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A single-cell atlas of the sexually dimorphic Drosophila foreleg and its sensory organs during development [1]

['Ben R. Hopkins', 'Department Of Evolution', 'Ecology', 'University Of California', 'Davis', 'California', 'United States Of America', 'Olga Barmina', 'Artyom Kopp']

Date: 2023-07

To respond to the world around them, animals rely on the input of a network of sensory organs distributed throughout the body. Distinct classes of sensory organs are specialized for the detection of specific stimuli such as strain, pressure, or taste. The features that underlie this specialization relate both to the neurons that innervate sensory organs and the accessory cells they comprise. To understand the genetic basis of this diversity of cell types, both within and between sensory organs, we performed single-cell RNA sequencing on the first tarsal segment of the male Drosophila melanogaster foreleg during pupal development. This tissue displays a wide variety of functionally and structurally distinct sensory organs, including campaniform sensilla, mechanosensory bristles, and chemosensory taste bristles, as well as the sex comb, a recently evolved male-specific structure. In this study, we characterize the cellular landscape in which the sensory organs reside, identify a novel cell type that contributes to the construction of the neural lamella, and resolve the transcriptomic differences among support cells within and between sensory organs. We identify the genes that distinguish between mechanosensory and chemosensory neurons, resolve a combinatorial transcription factor code that defines 4 distinct classes of gustatory neurons and several types of mechanosensory neurons, and match the expression of sensory receptor genes to specific neuron classes. Collectively, our work identifies core genetic features of a variety of sensory organs and provides a rich, annotated resource for studying their development and function.

Here, we use scRNA-seq to profile the sensory organs of the male D. melanogaster foreleg at 2 developmental time points that follow soon after the specification of sensory organ cells (24 h and 30 h APF) [ 11 ]. Using a fine-scale dissection technique, we specifically target the first tarsal segment (the “basitarsus”) to maximize the detection of rare sensory organ types, including the campaniform sensilla, chemosensory taste bristles, and sex comb teeth. We begin by examining the transcriptomic landscape of the tissues in which the sensory organs reside, constructing a spatial reference map of epithelial cells based on intersecting axes of positional marker expression, resolving joint-specific gene expression, and characterizing the distinct repertoires of expressed genes in tendon cells, hemocytes, and bract cells. We then focus on the nonneuronal component of the nervous system, describing the complement of glial cells present in the region, identifying and visualizing wrapping glia, surface glia, and a novel axon-associated cell population that is negative for the canonical glia marker repo and appears to contribute toward the construction of the neural lamella. We then resolve and validate a combinatorial transcription factor code unique to the neurons of each of mechanosensory bristles, campaniform sensilla, chordotonal organs, and the sex comb. We further identify and validate a transcription factor code unique to 4 transcriptomically distinct GRN classes, including known male- and female-pheromone sensing neurons, and recover this same code in a published adult leg dataset. With these annotations in place, we link a wide range of genes, including receptors and membrane channels, to specific neuron classes. Finally, we detail the transcriptomic differences that distinguish between sensory organ support cells, both within a single organ class (e.g., sheaths versus sockets) and between classes (e.g., chemosensory sheaths versus mechanosensory sheaths).

High-throughput single-cell RNA sequencing (scRNA-seq) technologies allow for the transcriptional profiles of many thousands of cells to be recorded from a single tissue. The advent of these technologies has precipitated an explosion of interest in cell type–specific patterns of gene expression. Over the last few years, “atlases” describing the cellular diversity of tissues [ 29 – 32 ], embryos (e.g., [ 33 – 35 ]), and whole adult animals (e.g., [ 36 – 38 ]) have been published for a variety of species. Through such work, regulators of development have been identified (e.g., [ 39 , 40 ]), novel cell types described (e.g., [ 41 , 42 ]), and the effects of age (e.g., [ 43 , 44 ]) and infection (e.g., [ 45 , 46 ]) on the gene expression profiles of individual cell types characterized. But in arthropods, tissues associated with the cuticle have presented a challenge to single-cell approaches because the cuticle prevents isolation of single cells from peripheral tissues without significant damage [ 38 , 47 ]. Although single-nuclei RNA-seq methods have been used in such tissues (e.g., [ 38 ]), scRNA-seq approaches generally offer substantially greater read and gene detection with reduced “gene dropout” and lower expression variability between cells [ 48 ]. Consequently, approaches that help characterize the transcriptomes of cuticle-associated cells are of significant value. In principle, the developmental window between pupa–adult apolysis—the separation of the pupal cuticle from the epidermis—and the formation of the adult cuticle (approximately 12 to 48 h after puparium formation (APF)) [ 49 ] provides a rare opportunity during which such cells might be accessible.

The function of sensory organs and their tuning to particular stimuli is not only a product of the neurons that innervate them. In each case, the sensory organ is a composite of multiple distinct cell types and dependent upon the involvement of glia to effectively relay detected signals to the brain. Different organ classes appear to share a common developmental blueprint, such that, despite variation in their form and function, mechanosensory bristles, chemosensory taste bristles, and campaniform sensilla each contain 4 homologous cell types [ 23 ]. In the bristle lineage, these are the neurons, which may vary in number between different sensory organ classes (such as between the polyinnervated chemosensory and monoinnervated mechanosensory bristles), along with 3 sensory support cells: the trichogen (shaft or, in campaniform sensilla, the dome), tormogen (socket), and thecogen (sheath). These sensory support cells bear features that clearly define the sensory capabilities of the organ. For example, the elongated shafts of mechanosensory bristles support the deflection-based mechanism through which stimuli are detected [ 10 ], the pore at the tip of chemosensory taste bristles enables the receipt of nonvolatile compounds [ 18 ], and the elliptical shape of many campaniform sensilla confers sensitivity to the direction of cuticular compression and strain ([ 24 ]; reviewed by [ 25 ]). But beyond these morphological features, our understanding of the wider, organ-specific contributions that support cells make to the specific sensory capabilities of each organ class remains poor [ 26 ]. Yet there is clear potential for their broader involvement in defining an organ type’s capabilities, given both their close physical associations with the neurons and, at least in taste bristles, their role in producing the lymph fluid that bathes the dendrites of GRNs and which is central to tastant detection [ 27 , 28 ].

(A) Anatomy of the Drosophila melanogaster foreleg. The first tarsal segment (ta1), the focal region of this study, is distal to the tibia. Two chordotonal organs (COs) are present outside of the tarsal segments (approximate positions shown in purple). One is situated in the proximal femur (FeCO) and the other in the distal tibia (tCO) [ 4 , 5 ]. (B) The ta1 of the D. melanogaster foreleg is enriched for a range of functionally and structurally diverse sensory organs. This region has the highest concentration of mechanosensory bristles of any part of the leg. Here, mechanosensory bristles are arranged in transverse rows on the ventral side, an arrangement thought to aid in grooming, and longitudinal rows on the anterior, dorsal, and posterior sides [ 9 ]. In males, the most distal transverse bristle row is transformed into the sex comb: The mechanosensory bristles, now “teeth,” are modified to be thicker, longer, blunter, and more heavily melanized, while the whole row is rotated 90° [ 11 , 12 ]. Males also show a sex-specific increase in the number of chemosensory taste bristles in ta1, bearing approximately 11 compared to the female’s approximately 7 [ 16 ]. Three campaniform sensilla are present in ta1, two on the dorsal distal end of ta1 and one on the proximal ventral side [Ta1GF and Ta1SF, respectively, using the nomenclature of 8]; no campaniform sensilla are present in the distal tibia, ta2, or proximal ta3. (C-E) Campaniform sensilla, mechanosensory bristles, and chemosensory bristles are all composed of modified versions of four core cell types: a socket (or “tormogen”), shaft/dome (or “trichogen”), sheath (or “thecogen”), and neuron [ 186 ]. The shaft and socket construct the external apparatus that provides the point of contact for mechanical or chemical stimuli and form a subcuticular lymph cavity that provides the ion source for the receptor current [ 142 , 187 ]. The sheath has glia-like properties, ensheathing the neuron and, as is thought, providing it with protection [ 187 ]. Ultimately, however, the contributions of these nonneuronal cells to sensory processing remain poorly characterized [ 26 ]. (C) Campaniform sensilla detect strain in the cuticle. They are singly innervated and capped with a dome, rather than a hair-like projection, which extends across the surface of the socket cell [ 25 ]. The dendrite tip attaches to the dome cuticle [ 187 ]. (D) Mechanosensory bristles detect deflection of the hair-like projection. They are innervated by a single neuron, the dendritic projections of which terminate at the base of the shaft. Specific to this bristle class, the most proximal epithelial cell to the developing sense organ is induced to become a bract cell [ 12 , 80 ]. Bract cells secrete a thick, pigmented, hair-like, cuticular protrusion. (E) The chemosensory taste bristles of the leg differ in their morphology from mechanosensory bristles, appearing less heavily melanized and more curved. They also house a pore at the terminus of the shaft and lack bracts. Each is innervated by a single mechanosensory neuron and 4 gustatory receptor neurons (GRNs) [ 16 ]. Figure created using Biorender.com .

All behavior rests upon the ability of animals to detect variation in the internal and external environments. In multicellular animals, the detection of such variation is a function performed by sensory organs. With much of its external surface covered by many different classes of sensory organs, the fruit fly Drosophila melanogaster has long been used to investigate the mechanisms through which animals sense the world around them. Much attention has focused on the eyes, antennae, and maxillary palps, but the male Drosophila forelegs, which perform wide-ranging roles in locomotion, grooming, and courtship (e.g., [ 1 – 3 ]), display a distinct repertoire of sensory organs ( Fig 1A and 1B ). Internal mechanosensory receptors known as chordotonal organs sense proprioceptive stimuli around leg joints [ 4 ] and substrate-borne vibrations [ 5 ] ( Fig 1A ). Campaniform sensilla, singly innervated, shaftless sensors embedded in the cuticle, detect and relay cuticular strain, allowing for posture and intraleg coordination to be maintained [ 6 – 8 ] ( Fig 1C ). Mechanosensory bristles line the surface of the leg, detecting contact by deflection of an external, hair-like process [ 9 , 10 ] ( Fig 1D ). This organ class isn’t uniform: In the males of a subset of Drosophila species, including D. melanogaster, some mechanosensory bristles are heavily modified to generate a “sex comb,” an innovation critical for male mating success [ 11 – 14 ] ( Fig 1B ). Finally, the foreleg also contains chemosensory taste bristles, which are sexually dimorphic in number and innervated by both multiple gustatory receptor neurons (GRNs) and a single mechanosensory neuron [ 15 – 17 ] ( Fig 1E ). As in other parts of the body, such as the labellum (e.g., [ 18 ]), a degree of functional diversity exists between chemosensory taste bristles on the legs. The tunings and sensitivities of these bristles to a wide panel of tastants vary in relation to both the pair of legs on which they’re housed and their position within a given leg [ 19 ]. This variation is, at least in part, achieved by restricting the expression of certain gustatory receptors to subsets of taste bristles [ 3 , 19 ]. A level below the bristles themselves, the multiple GRNs that innervate each bristle appear to perform distinct functions. Three distinct GRN classes involved in the detection and evaluation of conspecifics have been resolved in the leg, each of which makes a critical contribution to normal sexual behavior [ 20 – 22 ]. Ultimately, unraveling how this varied sensory apparatus is constructed through development and identifying the molecular basis of specialization in each sensory organ remain central objectives of developmental neurobiology.

Results

Epithelial cells express a signature of anatomical position We next turned to the largest portion of our dataset, the nonjoint epithelial cells. Here, we observed clear separation between dorsal and ventral cells. This separation was clearest when mapping the expression of H15 (ventral) and bi (dorsal) (Fig 3J). wg (ventral) and dpp (dorsal) showed a similar, albeit weaker separation (Fig 3K). Unlike the joints, anterior–posterior separation was also clear, as delineated by the expression of ci (anterior) and hh (posterior) (Fig 3L). A weaker signature of proximal–distal separation could also be discerned from the expression of bab2, which is absent from the tibia and increases in expression between ta1 and ta2 (reviewed in [60]) (Fig 3M). We also detected localized expression of rn, the expression of which in ta1 is limited to the distal region [61] (Fig 3M). Recovery of spatial patterning in epithelial cells has recently been demonstrated in Drosophila wing imaginal disc scRNA-seq data [62,63]. But unlike in wing discs, we find that in this region of the leg the anterior–posterior signature is stronger than the proximal–distal, which may be due to our sequencing only a small fraction of the proximal–distal axis (i.e., just one tarsal segment). We then used the intersecting axes of positional marker expression as a spatial reference map to assign clusters to regions of the dissected leg tissue (Fig 3N and 3O). We tested for DEGs by comparing each region to the remainder (Fig 3P). Many DEGs showed signatures of localized up-regulation rather than cluster-specific expression, as would reasonably be expected from a tissue composed of a single cell type (exceptions include lbl, CG13064, CG13065, and CG13046; S3S–S3V Fig). But the cluster enriched for the distal ta1 marker rn exhibited a more specific gene expression profile, including showing enriched expression of the effector of sex determination dsx (reviewed in [64]), consistent with the localization of the sex comb to this region [65,66]. Genes enriched here represent candidate components of the sex-specific gene regulatory network that drives sex comb rotation (S3W–S3AE Fig).

Pupal leg hemocytes form a uniform population We identified a single hemocyte cluster based on enriched expression of He, Hml, srp, and Nimrod-type receptor genes (Figs 4C, 4E and S4A–S4N) [67–69]. In our differential gene expression analysis, we observed strongly hemocyte-enriched expression of genes including CG31777, CG31337, CG3961, CG14629, CG4250, Mec2, CG42369, CG5958, Glt, and CG10621 (Fig 4B; see S4X–S4AG Fig for UMAPs of the first 5 genes). To test for the presence of hemocyte subtypes, we ran a wide panel of recently identified lamellocyte, crystal cell, and plasmatocyte subtype-specific genes against our data [70]. However, we saw no obvious subclustering in relation to these genes: They were either widely expressed among hemocyte cells, too patchily expressed to reflect a clear subpopulation, or absent from our dataset (S4O–S4W Fig). The absence of clear subclustering may reflect the rarity of these hemocyte subpopulations, that recovered cells are insufficiently differentiated at these time points to discriminate subclasses, that these notably fragile cells [71] lyse during tissue dissociation, or point to differences between the larval and pupal immune cell repertoire, for which there is some evidence [72,73]. PPT PowerPoint slide

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TIFF original image Download: Fig 4. The tarsus contains several types of nonsensory, nonepithelial cells. (A) Annotated UMAP plot of nonsensory cells. Cell labels provided in purple indicate populations that are discussed in this figure. Those in black are discussed in Fig 5. (B) Dot plot of the expression of top differentially expressed genes identified through comparisons between each named cluster and all remaining clusters in (A). (C-F) The nonsensory UMAP shown in (A) overlaid with expression of key marker genes for each cluster. Note that dsx (D) and dally (F) are expressed in a distinct subset of bract cells, which likely corresponds to sex comb bracts. (G) 24 h APF male pupal upper tarsal segments showing staining from 1151-GAL4 > UAS-mCherry.nls (magenta) and the neuronal marker anti-Futsch (green). 1151-GAL4 marks tendons [84]. The arrangement of tendon cells is clearly distinct from the paired nerve fibers that run along the same axis. (H-J) 24 h APF first tarsal segment and distal tibia from an 1151-GAL4 > UAS-mCherry.nls (magenta) male counterstained with anti-Vvl (green). Costaining is clearer in the levator and depressor tendons at the distal tibia/ta1 joint (marked with an arrow) than in the long tendon, which extends along the proximal–distal axis of the tarsal segments. This may be due to the greater concentration of tendon cells in this region and difficulties distinguishing between anti-Vvl staining in mechanosensory bristle cells (see Fig 6Q–6S) and tendon cells. Data and code for generating the scRNA-seq elements of this figure are available at https://www.osf.io/ba8tf. https://doi.org/10.1371/journal.pbio.3002148.g004

The induction of bract identity is accompanied by a transcriptomic shift away from epithelial cell profiles Most, but not all, mechanosensory bristles on the legs are associated with a bract cell [9,49]. We identified bracts based on the expression of the transcription factor pnt and aos, an EGF inhibitor selectively expressed in cells assuming bract fate [74–76]. Among our nonsensory clusters, we find 2 enriched for aos and pnt (Figs 4D, 4F, and S5A–S5D). Differential gene expression analysis comparing among our nonsensory cell clusters showed that the top markers of the major bract cluster also showed elevated expression in the minority cluster but that the top markers of the minority cluster were more specific in their expression (Fig 4B). The top DEGs for the minority cluster included several genes that were among the top markers of the putative sex comb bearing region identified in our epithelial cell analysis (rn, dsx, and dally; Fig 4D and 4F). Thus, these cells likely correspond to sex comb bracts. Given that bract identity is induced in epithelial cells, rather than emerging through the sensory organ precursor lineage, the natural comparison to make to identify putative determinants of bract fate is to compare bract cells with other epithelial cells [12,74,77–80]. Comparing the 2 bract clusters with the nonjoint epithelial cells, we observed several genes that were highly enriched in bracts and largely absent from epithelial cells (S5E Fig). It was common to find expression of some of these genes (e.g., CG33110, Nep2, CG32365, neur; S5F–S5P Fig) in bristle shaft and socket cells, which, considering the short bristle hair-like protrusion that bracts develop, may reflect their partially overlapping morphological characteristics. Ultimately, the distinct expression profile of the bracts suggests that induction of this identity in an epithelial cell is followed by remodeling of its transcriptome.

Tendon cells express the POU transcription factor vvl, but not sr, between 24 h and 30 h APF We identified a cluster of tendon cells based on the expression of Tsp, tx, and the joint marker drm (Fig 4B and 4E) [81–83]. To visualize the anatomical distribution of tendon cells in the focal leg region, we crossed the verified tendon marker line 1151-GAL4 [84] to UAS-mCherry.nls and counterstained with an antibody against the neuronal marker Futsch to distinguish between tendons and axonal trunks (Fig 4G). The dissected region contains the “long tendon,” which runs along the proximal–distal axis of the tarsal segments, as well as the distal portion of the “tarsus levator” and “tarsus depressor” tendons, which are housed in the distal tibia [84]. One of the top DEGs for our Tsp+/tx+ cells was the POU homeobox transcription factor vvl (Fig 4C). When counterstaining 1151-GAL4>UAS-mCherry.nls legs with an antibody raised against Vvl, we observed clear costaining, supporting a tendon cell identity for this cluster (Fig 4H–4J). Of the top DEGs we identified for tendon cells, none were entirely specific to this cluster when looking across all cells in the dataset. A common pattern was to see localized expression among epithelial cells (e.g., drm, Tsp, CG13003) or among sockets and shaft cells (e.g., tx and CG42326) (S6A–S6R Fig). Beyond the top 10 tendon cell DEGs, we detected significantly enriched expression of trol, which encodes the extracellular matrix proteoglycan Perlecan (validated using trol-GAL4; S6S–S6V Fig). Surprisingly, we did not detect enriched expression of the tendon-specifying transcription factor sr in this cluster, nor its tendon-specific downstream targets slow and Lrt [85,86] (S6W–S6AE Fig). Their absence may reflect the developmental time points that we sequenced or differences between this tibia/tarsus tendon population and more commonly studied populations in the embryo [87] and wings [88].

repo+ glia and Sox100B+ cells express distinct cell–cell communication gene repertoires Comparing between the repo+ glia and Sox100B+ cells, we observed cluster-specific expression of beaten path family genes. beat-IIa and beat-IIb were restricted to the Sox100B+ cluster, while beat-IIIc was enriched in repo+ cells (Fig 5AD and 5AF). beaten path genes are thought to act as neuronal receptors for sidestep gene family ligands expressed in peripheral tissues [107]. Thus, the differential expression of different subsets of beaten path family genes between repo+ and Sox100B+ cells point both to the importance of these genes in the nonneuronal component of the nervous system and to cell type–specific patterns of between-cell communication in the developing nervous system. These beat genes have been recorded elsewhere in the fly: In the visual system, beat-IIb is expressed in L3 and L4 lamina neurons, the glia beneath L5, and in the lamina neuropil, while beat-IIIc is expressed in a subset of retinal neurons [108]. Other cell communication pathway elements also showed cell type specificity. For example, we observed that the fibroblast growth factor (FGF) ligand ths was, among glia, largely restricted to svp+/repo+ cells (Fig 5AG). FGF signaling is known to underlie aspects of neuron–glia communication in Drosophila, although in these cases, the source of Ths is neuronal. In one example, Ths is thought to act in neurons as a directional chemoattractant for the migration of astrocytes and the outgrowth of their processes [109]. In 2 others, the release of Ths from olfactory neurons directs ensheathing glia to wrap each glomerulus [110], while Ths in photoreceptor neurons induces differentiation of glia in the developing eye [111]. The expression of ths in one of our glia populations, specifically the population we believe to correspond to the surface glia, is therefore surprising. It’s possible that FGF-mediated interactions between different glia populations guide their concerted differentiation and the development of the close physical associations they form. Similar interactions with neurons may also help guide the growth of neuronal projections in the vicinity of these glia.

A combinatorial transcription factor code for sensory neurons We recovered multiple distinct sensory neuron populations in our clustering analysis, each defined by the expression of a unique combination of transcription factors (Fig 6A and 6B). We recovered a similar clustering pattern in our analysis of male neurons from the Fly Cell Atlas (FCA) adult leg data (Fig 6G and 6H) [38]. In contrast to our pupal data, however, the FCA dataset is derived from all segments of all 3 pairs of legs and is single-nuclei, rather than single-cell. We failed to recover clear sex comb or MSNCB (mechanosensory neuron in chemosensory bristle) populations in the FCA data. But in their place, we recovered 3 populations apparently absent from our pupal dataset. These novel clusters were enriched for CG9650, a transcription factor we found to be enriched in joint and tendon cells, and showed cluster-specific, combinatorial expression of transcription factors, including erm and bab1 (Fig 6I and 6J). Consistent with signs of a joint identity, we believe these are likely to correspond to chordotonal neuron populations, a class that is absent from the first tarsal segment. The pupal and FCA neuron datasets did not integrate as well as our pupal datasets did to each other (S9I and S9J Fig). In light of the significant differences in sample preparation, age, cells versus nuclei, and dissected region between the datasets we opted to analyze them separately. PPT PowerPoint slide

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TIFF original image Download: Fig 6. Identification of a combinatorial transcription factor code for leg sensory neurons. (A) Annotated UMAP plot of neuronal cells from the integrated 24 h AFP and 30 h APF first tarsal segment dataset. GRN, gustatory receptor neuron; MSNCB, mechanosensory neuron in chemosensory bristle. See S9A–S9H Fig for details on the TkR86C+ mechanosensory neurons. (B) Dot plot showing the expression of a series of canonical neuronal markers (elav, nSyb, and para) and transcription factors across the major neuron class clusters labeled in the UMAP given in (A). Each cluster expresses a unique combination. The dotted lines separate the canonical neuronal markers and then the chemoreceptor from mechanoreceptor organs transcription factor markers. (C-F) UMAP plot of neuronal cells from the integrated 24 h AFP and 30 h APF first tarsal segment dataset overlaid with the expression of members of the transcription factor code depicted in (B). (C) Note how the MSNCB cluster branching off from the top of the mechanosensory neuron population is negative for both vvl and pros. (D) Ets65A is present in all non-GRN populations in the UMAP, while an effector of sex differentiation, dsx, is expressed in GRNs, sex comb neurons, and MSNCBs. (E) fru, the other effector of sex differentiation, is enriched in 2 GRN populations and sex comb neurons, while eyg is restricted to campaniform sensilla neurons. (F) CG42566 is the only nontranscription factor plotted. It is a top marker of MSNCBs and its expression in both MSNCBs and GRNs contributed to this cluster’s chemosensory bristle annotation. ham is enriched in mechanosensory neuron classes and 2 GRN populations. (G) Annotated UMAP plot of male neuronal cells subsetted from the Fly Cell Atlas single-nuclei RNA-seq leg dataset [38]. Note the presence of 3 clusters, annotated as “putative chordotonal,” which are absent from the pupal dataset—chordotonal organs are not present in the upper tarsal segments. No clear MSNCB or sex comb clusters could be resolved in this dataset. (H) As (B) but for the male neuronal cells subsetted from the Fly Cell Atlas single-nuclei RNA-seq leg dataset. Only those clusters present in the pupal single-cell data are shown. (I) UMAP plot of male neuronal cells subsetted from the Fly Cell Atlas single-nuclei RNA-seq leg dataset overlaid with expression of the mechanosensory neuron marker vvl (blue) and a top marker of the putative chordotonal organs, the predicted transcription factor CG9650 (red). (J) A subset of (I), showing only the putative chordotonal clusters overlaid with expression of 2 transcription factors, bab1 (blue) and erm (red). (K-V) Confocal images of 24 h APF male first tarsal segments. (K-M) Mechanosensory bristles from elav-GAL4 > UAS-mCherry.nls (magenta) stained with anti-Pros (green). Two elav-GAL4+ cells are present per mechanosensory bristle, one of which, the sheath, is Pros+. elav-GAL4 expression in the sheath is likely due to the legs being imaged soon after the division of the common pIIIb progenitor cell from which they derive (see also [113] and S9K–S9M Fig). MSN, mechanosensory neuron. (N-P) Two chemosensory bristles (circled) from elav-GAL4 > UAS-mCherry.nls (magenta) stained with anti-Pros (green). Note that each bristle includes 4 Pros+/elav-GAL4+ cells (the gustatory receptor neurons), 1 Pros+/elav-GAL4− cell (the chemosensory sheath cell), and 1 Pros−/elav-GAL4+ cell (the MSNCB, mechanosensory neuron in chemosensory bristle). (Q-S) Two chemosensory (CS) bristles and 1 mechanosensory (MS) bristle from elav-GAL4 > UAS-mCherry.nls (magenta) stained with anti-Vvl (green). Note that anti-Vvl staining is entirely absent from the CS bristle including, therefore, the mechanosensory neuron (MSNCB) that innervates it. Conversely, anti-Vvl staining is observed in all 4 constituent cells of a MS bristle. (T-V) The same stainings performed in (Q-S) but centered on the sex comb. Anti-Vvl staining is present in both the neuronal (elav-GAL4+) and nonneuronal cells of the sex comb. The “central bristle,” which develops from the same bristle row as the sex comb is labeled. (W-Y) Confocal images of 48 h APF male first tarsal segments showing the expression of fru-GAL4 (magenta) and anti-Futsch (green). fru-GAL4 expression is restricted to the sex comb and chemosensory (CS) neurons. The later 48 h time point was used as fru-GAL4 was undetectable up until 40 h and weak up until 48 h. (Z) Confocal image of the first tarsal segment from a 24 h male from eyg-GAL4 > UAS-GFP.S65T. Campaniform sensilla are marked with asterisks. The axonal projections can be seen as parallel lines running either side of the central autofluorescence. Note that some nonspecific fat body staining is also present in this image. (AA) Confocal image of a distal first tarsal segment campaniform sensillum from a 24 h male where eyg-GAL4 is driving the expression of UAS-mCherry.nls. The top and bottom image in this panel show the same sensillum but with different levels of saturation to variously highlight the domed structure (top) and the individual cells of the organ (bottom). (AB) As (Z) but showing an adult haltere. Note that the staining is restricted to the campaniform sensilla field on the pedicel (“Ped.”) and apparently absent from the field on the scabellum (“Sca.”). Data and code for generating the scRNA-seq elements of this figure are available at https://www.osf.io/ba8tf. https://doi.org/10.1371/journal.pbio.3002148.g006

Campaniform sensilla express the Pax family transcription factors eyg and toe In our pupal data, we resolved a small cluster of cells enriched for the Pax family transcription factors eyg and toe. Expressing UAS-GFP under the control of eyg-GAL4, we observed staining in the regions of the first tarsal segment that correspond to the positions of the campaniform sensilla: 1 proximal organ and 2 distal organs [8] (Fig 6Z). Repeating the experiment with a nuclear-localizing UAS-mCherry, we detected expression in 4 cells within each tarsal campaniform sensillum, which presumably correspond to the neuron, sheath, socket, and dome cell (Fig 6AA). Although the expression of eyg and toe were relatively low in the adult nuclei campaniform sensilla neuron cluster, we observed eyg-GAL4 activity in both adult legs (S9O Fig) and in the adult haltere (Fig 6AA and 6AB). In the haltere, eyg-GAL4 activity was detectable in the field of campaniform sensilla on the pedicel, but not the scabellum, raising the possibility that there exist distinct subtypes of campaniform sensilla that express unique gene repertoires.

The legs contain 4 gustatory receptor neuron (GRN) classes, each expressing a unique combination of the transcription factors acj6, fru, nvy, and fkh Our stainings showed that among neurons Pros was restricted to chemosensory bristles, suggesting that the multiple pros+ neuron clusters we detect in our scRNA-seq data represent subclasses of GRNs. Each GRN cluster expressed a unique combination of 5 transcription factors: pros+/acj6+/nvy+, pros+/acj6+/fkh+, pros+/acj6+/fru+, and pros+/fru+ (Fig 7A–7F). We validated these combinations using fru-GAL4 in conjunction with antibodies raised against Pros, Acj6, Nvy, and Fkh (Fig 7G–7AA). Multiple bristles are often closely associated within a single region of the leg, while the neurons themselves frequently overlap within a single bristle. Consequently, it wasn’t possible to definitively determine whether 1 cell of each GRN class was present in every ta1 bristle, but from our observations, this seems likely to be the case. Consistent with this, the numbers of cells recovered in each cluster generally appeared similar (Fig 7A). We recovered the same 4 populations, marked by the same transcription factor code, in the FCA full leg dataset, again recovering a similar number of cells in each GRN population (Fig 7AC). The expression of nvy was, however, far less extensive than in the pupal data, suggesting that expression drops off during later pupal development or that nvy is restricted to a subset of cells in this GRN class. Correspondence between the nvy+ cluster in the pupal and adult data was supported by additional marker genes, such as foxo and Fer1 (see below). Among neurons, the 4 GRN populations showed specific or enriched expression of Ir25a, Ir40a, Gluclalpha, RhoGAP102A, Snmp2, CG42540, CG13578, Tsp47F, and CG34342 (S10A–S10T Fig). Taken together, the recovery of the same 4 GRN classes across different time points, technologies, and dissected regions suggests that despite substantial between-bristle variation in receptor expression and sensitivity to given stimuli [19], just 4 core GRN classes might be present in the leg and that these classes are defined by combinatorial expression of a small set of transcription factors (Fig 7AB). PPT PowerPoint slide

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TIFF original image Download: Fig 7. Four gustatory receptor neuron (GRN) classes express a combinatorial transcription factor code and unique gene repertoires. (A) Annotated UMAP of the pupal integrated neuron data. GRN, gustatory receptor neuron; MSNCB, mechanosensory neuron in chemosensory bristle. The number of cells in each GRN cluster is presented. The numbers are generally similar between each GRN population, with the exception of the fru+ male-sensing GRNs. This population was closely associated with the fru+ female-sensing GRNs, more so than were any other 2 GRN subtypes, and the interface between them in UMAP space contained several cells bearing intermediate characteristics. Consequently, the discrepancy in cell numbers between fru+ GRN populations may reflect classification errors due to transcriptomic similarities. (B-F) The UMAP shown in (A) overlaid with the expression of 5 transcription factors (pros, acj6, nvy, fkh, and fru) that are expressed in unique combinations in each of the 4 GRN clusters. (G-AA) Testing the GRN transcription factor code derived from the scRNA-seq data on 24 h APF male first tarsal segments. (G-I) anti-Nvy (green) and anti-Pros (magenta). Note that 1 Pros+ cell is partially obscuring the Pros+ sheath cell, which has a distinct, elongated morphology. (J-L) Anti-Pros (green) and anti-Fkh (magenta). (M-O) Anti-Pros (green) and fru-GAL4 > UAS-mCherry.nls (magenta). (P-R) Anti-Acj6 (green) and fru-GAL4 > UAS-mCherry.nls (magenta). (S-U) Anti-Acj6 (green) and anti-Fkh (magenta). (V-X) Anti-Nvy (green) and anti-Acj6 (magenta). (Y-AA) Anti-Nvy (green) and anti-Fkh (magenta). (AB) A schematic summarizing the expression patterns of each transcription factor across GRNs, along with a selection of other genes detected in each subtype. Gene names are colored pink, green, or blue when shared across multiple GRN subtypes. (AC-AH) Recovery of the same transcription factor code in the Fly Cell Atlas single-nuclei adult male leg neuron data. Note that in the adult data, nvy is barely detected. Correspondence between the nvy+ cluster in the pupal and adult data was supported by additional marker genes, such as foxo and Fer1 (see S11B, S11C, S11H, and S11I Fig). (AC) As in (A), the number of cells in each GRN population is presented. In this dataset, a subregion of what unsupervised clustering labeled as the fru+/acj6− population showed acj6 expression, suggestive of a classification error. This conclusion is further supported by the VGlut, ppk25, and ppk10 data below (see S10Y–S10AB Fig). We therefore manually labeled these as part of the fru+/acj6+ cluster. As in the pupal data, the interface between the 2 fru+ populations appeared particularly close. (AI) A dot plot summarizing the expression of a selection of top differentially expressed genes for each cluster that we identified in the Fly Cell Atlas single-nuclei adult male leg neuron data. Data and code for generating the scRNA-seq elements of this figure are available at https://www.osf.io/ba8tf. https://doi.org/10.1371/journal.pbio.3002148.g007

Male- and female-sensing GRNs express distinct receptor repertoires Previous work has shown that there are 2 fru+ GRNs per leg chemosensory bristle [21,22]. Both express the ion channels ppk23 and ppk29, while one additionally expresses ppk25 and VGlut [21,22,123–126]. These ppk25+/VGlut+ neurons respond to female pheromones, while the ppk25−/VGlut− cells respond to male pheromones [22]. In the pupal data, we observe ppk23 in both fru+ populations, with a small number of ppk29+ cells also distributed across both (S10U and S10V Fig). Although ppk25 was absent in our dataset, we observed a sharp divide between fru+/acj6+ and fru+/acj6- cells based on VGlut expression (S10W Fig). VGlut is restricted to the fru+/acj6− population, identifying these as the female-sensing neurons and the fru+/acj6+ cells as male-sensing. Receptor expression was more readily detected in the adult nuclei data, suggesting that receptors are generally expressed later in development than the time points we sequenced. In the adult dataset, we again observed that VGlut was restricted to the fru+/acj6− population and recovered the previously documented expression patterns of ppk23, ppk25, and ppk29 (S10X–S10AA Fig). However, we also observed GRN-specific expression patterns of several other ppk genes. While ppk25 was restricted to female-sensing cells, ppk10 and the less frequently detected ppk15 were restricted to male-sensing cells (S10AB–S10AD Fig). These therefore represent candidate male pheromone receptors. Conversely, Ir21a and CG46448 appeared to be largely restricted to female-sensing neurons (S10AE–S10AF Fig). CG46448 is adjacent to VGlut on chromosome 2L, so their apparent coexpression in female-sensing neurons points to the possibility of their transcriptional control by shared cis-regulatory elements. These genes aside, the expression differences we observed between male- and female-sensing neurons were generally limited, but several genes were common to both pheromone-sensing populations, including the transcription factors tup and svp, the predicted sulfuric ester hydrolase CG8646, CG14007, and the glycine transporter GlyT, the latter suggesting that these neurons are glycinergic [127] (S10AG–S10AK Fig).

nvy+ GRNs correspond to a sexually dimorphic population required for normal mating behaviour The nvy+ GRNs showed specific expression of a further set of transcription factors in the pupal data, namely Fer1, foxo, and CG7786 (S11A–S11D Fig). At these time points, few other genes showed enrichment in the nvy+ GRN cluster—exceptions include the phosphodiesterase Pde6 and G-protein–coupled receptor TrissinR (S11E and S11F Fig). To further probe the identity of these neurons, we turned to the adult dataset. The transcription factor code identified in the pupal data largely persisted in the adult data, although each was detected in fewer cells and CG7786 was absent (Figs 7AF and S11G–S11J). AkhR and Gr64a were among the top markers of the nvy+ GRNs in the adult data (S11M and S11N Fig). But like the other 5 Gr genes we detected across the 2 datasets, Gr64a was present in only a handful of cells (S11O–S11T Fig). The sparse detection of Grs may stem from gene dropout due to low abundance or reflect their restricted expression among GRNs of the same class. Ir52a, Ir52b, Ir52c, and Ir52d were also among the top markers of the nvy+ GRN cluster (S11U–S11X Fig). With the exception of Ir52b, these ionotropic receptor (IR) genes have been well characterized: They are known to be largely coexpressed in a subset of leg taste bristles enriched in the first tarsal segment, to show quantitative differences in expression between males and females, to show sexual dimorphism in their projections (they cross the midline in a commissure in males), to be required for normal sexual behavior, and to be expressed in neurons distinct from those involved in sweet or bitter sensing [20,128]. Ir52c and Ir52d are further known to be restricted to the forelegs [20]. Despite their sexually dimorphic characteristics, the neurons expressing these receptors are mutually exclusive from those expressing fru-LEXA within the same bristle [20]. This accords with the scRNA-seq data, where fru appeared largely restricted to the male- and female-sensing populations (Fig 7F and 7AH). Sexual dimorphism in these neurons is therefore instead likely driven by dsx; indeed, at least some Ir52c+ neurons have been shown to descend from a dsx+ lineage [20]. In the adult dataset, dsx expression was patchy among GRNs, appearing enriched in the male- and female-sensing populations (S11Y Fig). In the pupal data, dsx was widely detected across all GRN clusters (S11Z Fig). The discrepancy in the extent of dsx expression in GRNs between the datasets may reflect the differences in the dissected regions: While most male and female pheromone-sensing GRNs may be dsx+ regardless of position, it may be that dsx expression is restricted among nvy+ and fkh+ neurons to those in the regions of the foreleg more likely to contact a mate than food—i.e., the foreleg upper tarsal segments. Restriction of dsx expression to a subset of neurons within a GRN class would provide a mechanism through which an additional layer of between-bristle variation in activity could be achieved.

Distinct and shared modules of gene expression in nvy+ and fkh+ GRNs The 3 GRN populations that we have discussed—male-sensing, female-sensing, and nvy+—match known populations in the literature. However, we were unable to find mention of a population that resembled our fourth, which was pros+/acj6+/fkh+. Although none of the top 20 DEGs obtained from a comparison with the 3 other GRN populations showed specific expression in the pupal data, there were intriguing similarities with the nvy+ cluster: CAH2, jus, and Glut4EF each looked specific to or highly enriched in both the nvy+ and fkh+ GRNs (S11AA–S11AC Fig). But in the adult dataset, these specific differences were reduced or lost, suggesting that the variation we observed between GRNs in the pupal data may reflect heterochronic differences or that these particular between-GRN developmental differences are lost in adulthood (S11AD–S11AF Fig). Nonetheless, the adult dataset presented its own similarities between fkh+ and nvy+ GRNs: Expression of Ir76b, which is known to be widely expressed among olfactory and gustatory receptor neurons and is thought to form heteromeric complexes with more selectively expressed Ir’s, was restricted to fkh+ and nvy+ GRNs (S11AG Fig) [20,129–131]. The restricted expression of Ir76b contrasts with that of another such coreceptor, Ir25a, which we found broadly expressed across all 4 GRNs (S11AH Fig). Although detected in fewer cells, Ir40a, which is known to be coexpressed with Ir25a in the antennal sacculus, was similarly broadly expressed [132] (S11AI Fig). The fkh+ population also had something in common with the female-sensing GRNs: Across both datasets, the extracellular matrix proteoglycan gene trol—which we also observed in tendon cells (S6S–S6V Fig)—was enriched in fkh+ and female-sensing GRNs, showed patchy, low-level expression in male-sensing GRNs, and was essentially absent from nvy+ GRNs (S12A–S12D Fig). We recovered this pattern in trol-GAL4 > UAS-mCD8::GFP [133] first tarsal segments counterstained with anti-Pros: Of the Pros+ cells in a single chemosensory bristle, at least 2 were strongly trol-GAL4+ and at least 2 were trol-GAL4− (including the sheath) (S12E–S12G Fig). In some bristles, the remaining Pros+ cell was trol-GAL4+ and in others trol-GAL4−. trol performs several roles during the assembly of the nervous system [134,135]—why it should be limited in its expression among GRNs is unclear. Alongside these shared modules of gene expression, we identified several uniquely expressed genes in fkh+ GRNs, including Tbh, which encodes the key limiting enzyme in octopamine synthesis and therefore suggests that these neurons are octopaminergic (S11AJ and S11AK Fig) [136]. Although very sparsely detected, we also observed Ir60a to be limited to fkh+ neurons (S11AL Fig).

Potential heterochrony in the gene expression profiles of sensory organ neurons In attempting to identify subtype-specific genes among subclasses of neurons, we observed that genes that appeared cluster-specific in the pupal stages sometimes showed widespread expression in the adult nuclei data. For example, DCX-EMAP, a gene that has been implicated in mechanotransduction in both campaniform sensilla and the chordotonal receptors of the Johnston’s organ [137], switches from being exclusive to campaniform sensilla neurons in the pupal data, to being widespread among both campaniform sensilla and mechanosensory neurons in the adult data (S13C and S13G Fig). Several other genes, such as nAChRalpha7, Ccn, CG1090, CG17839, and CG34370, showed a similar pattern (S13A–S13N Fig). Analogously, MSNCBs and sex comb neurons, identifiable as a vvl- and vvl+/fru+/rn+ subpopulation of mechanosensory neurons, respectively, appeared to develop ahead of the major body of mechanosensory neurons that innervate mechanosensory bristles, as evidenced by the expression patterns across datasets of genes such as sosie, CG31221, and dpr13 (S13O–S13T Fig). In these cases, the broadening of expression between pupal and adult datasets is suggestive of heterochronic differences (i.e., a difference in rate, timing, or duration) in development between the neurons of different sensory organ classes, such that the neurons in mechanosensory bristles lag behind those in chemosensory bristles, campaniform sensilla, and the sex comb. This hypothesis is consistent with documented between-organ variation in developmental timing: Chemosensory bristles are known to be specified earlier than all but the largest mechanosensory bristles [115,116].

Mechanotransduction neurons from different external sensory organ classes express largely shared gene repertoires Heterochronic differences between sensory organs complicate the identification of organ-specific genes in the pupal data: Genes that appear unique at one stage may be widespread at a later time point. For that reason, we initially focused on the adult nuclei dataset to identify genes enriched in specific non-GRN neuron populations (Fig 7AI). The majority of the top mechanosensory neuron markers that were returned from a DGE analysis comparing these cells to all other neurons appeared nonspecific, being expressed in one or more additional clusters (e.g., Calx, Fife, Dop2R, KrT95D, CG4577, and Ten-m; S14A–S4M Fig). The same applied to the top campaniform sensilla markers, but in this case, despite their lack of complete specificity, many showed a relatively restricted expression profile that extended across both campaniform sensilla and chordotonal organs (e.g., dati, unc79, CG42458, TyrR, Cngl, CARPB, and beat-VI; S14N–S14Z Fig). This pattern is suggestive of certain molecular commonalities between campaniform sensilla and chordotonal organ neurons, commonalities that make them distinct from other mechanotransduction neurons. In further pursuit of genes specific to each of mechanosensory neurons and campaniform sensilla, we tried another approach: identifying top markers of each of these clusters in the pupal data and mapping their expression in adults to determine whether their expression remains cluster specific. Of these, the transcription factor Ets65A, which in the pupal data was restricted to mechanosensory neurons, sex comb neurons, MSNCBs, and campaniform sensilla neurons, remained restricted to mechanosensory neurons and campaniform sensilla neurons in the adult data (S14AA and S14AB Fig). Ets65A therefore represents a candidate regulator of mechanosensory identity in external sensory organ neurons. We applied this same pupal identification and adult mapping approach in sex comb neurons and MSNCBs, populations that didn’t form distinct clusters in the adult data. No individual gene showed clearly restricted expression to either, but there was strong enrichment of genes that were otherwise patchily expressed across cells. shakB was strongly enriched in sex comb neurons relative to other mechanosensory bristles in the pupal data and widely present across putative chordotonal and campaniform sensilla neurons in the adult data (S14AC and S14AD Fig). In MSNCBs, CG42566, and to a lesser extent CG33639, appeared enriched relative to other mechanosensory populations, with both also detected across GRNs, consistent with a chemosensory bristle origin (S14AE–S14AH Fig). Collectively, MSNCBs and sex comb neurons showed clear enrichment of both effectors of the sex determination pathway: fru and dsx (S14AI–S14AL Fig). The specificity of this enrichment was greater in the case of dsx, which across both neuronal datasets was largely absent outside of the comb, GRN, and MSNCB clusters; fru was surprisingly widespread in the adult data, including expression in the putative chordotonal clusters and campaniform sensilla. The expression of these 2 transcription factors provides a clear regulatory mechanism through which the transcriptomic profiles and activity of these derived mechanosensory populations could readily diverge from other mechanosensory bristles—and do so in a sex-specific manner. But ultimately, determining whether any of the transcriptomic differences between the mechanotransduction neurons that innervate each of mechanosensory bristles, chemosensory bristles, the sex comb, and campaniform sensilla translate into functional differences in operation or sensitivity requires further work. It’s clear from these data that some differences between mechanotransduction neurons innervating different organ classes are present, but they appear minor and on the whole less clear cut than between GRN populations.

Putative chordotonal organ neuron subtypes express specific gene repertoires In the FCA adult data, we recovered 3 putative chordotonal organ neuron clusters that were absent from our first tarsal segment dataset because these organs fall outside the dissected area. We refer to the largest as “putative chordotonal” and the 2 smaller clusters as erm+ and bab1+ putative chordotonal based on a transcription factor they were each specifically enriched for. Many of the genes we identified as specifically enriched in the putative chordotonal organs in our DGE analysis were absent from the pupal dataset, consistent both with their organ specificity and the absence of chordotonal organs from our dataset. Along with the genes shared among campaniform sensilla neurons and chordotonal organ neurons discussed in the previous section (S14N–S14Z Fig), we identified genes specific to or highly enriched in all chordotonal populations (S15A–S15K Fig), as well as those specific to subpopulations (S15–S15V Fig). The transcriptomic distinctiveness we detect between putative chordotonal neuron clusters aligns with previous work that has identified multiple, functionally distinct neurons in a single chordotonal organ (e.g., “Type A” and “Type B” neurons;, [4,5]). The next step will be to match the distinct clusters we recover to these different chordotonal neuron classes. In turn, that would raise secondary questions, such as whether each neuron class is present in each of the several chordotonal organs that are housed within the leg.

The support cells within a mechanosensory organ each express a distinct gene repertoire Campaniform sensilla, chemosensory bristles, and mechanosensory bristles each consist of 4 distinct cell types generated through asymmetric divisions of a sensory organ precursor (SOP). The first division gives rise to a progenitor of the socket (tormogen) and shaft (trichogen) cells, while the other to a progenitor of the sheath (thecogen) and neuron(s) (reviewed in [138]) (Fig 8A). Few marker genes are known for the nonneuronal SOP descendants and, to the best of our knowledge, none that definitively separate the same cell type between different organs (e.g., mechanosensory versus chemosensory sockets) [26]. Those markers that are known include the following: Su(H) and Sox15, which specifically accumulate in socket cell nuclei [139,140]; sv (Pax2), which although initially expressed in all bristle cells during the mitotic phase of development is eventually restricted to the shaft and sheath [141]; nompA, which is specifically expressed in sheath cells where it is required to connect dendrites to the shaft [142]; and pros, which is expressed in sheath cells (Fig 6K–6P; [113]). PPT PowerPoint slide

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TIFF original image Download: Fig 8. Distinct and shared modules of gene expression between sensory organ support cells. (A) The 4 constituent cell types of external sensory organs, such as the mechanosensory bristle in this schematic, originate through asymmetric divisions of a sensory organ precursor (SOP) cell (reviewed in [138]). The SOP divides to produce a pIIa and pIIb daughter cell. pIIa further divides to generate a socket and shaft cell. In the notum, where it’s been studied, pIIb divides into a pIIIb cell and glial cell, the latter of which enters apoptosis soon after birth [188]. pIIIb further divides to produce the sheath and neuron. To the best of our knowledge, whether the pIIb glial division occurs in the leg remains untested. (B) Annotated UMAP plot of the bristle cells from the integrated 24 h AFP and 30 h APF first tarsal segment dataset. The campaniform support cluster included Su(H)+ cells, which suggests that it corresponds to socket cells, but it’s possible that it includes a mix of campaniform sensilla accessory cell types. (C-F) The UMAP shown in (B) overlaid with the expression of a series of marker genes, either previously published or demonstrated in this study, for different sensilla classes or accessory cell types. (G) A dot plot summarizing the expression patterns of a selection of genes identified as being differentially expressed in each of the clusters given in the UMAP shown in (B). Dotted lines separate the 3 major classes of sensory support cell. Of the socket markers, CG31676 is known to be expressed in a subset of olfactory projection neurons [133]; nw is a C-type lectin-like gene; stan, a cadherin that controls planar cell polarity [189]; and nrm, ed, and hbs are cell adhesion molecule genes [143–147]. Of the shaft markers, CG9095 encodes a C-type lectin-like gene; disco-r encodes a transcription factor; dUTPase encodes a nucleoside triphosphate; spdo encodes a transmembrane domain containing protein that regulates Notch signaling during asymmetric cell division [190–192]; and sha encodes a protein involved in the formation of bristle hairs [148]. Aside from pros and nompA, the top markers of the sheaths include the following: the transcription factors Glut4EF, pnt, and SoxN; jv, which encodes a protein involved in actin organization during bristle growth [149]; qua, which encodes an F-actin cross-linking protein [150]; and the midline glia marker wrapper, which encodes a protein involved in axon ensheathment [99–101]. (H-S) The UMAP shown in (B) overlaid with the expression of genes identified in this study as markers of sensory organ support cell subtypes. Data and code for generating the figure are available at https://www.osf.io/ba8tf. https://doi.org/10.1371/journal.pbio.3002148.g008 Based on these markers, we identified major shaft, socket, and sheath clusters in our data (Fig 8B–8F). The large size of each of these relative to other clusters in the support cell dataset indicates that they belong to the dominant sensory organ in the first tarsal segment: mechanosensory bristles. Further evidence for a mechanosensory origin comes from the observation that each of these clusters was vvl+, which we previously observed to be expressed in all mechanosensory, but no chemosensory, bristle cells (Fig 6Q–6S). Among the DEGs we identified for each of the mechanosensory shaft, socket, and sheath clusters were many that reflected the biology of these sensory support cells (Fig 8G–8M): the cell adhesion molecule genes nrm, ed, and hbs [143–147] in socket cells; sha, which encodes a protein involved in the formation of bristle hairs [148] in shaft cells; and, in the sheath, a duo of genes, qua and jv, involved in the organization of actin during bristle growth [149,150]. Another nod to the biology of the sheath came in its enrichment for wrapper, which encodes a protein known to be involved in axon ensheathment [101]. Classically used as a marker of midline glia [99–101], the expression of wrapper in sheaths reinforces their glia-like properties, despite not expressing repo or gcm. Moreover, it points to general, shared elements in the mechanisms of ensheathment of neuronal processes between these cell types. The enriched expression in sheaths of a trio of transcription factors—Glut4EF, pnt, and SoxN, of which the latter was completely restricted to sheaths in our dataset—provide a potential route to regulatory divergence from other support cells and glia.

Homologous support cells show transcriptomic divergence between sensory organ classes We observed that homologous support cells in different sensory organ classes express both shared and distinct gene repertoires. The top markers for each of the mechanosensory socket, shaft, and sheath clusters were enriched in a set of smaller clusters: 3 clusters showed socket-like profiles, 2 shaft-like profiles, and 1 sheath-like profile (Fig 8G). These minor clusters therefore appear to be sensory organ cell subtypes from sensilla classes that are less abundant in the first tarsal segment than are mechanosensory bristles. Our visualization of eyg-GAL4 > UAS-GFP revealed eyg-GAL4 expression in all 4 cells in a campaniform sensillum (Fig 6AA), so the presence of eyg and toe in the smallest cluster suggest that these cells correspond to a campaniform sensilla population. This cluster uniquely expressed the transcription factor mirr (Fig 8N) and the CPLCP cuticle protein family gene Vajk4, and was Su(H)+, suggesting a socket cell identity, but no further eyg+/toe+ clusters were present. Several explanations for why these are plausible: (a) this cluster contains a mix of all campaniform sensilla support cells; (b) only the sockets are transcriptomically distinct enough to cluster separately; and (c) only the sockets were recovered in sufficient numbers to cluster separately. That a small, coclustering group of eyg+ sheath cells were present in the mechanosensory sheath population, rather than forming their own distinct cluster, provides some support for (b) and (c) (Fig 8D). Ultimately, the presence of eyg and toe across the constituent cells of campaniform sensilla suggest that these genes may be master regulators of campaniform sensillum identity. The minor sheath population was enriched for CG42566, a gene we previously found to be specific to MSNCBs and GRNs among the neurons (S14AE and S14AF Fig), suggesting that this cluster represents descendant cells of the chemosensory pIIb lineage and, therefore, the chemosensory sheath cells specifically. As well as several poorly characterized genes, this cluster showed enriched or unique expression of the lipid-binding protein encoding gene NLaz (Fig 8O), the chitin-binding protein encoding gene Gasp, and Side-VIII. As we observed with neurons, at least some of the differences between mechanosensory and chemosensory sheaths appear to be heterochronic, with chemosensory sheaths developing ahead of their mechanosensory homologs: Several genes, including nompA and wrapper, were widely detected among chemosensory sheath cells, but in mechanosensory sheaths showed localized expression in a region enriched for cells from the 30 h dataset (S16A–S16J Fig). Based on the expression of Su(H), Sox15, and sv, the remaining clusters have apparent socket and shaft identities. Given the representation of different sensilla classes in the first tarsal segment, the most likely classification for the vvl− clusters are chemosensory sockets and shafts. Except for the extracellular protease gene AdamTS-B, which was heavily enriched in both clusters (Fig 8P), there was no clear transcriptomic link between them. Among the top markers for each, the sv+ putative shaft cluster showed strong enrichment for mtg, which encodes a chitin binding domain-containing protein that’s required to drive postsynaptic assembly [151], while the Su(H)+/Sox15+ putative socket cluster showed unique expression of CG43394 (S17 Fig) and Ance-3 (Fig 8Q), which encodes a predicted membrane component orthologous to human ACE2, the receptor for SARS-CoV-2 [152].

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