this stage of oogenesis appear to respond by activating this homeodomain transcription factor. It is still not clear whether the early expression of mirr in the germarium requires the Grk/Egfr signaling. Grk/Egfr signaling activity does not always activate the mirr gene, since mirr is not expressed in posterior follicle cells at stage 7 of oogenesis, when grk signaling occurs at the posterior of the egg chamber. Other factors must, therefore, determine the way in which follicle cells, in different positions and with different developmental histories, respond to the grk signal (Zhao, 2000).

fringe (fng), which encodes a glycosyltransferase-like secreted protein, is involved in different developmental processes, such as the development of the wing and the eye and oogenesis. Its expression at stage 10 of oogenesis is restricted to the ventral and posterior follicle cells, and it is not observed in those cells that express mirr. So the expression patterns of mirr and fng are complementary, and both these patterns are defined by the position of Egfr activation in response to the Grk signal (Zhao, 2000).

To test whether the complementary expression of mirr and fng depend upon Egfr signaling at stage 10, their expression patterns were examined in flies expressing activated or dominant negative forms of Egfr. When the activated form of Egfr (DER^AF or lambdatop) is expressed in the anterior follicle cells, all those cells now express mirr, but not fng. When the dominant negative form of Egfr (DER ^DN) is expressed in those follicle cells surrounding the oocyte, but not the centripetal follicle cells, the anterior-dorsal follicle cells no longer express mirr. The expression domain of fng expands to include the anterior-dorsal cells in these mutants. Thus, the expression of both mirr and fng are either positively or negatively regulated by the activation of Egfr, and a complementary expression pattern is maintained in all these experiments. Experiments show that Grk/Egfr signaling is required to activate the expression of mirr in anterior-dorsal and centripetal follicle cells, which in turn represses the expression of fng in those cells. As a result, fng is only expressed in the posterior and ventral follicle cells, where it is required for the normal morphogenesis of the follicle cell layer (Zhao, 2000).

fng expression is restricted to specific regions of wing discs, eye discs and follicle cells of the egg chamber. The repression of fng expression in anterior-dorsal follicle cells in wild-type egg chambers by mirr possibly uses a similar mechanism to that observed in eye development, where it is required for the modulation of the dorsal-ventral boundary established by Notch activation. As a secreted protein, Fng modulates the binding of Notch to its ligands at the dorsal-ventral boundary. Ectopic expression of fng induces new dorsal-ventral boundaries in the wing disc and can reverse the planar polarity of photoreceptor clusters in the eye disc. Interfering with fng expression using antisense RNA experiments and mitotic clones, causes abnormalities in epithelial development in the egg chamber and defects in the positioning of the chorionic appendages. Vertebrate Fng homologs are similarly involved in mediating the signals between dorsal and ventral cells during limb development. These findings suggest that the boundary between fng-expressing and non-expressing cells is important in pattern formation, and the restricted expression pattern is regulated by mirr, at least in eye development and oogenesis in Drosophila. In this way, mirr is controlling epithelial morphogenesis via fringe and possibly other targets. As yet it is not known whether mirr directly represses fng transcription or whether there are other genes in the pathway between mirr and fng (Zhao, 2000).

fng affects eggshell patterning. The Gal4/UAS system was used to misexpress mirr, and this results in abnormalities of the chorion. When mirr is ectopically expressed at low levels in follicle cells surrounding the oocyte, there are enlargements of the dorsal appendages in the eggs, some of which (2%) become multiple pseudo-dorsal appendages. During oogenesis, when the ectopic expression of mirr is driven by the Gal4 line C710, 95% of eggs laid by these females have no chorion, suggesting that ectopic expression of mirr can repress the expression of the genes in the ventral cells needed for secretion of the chorion over the ventral regions of the egg. Thus mirror is needed for the correct dorso-ventral patterning of the eggshell which is secreted by the follicle cells. How much of this is mediated via fng, and how much through other target genes and pathways is not known (Zhao, 2000).

Eggshell polarity and embryonic polarity are closely integrated and grk signaling initiates both these processes. Some genes downstream of the grk signal affect patterning in both the eggshell and embryo, e.g. Egfr, while others just affect the eggshell, e.g. BR-C. Therefore, it was asked whether mirr also affects patterning in the embryo. Both patterning events can be monitored in individual eggs by observing the phenotype of the egg chorion, which is secreted by the follicle epithelial layer, and by the cuticle pattern of the resulting embryo. In those eggs with overgrowths of the dorsal appendage, induced by misexpression of mirr, the denticles of the resulting embryos are partially or completely lost, showing a dorsalized phenotype. This suggests mirr affects both patterning systems (Zhao, 2000).

To further investigate how the dorsal-ventral polarity of embryos is affected by ectopic expression of mirr, expression of the twist (twi) gene was used as a marker for ventral embryonic cells. After Egfr activation in anterior dorsal follicle cells, 11 dorsal-group maternal genes are later involved in the establishment of a gradient of nuclear Dorsal (DL) protein in the ventral cells of the embryo. This in turn activates the expression of twi in the whole ventral domain of the wild-type embryo. In those eggs laid by females with ectopic expression of mirr driven by the Gal4 line T155, the central expression domain of twi is reduced and in some embryos completely disappears. The terminal domains of twi at both ends of the embryos are rarely affected. Similar phenotypes are observed when DER^AF is ectopically expressed, but the frequencies of abnormalities are very different. Of the eggs laid, 98% have multiple dorsal appendages, rather than the 2% observed following ectopic expression of mirr. Also, most eggs lose the main domain of twi expression following ectopic expression of DER^AF. The terminal regions of twi expression still remain, using this Gal 4 driver for DER^AF (Zhao, 2000).

Further evidence that mirr is responsible for the repression of ventral genes that direct the dorsal-ventral polarity of the embryo comes from experiments in which mirr is ectopically expressed in the posterior of the egg chamber. Ectopic expression of DER^AF in this region causes the loss of both posterior embryonic segments and the posterior twi expression domain. When mirr is ectopically expressed in the posterior follicle cells, a similar phenotype is observed, with both posterior embryonic denticle belts and the twi expression domain missing. This clearly shows that mirr function is critical for the dorsal-ventral axis of the embryo and that ectopic expression of mirr can lead to an abnormal localization of the ventral signal, which is required to trigger the initiation of the embryonic ventral pattern (Zhao, 2000).

One of the dorsal group of maternal genes, pipe, is negatively regulated by mirr since pipe expression is expanded in mirr minus clones. Since pipe is required for the activation of twi via Dorsal, it is likely that mirr affects twi expression by repressing the expression of pipe in dorsal follicle cells. The downstream targets of mirr, in addition to pipe, need to be further identified to understand how mirr executes these developmental decisions in response to Grk/Egfr signaling. It is possible that mirr is required for the repression of pipe and windbeutel (wind) along with fng in the anterior-dorsal follicle cells. The ventrally-localised activities of these genes then cooperatively generate an extracellular ventral signal. There is evidence that CF2, which is expressed in the ventral and posterior cells in a pattern similar to fringe, regulates pipe and wind, and is responsible for the ventral signal produced in follicle cells that determines the embryonic axis. One might speculate that mirr could repress CF2 in anterior/dorsal follicle cells. However, it is observed that CF2 transcripts are expressed in the mirr expressing cells, and the ventrally-localized protein distribution of CF2 is translationally regulated. Since mirr encodes a transcription factor it cannot be directly responsible for the lack of CF2 protein in anterior/dorsal follicle cells, but could indirectly cause a repression of its translation in the anterior-dorsal cells via another gene. Alternatively CF2 protein could be upstream of mirror expression and repress transcription in ventral cells (Zhao, 2000).

When the mirr mutant phenotype was examined, some unusual results were found, namely that one or two mirr alleles affect only the eggshell, but not the embryonic cuticle, when heterozygous or heteroallelic. All mirr mutant alleles are homozygous lethal, therefore homozygous mutant adults cannot be obtained for analysis. One of the mirr alleles (mirr^P1), when heterozygous, balanced with TM3 (mirr^P1/TM3), lays either eggs with no dorsal appendages or with a pair of very tiny dorsal appendages, thus showing that some mirr alleles can have either dominant effects on the egg-shell or they interact with other genes in this genetic background. Since this phenotype is not observed in mirror/+, the latter is more likely. Other stocks carrying this TM3 balancer do not show this phenotype under similar conditions, and TM3/TM6 females also lay eggs with normal chorions. Regardless of how this is controlled, the heterozygous mirr^P1/TM3 females show reduced expression of mirr and expanded expression of fng in some egg chambers at stage 10 of oogenesis, and lay eggs showing the normal expression pattern of twi. The resulting embryos have normal denticle patterns, and 99% hatch. Thus the mirr P1 allele in a TM3 background affects epithelial morphogenesis, but not embryogenesis. To test further the effects of mirr mutants on eggshell and embryonic development, different genetic combinations of mirr mutants were investigated. Some heteroallelic combinations are lethal, but others generate some adult females. It is suggested that the effect of mirr on the eggshell and embryo can be separated and this either involves mirr functioning separately in two different genetic pathways, and/or different sensitivities to the level of mirr expression for these two functions. The possibility that there are background mutations in the mirr stocks causing the eggshell phenotype cannot be ruled out. However, the possibility that mirr has more than one function is supported by the recent finding that mirr regulates equator formation in eye development by two mechanisms; creating a boundary of fng expression, and reducing the mixing of dorsal and ventral cells at the equator (Zhao, 2000).

In summary, mirr functions as a transcription factor linking the Grk/Egfr signaling in oogenesis to the formation of the dorsal-ventral axis of the egg chamber and eggshell, by modulating the Fringe/Notch/Delta pathway, and by affecting the establishment of the signal from ventral cells to set up the embryonic dorsal-ventral axis. mirr expression in anterior-dorsal follicle cells is activated by Grk/Egfr signaling at stage 9-10 of oogenesis. It then represses the expression in dorsal follicle cells of those genes that are required only in ventral follicle cells. The activity of these genes in the ventral follicle cells are required either for the formation of the eggshell and its dorsal-ventral pattern, or for the initiation of dorsal-ventral axis formation in embryogenesis (Zhao, 2000).

The cellular diversity and transcription factor code of Drosophila enteroendocrine cells

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, 2018). 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).

GENE STRUCTURE

araucan and caupolican are transcribed in the same direction on the chromosome and are separated by 12 kb. mirror is located in chromosomal region 69D, proximal to caupolican, that is, closer to the centromere (Gómez-Skarmeta, 1996).

Transcript size - 3.5 kb

Exons - 6

PROTEIN STRUCTURE

Amino Acids - 641

Structural Domains

The homeodomains most similar to those of Mirror, Araucan and Caupolican have been found in the human sequence R46202 and in several mouse proteins. The next most similar homeodomains, those of the human Pbx and Drosophila Exd proteins, have only 37% identity. The Mrr homeodomain contains 3 additional amino acids between helix 1 and 2 that are not present in most homeodomains but are present in PBX1 and Exd. Mirror, Ara, and Caup homeodomains are nearly identical. The homeodomain of Drosophila Mirror (McNeill, 1997) has 57 out of 60 amino acids identical to the homeodomains of Caup and Ara. In the amino-terminal domain, these proteins have a highly conserved Notch interaction domain, which has been proposed to be involved in protein-protein interactions. The Notch interaction domain of the homeodomain proteins is homologous to a stretch of amino acids in Xenopus, rat and Drosophila Notch. This putative protein interaction domain is similar to the central part of the epidermal growth factor repeats of the Notch protein. C-terminal to the novel homology region are two potential phosphorylation sites for mitogen-activated kinase (Rolled in Drosophila). Mirror also possesses an acidic activation domain just C-terminal to the homeodomain (Gómez-Skarmeta, 1996 and McNeill, 1997).

date revised: 26 MAY 97 

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