Gut-Neuron Interaction Via Hh Signaling Regulates Intestinal Progenitor Cell Differentiationin

Gut-neuron Interaction via Hh Signaling Regulates Intestinal Progenitor Cell Differentiationin Drosophila

Hui Han1, Chenyu Pan1, Chunying Liu1, Xiangdong Lv1, Xiaofeng Yang1, Jialin Fan1, Yuanxin Xia1, Yue Xiong1, Yi Lu1, Wenqing Wu1,Junhai Han2, Zhaocai Zhou1, Hai Jiang1, Lei Zhang1, Yun Zhao1*

1 State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai, 200031, P.R. China

2 Institute of Life Sciences, the Key Laboratory of Developmental Genes and Human Disease, Southeast University, 2 Sipailou Road, Nanjing, 210096, P.R. China

*Correspondence to: Yun Zhao (Tel: 86-21-54921618; E-mail: ).

Contents

Supplementary Figures (S1-S12)

Supplemental Experimental Procedures

Supplementary Information,Figure 1 Locations of Neurons and progenitor cells in intestine.

(A-B’’’) Immunostaining of neurons and progenitor cells in intestine.Progenitor cells (esglacZ+: red ;Arm+/Pros-: blue staining in cytoplasm without blue staining in nucleus) are detected in the basal layer (B-B’’) while not being detected in the outside of intestine (A-A’’). Neurons (EGFP+: green)are detected in the layer outside epithelium cells(A’’’) while few signal is detected in the basal layer(B’’’). In all the confocal imaging, nuclei aremarked with DAPI (white).

Supplementary Information, Figure 2 Neuron ablation induces an accumulation of progenitor cells and promotes proliferation of ISCs.

(A and B) Immunostaining of neurons in intestinesof control (A) and elavGal4UAS-grimts(B) flies. Overexpression of UAS-grim with elavGal4for 7 days causes neuron ablation. In all the confocal imaging, nuclei aremarked with DAPI (white).

(C) qPCR analysis of intestine progenitor-specific marker esg under indicated experimental conditions. The results showed hereare Means ± SD from 3 independent experiments. In each experiment, at least 10 intestines were pooled together.

(D-F’) Immunostaining of progenitor cells (Arm+/Pros-: green staining in cytoplasmand no green staining in nucleus) and dividing intestinal stem cells (arrows; PH3+: red) in control (D),hid (E and E’) andrpr (F and F’)overexpressing flies.

(G and H) Immunostaining of ISCs (Delta+: green staining incytoplasm) and EEs (Pros+: green staining in nucleus) in control (G) and hid overexpressing(H) flies.

(I and J) Immunostaining of ECs (Pdm1+: green) in intestines of control (I) and hidoverexpressing(J) flies.

Supplementary Information, Figure 3 Neuron ablation by pdfGal4 and ok371Gal4 induce similarphenotypes as does elavGal4.

(A-A’’’) elavGal4 (A and A’; GFP: green) is also expressed in intestinal progenitor cells (A’’, arrowhead; esglacZ: red and Arm+Pros-: blue). EEs are marked with arrows (A’’’; Pros+: blue).

(B-C’) Immunostaining of intestinal progenitor cells (Arm+/Pros-: green staining in cytoplasm) and EEs (Arm+/Pros+: green staining in nucleus) in pdfGal4 (B-B’), and neuron ablation (C-C’) flies. After neuron ablation bypdfGal4 driving UAS-grimts, there is an accumulation of progenitor cells.

(D-E’)Immunostaining of intestinal progenitor cells (Arm+/Pros-: green staining in cytoplasm) and EEs (Arm+/Pros+: green staining in nucleus) in ok371Gal4 (D-D’), and neuron ablation (E-E’) flies. After neuron ablation by ok371Gal4 driving UAS-grimts, there is an accumulation of progenitor cells.

(F-F’)Immunostaining of ISCs with mitotic activity (arrows; PH3+:red). The proliferation rate is dramatically increased after neuron ablation using pdfGal4.

(G-H) qPCR analysis of the ISC-specific marker delta(G) and progenitor cell-specific marker esg (H). After pdfGal4mediated neuron ablation by driving UAS-grimts overexpression, the expression level of deltaand esgin total intestine is significantly increased. In each experiment, at least 10 intestines were pooled together for analysis.

Supplementary Information, Figure 4 Downregulation of Hh signaling with ptcGal4ts induces similar phenotypes as those induced by neuron ablation, while upregulation of Hh signaling in neurons induces opposite phenotypes.

(A-C’) Immunostaining of intestinal progenitor cells (Arm+/Pros-: green staining in cytoplasm;esglacZ+: red) and EEs (Arm+/Pros+: green staining in nucleus) in control (A-A’), ci RNAi (B-B’), ptc RNAi (C-C’) flies. After ci RNAi, there are many abnormal progenitor cell marker-positive cells with large nuclei, resembling ECs (arrowheads). In all the confocal imaging, nuclei aremarked with DAPI (white).

(D-F’) Immunostaining of ISCs (arrowheads;Delta+: red staining in cytoplasm) and EEs (arrows; Pros+: red staining in nucleus) in the midguts of control (D-D’), ci RNAi (E-E’) and ptc RNAi (F-F’) flies.

(G-I) Immunostaining of EBs (Gbe-Su(H)-lacZ+: green) in control (G), ci RNAi (H), ptc RNAi (I) flies. After ci RNAi, many EB marker-positive cells show an EC-like phenotype (big nuclei; arrowheads; H).

(J and K) Cross section immunostaining of ECs (Pdm1+: green) in control (J) and ci RNAi (K) flies.

(L-N) Longitudinal section of ECs (large nuclei: white) in intestines of control (L), ptc RNAi (M) and UAS-ptc overexpressing (N) flies. Progenitor cellsaremarked with Arm+/Pros- (green staining in cytoplasm).

Supplementary Information, Figure5 Neuronal Hh signaling is essential for the regulation of intestinal homeostasis.

(A-C’) Immunostaining of neurons in intestines of elavGal4 control (A-A’), neuron specificci RNAi (B-B’), and ptc RNAi (C-C’)flies. Overexpressing ci RNAi or ptc RNAi for 2 weeks doesnot induce neuron depletion. Neurons were marked with UAS-syt-EGFP (green) and Pdf (red). In the all confocal imagings, nuclei aremarked with DAPI (white).

(D) qPCR analysis of intestine progenitor-specific marker delta under indicated experimental conditions. The data depicted hereare Means ± SD from 3 independent experiments. In each experiment, at least 10 intestines were pooled together. ***p<0.005.

(E)Statistic analysis about therelative percentage of progenitor cells (PCs) and ECs from TEM. The resultsare the average data of two independent experiments.

(F-H’) Immunostaining of progenitor cells (Arm+/Pros-: green staining in cytoplasmand no green staining in nucleus) in intestines of control (F-F’), neuron specific smo RNAi (G-G’) and neuron specific overexpression of UAS-ci1031(H-H’) flies. Overexpression of smo RNAi in neurons induces an accumulation of progenitor cells while overexpression of UAS-ci103 in neurons induces a reduction of progenitor cells.

(I-K) Immunostaining of EBs (Gbe-Su(H)-lacZ+: green) in intestines of control (I), neuron specific smo RNAi (J) and neuron specific overexpression of UAS-ci103 (K) flies. Overexpression of smo RNAi in neurons inducesan accumulation of EBs (J) while overexpression of UAS-ci103in neurons induces a decreased number of EBs (K).

(L-N) Immunostaining of ECs (Pdm1+: green) in intestines of control (L), neuron specific smo RNAi (M) and neuron specific overexpression of UAS-ci103 (N) flies. Overexpression of smo RNAi in neurons causes a reduction of ECs (M) while overexpression of UAS-ci103 in neurons causes an accumulation of ECs (N).

(O) qPCR analysis of intestine progenitor-specific marker esg under indicated experimental conditions. The depicted dataare Means ± SD from 3 independent experiments. In everyexperiment, at least 10 intestines were pooled together. ***p<0.005.

(P-Q’) Immunostaining of ISCs (Delta+: red staining in cytoplasm) and EEs (Pros+: red staining in nucleus) in control (P and P’) and neuron specific smo RNAi (Q and Q’) flies. Overexpression of smo RNAi in neurons increases the number of ISCs and EEs.

(R and R’) Longitudinal section immunostaining of midgut in ptc RNAi flies. ECs arelabeled with Pdm1 staining(red).

(S-V) TUNEL assay in intestines of control (T), neuron specificci RNAi (U) and smo RNAi (V) flies. Overexpression ci RNAi or smo RNAi in neuronsdoesnot induce an increased apoptosis level (TUNEL assay: green). DNase I treatment was used as a positive control in TUNEL assay (S).

Supplementary Information, Figure6Neuronal Hh signaling is involved in the differentiation towards ECs.

(A-A’’) BrdU tracing experiment in the intestineof elavGal4 control flies. About half of the BrdU+ (red) cells are progenitor cells (arrows;esglacZ+: green). Anotherhalf of the BrdU+ (red) cells are ECs (arrowheads; esglacZ-: big nuclei).

(B-B’’) BrdU tracing experiment in the intestine of neuron-specific ptc RNAi flies. Almost all of the BrdU+ (red) cells are ECs (arrowhead;esglacZ-; big nuclei).

(C-C’’) BrdU tracing experiment in the intestine of neuron-specific ci RNAi flies. Almost all of the BrdU+ (red) cells are progenitor cells (arrows;esglacZ+: green).

Supplementary Information, Figure 7 Neuronal Hh signaling works in a dose-dependent manner.

(A-A’) Immunostaining of Ptc in the intestine. Ptc is marked with red (A’). Intestinal progenitor cells are marked with esgGal4 > UAS-GFP (green). There is no Ptc located in intestinal progenitor cells.

(B-B’’’) Immunostaining of Ci and Smo in the intestine. Intestinal progenitor cells are marked with esgGal4 > UAS-GFP (green) (B’); Ci is marked with red (B’’); Smo is marked with blue (B’’’). There is no Ci and Smo signal in intestinal progenitor cells.

(E-H) Immunostaining of intestinal progenitor cells (GFP: green) in intestines of control (E), ci RNAi overexpression (F), smo RNAioverexpression (G)and ptc RNAi overexpression flies (H) driven by esgGal4ts. Overexpression of ci/smo/ptc RNAiin intestinal progenitor cells does not induce dramatic changes of progenitor cells.

(I-K’’) Immunostaining of intestinal progenitor cells (Arm+/Pros-: green staining in cytoplasm and no green staining in nucleus), EEs (Arm+/Pros+: green staining in nucleus) and mitosis ISCs (PH3+: red) in intestines of control (I and I’), neuronal UAS-smoSD123overexpression (J and J’) and neuronal UAS-smo overexpression driven by elavGal4(K–K’’) flies. Overexpression of UAS-smoSD123 induces a reduction of intestinal progenitor cells while UAS-smo induces an accumulation of progenitor cell and an increased proliferation rate (K’’).

(L) Quantitative analysis of dividing ISC cells (PH3+) in the intestines of control, neuronalUAS-smooverexpression and neuronalUAS-smoSD123overexpression flies. Overexpression of UAS-smo in neurons induces an increased proliferation rate, on the other hand, overexpression of UAS-smoSD123 in neurons induces a decreased proliferation rate.

Supplementary Information, Figure8 Hh is involved in the regulation of intestinal homeostasis.

(A-C’) Longitudinal section immunostaining of ECs (myoIAGal4ts>GFP+: green) in intestine of control (A and A’), EC-specific hh RNAi (B and B’) and EC-specific UAS-hh overexpressing (C and C’) flies.

(D)Immunostaining of ISCs with mitotic activity (arrows; PH3+: green). Proliferation rate is dramatically increased after hh RNAi is induced in ECs.

(E-G’) Immunostaining of intestinal progenitor cells (Arm+/Pros-: green staining in cytoplasm and no green staining in nucleus) and EEs (Arm+/Pros+: green staining in nucleus) in intestines of control (E and E’), hh overexpression (F and F’) and hh RNAi (G and G’) flies. Overexpression of hh does not induce dramatic changes of progenitor cells while hh RNAi induces an accumulation of progenitor cell. In all the confocal imaging, nuclei aremarked with DAPI (white).

(H-J’) Immunostaining of ECs (Pdm1+: green) in intestines of control (H and H’), hh overexpression (I and I’) and hh RNAi (J and J’) flies. Overexpression of hh induces an accumulation of ECs while hh RNAi induces a reduction of ECs.

(K)Quantitative analysis of dividing ISC cells (PH3+) in the intestines of control,hh RNAi and UAS-hhflies. Shown are Means ± SD, ***p<0.005.

Supplementary Information, Figure 9 Neuronal ci RNAi could cover phenotypes caused by EC-hh overexpression.

(A-D’)Immunostaining of intestinal progenitor cells (Arm+/Pros-: green staining in cytoplasm and no green staining in nucleus) and EEs (Arm+/Pros+: green staining in nucleus) in intestines of ci RNAi in ECs (A and A’), hh overexpression in ECs and neurons (B and B’), ci RNAi in ECs and neurons (C and C’) andci RNAi,hh overexpression in ECs and neurons (D and D’) flies. While overexpression of ci RNAiin ECs could not induce dramatic changes in intestine, overexpression of ci RNAi in ECs and neurons caused similar phenotypes as does ci RNAi in neurons. ci RNAi in ECs and neurons could cover the phenotypes caused by hh overexpression. In all the confocal imaging, nuclei are marked with DAPI (white).

Supplementary Information, Figure10 JAK/STAT responds to neuronal Hh signaling.

(A-C’)Immunostaining of 10 X stat92E-GFP in control (green; A and A’), neuronal ci RNAi (green; B and B’) and neuronal ptc RNAi(green; C and C’)flies.RNAis were driven by elavGal4.

Supplementary Information, Figure 11 Neuronal Hh signaling is required for intestinal homeostasis after DSS treatment.

(A-B’’) Immunostaining of intestinal progenitor cells (Small nuclei; Arm+/Pros-: green) and ECs (Pdm1+: red) in DSS-induced control gut (A-A’’) and neuronal Hh activation gut (B-B’’), in which ptc RNAi was overexpressed driven by elavGal4. Neuronal Hh activation partially rescues the phenotypes caused by DSS treatment.

(C-E) Immunostaining for cilacZ (green; C) in brain of cilacZ flies. Smo (green; D) and Ptc(green; E) staining in brains of yw flies.

Supplementary Information, Figure 12 Neuronal ablation and abnormal of neuronal hh signaling induced different phentypes of food intake.

(A)Prandial behavior analyzed in the CAFEAssay. Neuron ablation causes a decrease of food consumption, and abnormal of neuronal Hh signaling has no detectableeffect on it.

(B-E) Digestion situation of control, neuronal ci RNAi, neuronal ptc RNAi and neuron ablation flies. After 1 hour of food intake, food has passed through entire gastrointestinal tract in control, neuronal ci RNAi and neuronal ptc RNAi flies.However, food has not reached the posterior midgut in neuron ablation flieseven after 2 hours of food intake.

Supplemental Experimental Procedures

Immunofluorescence staining

The entire gastrointestinal tract was taken and fixed in 1XPBS (10mM NaH2PO4/Na2HPO4, 175mMNaCl, pH 7.4) plus 4% EM grade formaldehyde (Sigma) for 1h.For Delta staining, thefixation time was reduced to 30 min. Afterfixation,intestineswererinsedtwotimesquickly in PBST,washed20minfor 3 timeswith PBST (1XPBS and 0.1% Triton X-100),andblockedfor1 hinPBST with2%normalhorseserum. Then Samples were incubated with primary and secondary antibodies in a solution containing PBST and 2% BSA for 2 h, however, the stainingtime forcy3-conjointed-Phalloitin was15 min andfor DAPI was5 min.

qPCR

Primers used are as following.

delta: forward 5’ TTCATTTGGACACTCGACTTGCT3’

reverse 5’ AAACGCATTGATTGGGCTTGTC3’

esg: forward 5’ GCCAAGAAGGACAAGAAC3’

reverse 5’ CACCCAACGAAACATAGG3’

rpl32: forward 5’ GCTAAGCTGTCGCACAAATGG3’

reverse 5’CGCTTGTTCGATCCGTAACC3’

CAFE Assay

Theexperiment was done as previously reported2. Calibrated glassmicropipettes (5ul, catalog no. 53432-706; VWR,West Chester,PA) were used.

Intestinal Assays using Food Dyes

Food with dyes was made by adding 0.5%Bromocresol purple sodium salt (17492, Sigma) to instant food. Flies were starved for 6 hours then fed with food with dyes. After fed for 10 minutes, chose the flies whose belly has turned red for experiments. Then dissected gastro-intact after fed them for 1hour and 2 hours.

References

1.Chen, Y., Goodman, R.H. & Smolik, S.M. Cubitus interruptus requires Drosophila CREB-binding protein to activate wingless expression in the Drosophila embryo. Molecular and cellular biology20, 1616-1625 (2000).

2.Ja, W.W. et al. Prandiology of Drosophila and the CAFE assay. Proc Natl Acad Sci U S A104, 8253-8256 (2007).