Cellular and molecular organization of the Drosophila foregut

Significance We characterized the adult Drosophila foregut using transcriptomics to provide a genetically tractable model for analyzing how animal foreguts coordinate digestion and immune response with sensory information. Single-cell RNA sequence analysis was strengthened using tagged endogenous genes as reporters to validate candidate cell types and enable cell-type-specific genetic disruption. Neuroendocrine cells coordinate gut activity with nutrition, the microbiome, and circadian cycles. The proventriculus (PV) secretes the peritrophic matrix (PM) that lines the gut, controlling digestion and microbiome interaction. Esophageal and salivary gland–secreted proteins allow us to identify candidate host proteins constituting a foregut commensal niche for specific bacterial species. Overall, foregut structure and function have likely been conserved during animal evolution.


Drosophila stocks, husbandry, and crosses
Drosophila were cultured on the standard laboratory fly food at room temperature (23 to 25 °C).Genes driving Gal4 were examined after crossing to UAS-GFP reporters.
Genes fused to GFP were examined directly.Drosophila stocks used in the study are summarized in the key resources table.

Immunofluorescence and light microscopy
Drosophila guts were dissected in phosphate-buffered saline (PBS) followed by immediate fixation in 4% paraformaldehyde in PBS (made from Paraformaldehyde 16% Aqueous Solution EM Grade, Electron Microscopy Sciences, Sku#15710) for 90 minutes at room temperature.Samples were then washed and permeabilized three times in PBST (0.1% Triton X-100 in PBS) for 15 minutes each.Samples were stained with DAPI (2 µg/mL in PBST) for 10 minutes at room temperature, and then washed three times in PBST for 15 minutes each.Samples were equilibrated in 50% glycerol in PBST for at least 30 minutes, and then mounted in mounting media (4% propyl gallate, Sigma-Aldrich, Sku#02370, and 80% glycerol in PBST).All the incubation and washing steps were performed on a nutator.Images were acquired with a Leica TCS SP8 confocal microscope or a Leica Stellaris 8 DIVE multiphoton confocal.

IMARIS nucleus quantification
Proventriculus cell quantification was performed using the spot detection function of the IMARIS software (v10.0.0).Specifically, the raw Z-stack data was reconstructed into a 3D object from which spots were generated by automated spot detection based on the DAPI signal.Cell type boundaries were determined based on the cellular identities and later verified by transgenic labeling.The results were validated manually and multiplets were removed when necessary.

Ex vivo live imaging
Ex vivo foregut live imaging was conducted by following Marchetti, Zhang, and Edgar (2022) with minor modifications as noted.Briefly, Drosophila gut tissues were freshly dissected and mounted in agarose pads made of 1% low gelling temperature agarose (Sigma-Aldrich, Sku#A9414) in modified Schneider's medium without additives.
About 120 µL of the complete culture medium with no isradipine was carefully added to the dish.See Marchetti, Zhang, and Edgar (2022) for the detailed recipe.Live imaging was performed on a Leica DMI8 inverted microscope equipped with the Hamamatsu Flash4 v3 sCMOS camera.Images were acquired using the DIC mode at 100 fps.The movie was generated from the raw data using the VisiView software (Visitron Systems, v6.0.0.18),IMARIS (v10.0.0), and Adobe Premiere (v22.64).

RNA fluorescence in situ hybridization (RNA-FISH)
RNA-FISH probes were designed using the Stellaris Probe designer (https://www.biosearchtech.com/support/tools/design-software/stellaris-probe-designer)and synthesized with the Stellaris Probe Technology (Biosearch Technology Inc.).The probe sequences are summarized in Dataset S8.RNA-FISH experiments were performed by following the Stellaris RNA-FISH D. melanogaster Wing Imaginal Discs protocol (Biosearch Technology Inc.).Briefly, dissected proventriculus tissues were fixed in 4% PFA in PBS and permeabilized in 70% ethanol in PBS overnight at 4 °C.Samples were washed in 1 mL of Wash Buffer A (Cat#SMF-WA1-60) for 5 minutes at room temperature, and then incubated in 100 µL of Hybridization Buffer (Cat#SMF-HB1-10) containing probe (1 µL of 5 nmol probe stock solution to 100 µL of Hybridization Buffer) for 10-16 hours at 37 °C.Wash Buffer A and Hybridization Buffer contain 10% (v/v) formamide.Samples were washed in 1mL of Wash Buffer A for 30 minutes at 37 °C followed by DAPI nuclear stain (2 µg/mL DAPI in Wash Buffer A) for 30 minutes at 37 °C.Afterward, samples were washed in 1 mL of Wash Buffer B (Cat#SMF-WB1-20) for 5 minutes at room temperature and finally mounted in mounting media (4% propyl gallate, Sigma-Aldrich, Sku#02370 and 80% glycerol in PBST).All the steps were performed on a nutator.Images were acquired with a Leica TCS SP8 confocal microscope.

Electron microscopy
Samples for electron microscopy were prepared as described in Dodge et al. 2023 with minor modifications as noted.Drosophila foreguts were dissected and fixed in 1% paraformaldehyde (made from Paraformaldehyde 16% Aqueous Solution EM Grade, Electron Microscopy Sciences, Sku#15710), 3% glutaraldehyde (made from Aqueous Glutaraldehyde EM Grade 25%, Electron Microscopy Sciences, Sku#16220), 2 mM Calcium Chloride, and 0.1 M Cacodylate buffer (pH 7.4) for one hour at room temperature and then overnight at 4 °C.After fixation, samples were quenched in 50 mM Glycine, 0.1 M Cacodylate buffer for 15 minutes, washed with 0.1 M Cacodylate buffer 2 times, and post-fixed with 1% osmium tetroxide (made from Osmium Tetroxide 4% Aqueous Solution, Electron Microscopy Sciences, Sku#19150) and 1.5% potassium ferrocyanide in 0.1 M Cacodylate buffer for 1 hour at room temperature.Samples were washed with Cacodylate buffer and then 0.05 M Maleate buffer (pH 5.15) followed by en bloc staining with 0.5% (w/v) uranyl acetate (Electron Microscopy Sciences, Cat#22400) in 0.05 M Maleate buffer (pH 5.15) for 1 hour.After washing in water 3 times, samples were dehydrated using 30%, 50%, 75%, 85%, 95%, and 100% ethanol in series, followed by four rinses in propylene oxide.After dehydration, samples were infiltrated and embedded in EMbed 812 resin (Electron Microscopy Sciences, Sku# 14900) following the manufacturer's protocols.The resin blocks were oriented and cross-sectioned using a Leica EMUC7 ultramicrotome.Ultrathin sections at about 85 nm thickness were collected at every 25-30-micron interval and examined using a Hitachi HT7800 transmission electron microscope operated at 80KeV.Digital images were acquired by using an AMT NanoSprint 12 camera.

Single cell RNA sequencing (scRNAseq)
Proventricular and associated tissues were dissected in cold PBS from adult mated female flies (w[1118]; P{w[+mC]=UAS-GFP.nls}/+;PBac{w[+mC]=IT.GAL4}bond[1385-G4]/+) reared with a conventional gut microbiome.Tissue-cutting sites were at the anterior esophagus before its entry point into the proventriculus, the anterior midgut boundary, and the crop duct entering the base of the crop (Figure S1A).Dissected tissues were kept in PBS on ice until about 60-70 pieces were collected.Each proventriculus was delaminated from a triple-layer epithelium into a monolayer by gentle tugging with forceps before further dissociation (Figure S1B).The tissue suspension was processed with a combined mechanical and enzymatic dissociation (8-10 mg/mL Gibco Collagenase, Type IV, powder, Thermo Fisher, Cat#17104019 in PBS) for about 30 minutes at room temperature.After the dissociation, 150 µL FBS was added to quench the reaction.The cell suspension was filtered through a 20 µm syringe (BD,Cat#340595).The cells were spun down at 500 g for 5 minutes at 4 C following supernatant removal.The cell pellets were washed in 1 mL of cold PBS with 0.08% BSA with a gentle resuspending.The cells were spun down again at 500 g for 5 minutes at 4 C and resuspended in 40 µL PBS with 0.08% BSA.A small portion of the cell suspension was examined on a hemocytometer from which single cells with various sizes and morphologies were observed.Libraries were prepared using 10X Genomics products including the Chromium Next GEM Single Cell 3' Kit v3.1 (PN1000128), Chromium Next GEM Chip G Single Cell Kit (PN1000127), and Single Index Kit T Set A (PN1000213) and following the manufacturer's protocol (CG000204 Rev D).Qubit dsDNA Quantification Assay Kit (Thermo Fisher, Cat#Q32850) and the Agilent High Sensitivity DNA Kit (Agilent, Cat#5067-4626) on the 2100 Bioanalyzer System were used at the appropriate steps for quantification and quality control.Sequencing was conducted on the Illumina NextSeq500 using 28x91 bp reads with 8 base indexing, as per the 10X Genomics protocol.

Analyses of scRNAseq
Reads were processed using Cell Ranger (v6.0.1) following the 10X Genomics protocol.The Drosophila melanogaster genome BDGP6.22Ensemble release 98 was used as the reference genome.A piece of GFP CDS sequence (Dataset S7) was added as an artificial chromosome in the reference genome to allow GFP expression detection.
Further bioinformatic analyses were performed using the Seurat package (v4.3.0)following the published instructions from the Satija lab in R (v4.1.2).Putative cells with a high ratio of mitochondrial gene expression (mtRNA > 8%) or/and abnormally high UMI (UMI > 5000) were eliminated.Triplicate datasets were integrated using the Seurat integration function.The initial clustering identified 18 putative cell clusters (Fig 2A).
One initial cluster (#2) with an abnormally higher average UMI than the others and mixed molecular identities was removed.A comprehensive re-clustering was conducted to further improve the signal sensitivity by examining the analysis with different numbers of principal components and the resolution parameter (PCs = 30, resolution = 0.3).Subclustering was introduced to distinguish a few clusters containing multiple cell types.
These steps were essential to identifying the balance of over-and under-clustering and maximizing the detection of true biological signals.Overall, the analyses determined 18 final cell clusters which were further validated by transcriptomic analyses and genetic labelling.Plots were generated using the ggplot2 package (v 2_3.4.3).

Bulk messenger RNA sequencing (RNAseq)
Flies used for RNAseq were reared axenically.Flies except the axenic proventriculus control samples were inoculated with a dose of Lactiplantibacillus plantarum (LpWF, Obadia et al. 2017 andDodge et al. 2023), one of the Drosophila melanogaster core gut microbiome species, during adulthood.For LpWF inoculation (Obadia et al. 2017 andDodge et al. 2023), an equivalent of 10 5 -10 6 CFU/fly LpWF cell suspension in PBS was spread on the top of the fly food where adult flies were housed.
For the proventriculus isolation, about 30 pieces of the proventriculus tissue (with minimum tissues from crop ducts) from adult mated female flies were dissected in cold PBS.For crop isolation, about 30 pieces of the crop tissue (including crop ducts) from adult mated female flies were dissected in cold PBS.Excess PBS was removed as much as possible from the tissue collection after a quick spin-down.Samples were flash frozen in liquid nitrogen and stored at -80 °C until enough samples were collected.RNA extractions were performed using the Qiagen RNeasy Mini Kit (50) (Qiagen,Cat#74104) and Qiagen RNase-Free DNase Set (50) (Qiagen, Cat# 79254) and following the manufacturer's protocols.
For the salivary gland isolation, about 20 pairs of salivary gland tissues from adult mated female flies were dissected in cold PBS.Excessive PBS was removed as much as possible from the tissue collection after a quick spin-down.Samples were resuspended in an appropriate amount of TRIzol Reagent (Thermo Fisher, Cat#15596026) following the manufacturer's protocols.Samples were flash frozen in liquid nitrogen and stored at -80 °C until enough samples were collected.RNA extractions were performed using the TRIzol Reagent (Thermo Fisher, Cat#15596026) and following the manufacturer's protocols.

Generation of transgenic flies
The Muc68D-tag line was generated using the plasmid containing the Muc68Dtag construct (ConstructID#35649249831002394_H07) generated by Sarov et al. (2016).
The plasmid was injected into the embryos of the recipient fly line with germline phiC31expression and the attP40 landing site (yw, nos-PhiC31; attP40).Flies with successful recombination events as indicated by the dsRed phenotypic marker were selected and balanced.
The vir-1-GFP line was generated based on the MiMIC line MI04501 whose insertion was located in the first vir-1 coding intronic region by following the MiMIC RMCE protocol (Li-Kroeger et al. 2018).

Reanalysis of esophagus X-ray micro-computed tomography (XR µCT) data
The X-ray micro-computed tomography (XR µCT) data for the adult fly esophagus from Dodge et al. (2023) was reanalyzed using IMARIS software (v10.0.0).Specifically, samples with LpWF colonization, and LpWF and Acetobacter indonesiensis (Ai) co-colonization were examined.Samples were reoriented and resampled with the esophagus perpendicular to the plane of the screen using the free rotation function.

Reagent or
Dataset S5.Cell type specific gene expression and associated transgenic lines identified.

Figures
For all RNAseq experiments, purified total RNA samples were quantified and examined using the Qubit RNA High Sensitivity (HS) Assay Kit (Thermo Fisher, Cat#Q32852) and the Agilent RNA 6000 Pico Kit (Agilent, Cat#5067-1513) on the 2100 Bioanalyzer System for the quantification and quality control.The TruSeq Stranded Total RNA Library Prep Human/Mouse/Rat, 48 Samples kit (Illumina, Cat#20020596) was used for proventriculus and crop samples, and the TruSeq Stranded mRNA Library Prep, 48 Samples kit(Illumina, Cat#20020594)  was used for salivary gland samples, following the manufacturer's protocols.The indexes used were from TruSeq RNA Single Indexes Set A, 12 Indexes, 48 Samples kit(Illumina, Cat#20020492).Sequencing was conducted on the Illumina NextSeq500 using 75bp single-end reads.

Figure S2 .
Figure S2.Electron microscopical analysis reveals PV outer wall cells and the PM.

Figure S4 .
Figure S4.The crop expands supporting transient food storage.