Developmental Biology

Spatiotemporal WNT and BMP gradients orchestrate regional enteroendocrine cell diversity along the Drosophila midgut

Jiaying Lv
Xingting Guo author has email address
Rongwen Xi author has email address

School of Life Science, Tsinghua University, Beijing, China
National Institute of Biological Sciences, Beijing, China
Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing, China

https://doi.org/10.7554/eLife.108542.1

Open access
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Figures and data

The distribution pattern of EE subtypes along the length of adult Drosophila midgut.
(A-F) Midguts of CCHa2-LexA>GFP flies stained with anti-Pros (blue), anti-AstC (white) and anti-Tk (red) antibodies 10 days after eclosion. Separated color channels for the insets from different gut regions in (A) were enlarged and displayed in (B-F), showing EE distribution characteristic of different gut regions. Scale bar represents 300 μm. (G) Quantification of the three major EE subtype percentage in different gut regions of flies 10 days after eclosion. Error bars represent Mean ± SEM, n=5. (H) Quantification of EE subtype percentage in the R4bc region of flies 10 days after eclosion. Error bars represent Mean ± SEM, n=5. (I) A schematic diagram showing the distribution patterns of the three EE subtypes along the Drosophila midgut.

Choices of symmetric or asymmetric division of EEPs contribute to regional EE diversity.
(A-E) Midguts stained with anti-Pros (blue) and anti-AstC (white) antibodies in CCHa2-LexA>GFP, Tk-Gal4>RFP flies fed with 5% DSS for 2-3 days. EE pairs were pointed by arrow head pairs. Scale bar represents 25 μm. (F) Quantification of different EE subtype pair percentage in different gut regions. (R1a: n=14; R1b-R2a: n=7; R2b: n=25; R2c: n=25; R3-R4c: n=48; R4b: n=64; R4c-R5: n=71) (G) Quantification of different EE subtype pair percentage in the R4b region. (n=64) (H) Speculation of EE subtype percentage in different gut regions by taking the percentage of EE subtype pairs from (F) to generate EEs. (I) Speculation of EE subtype percentage in the R4b region by taking the percentage of EE subtype pairs from (G) to generate EEs.

Notch is dispensable for the specification of both class I and III EE subtypes.
(A-E) Midguts stained with anti-Pros (green), anti-AstC (blue) and anti-Tk (white) antibodies in esg-Gal4^ts>Notch RNAi, CCHa2-LexA>mCherry flies shift to 29 °C for 5-10 days after eclosion. Scale bar represents 25 μm.

WNT signaling gradient orchestrates regional class II EE specification.
(A) Midguts of fz3-RFP flies stained with anti-RFP (red) antibodies 5 days after eclosion. (B-G) Midguts stained with anti-AstC (blue), anti-Tk (white) and anti-CCHa2 (red) in the following genotypes of flies driven by esg-Gal4^ts for 8 days since 48h APF: WT (B-C), UAS-arm^S¹⁰ (D), UAS-nkd (E), UAS-Tcf^DN(F) and UAS-Tcf RNAi (G). (H-I) Quantification of the three major EE subtype percentage in R1a (H) and R4b (I) of WT, UAS-nkd, UAS-Tcf RNAi and UAS-arm^S10flies. Error bars represent Mean ± SEM, ****p <0.0001 (two-way ANOVA). (J-K) Midguts stained with anti-AstC (blue), anti-DH31 (green), anti-CCHa2 (red) and anti-Tk (white) in the following genotypes of flies driven by esg-Gal4^ts for 10 days since 48h APF: WT (J-J’) and UAS-arm^S10 (K-K’). (L-M) Midguts stained with anti-AstC (blue), anti-NPF (green), anti-CCHa2 (red) and anti-Tk (white) in the following genotypes of flies driven by esg-Gal4^ts for 10 days since 48h APF: WT (L-L’) and UAS-arm^S10 (M-M’). (N-O) Midguts stained with anti-AstC (blue), anti-AstA (green), anti-CCHa2 (red) and anti-Tk (white) in the following genotypes of flies driven by esg-Gal4^ts for 10 days since 48h APF: WT (N-N’) and UAS-arm^S10 (O-O’). (P-Q) MARCM clones co-stained with anti-AstC (blue), anti-Tk (white) and anti-CCHa2 (red). (P-P’’’) WT FRT82B clone, (Q-Q’’’) Apc^G10Q8 FRT82B MARCM. Scale bar in (A): 500 μm; others: 50 μm.

BMP signaling negatively regulate class III EE differentiation.
(A) Midguts of dad-LacZ flies stained with anti-LacZ (red) antibodies before eclosion. (B-E) Midguts stained with DAPI (blue), anti-2B10 (red) and anti-CCHa2 (white) in the following genotypes of flies driven by esg-Gal4^ts for 8 days since 48h APF: WT (B), U-dad (C), U-Tkv^ACT (D-E). (F-R) Midguts stained with anti-AstC (blue), anti-Tk (white) and anti-CCHa2 (red) in the following genotypes of flies driven by esg-Gal4^ts for 8 days since 48h APF: WT (F, J and M-O), UAS-dad (G and K), UAS-mad RNAi (H and L) and UAS-Tkv^ACT(I and P-R). (S-W) Quantification of the three major EE subtype percentage in R1a (S), R2 (T), R3 (U), R4b (V) and R4c (W) of UAS-dad, UAS-mad RNAi and UAS-Tkv^ACT flies. Error bars represent Mean ± SEM, *p <0.05; **p <0.01; ***p <0.001; ****p <0.0001 (two-way ANOVA). (X-Y) Midguts stained with anti-AstC (blue), anti-NPF (green), anti-CCHa2 (red) and anti-Tk (white) in the following genotypes of flies driven by esg-Gal4^ts for 10 days since 48h APF: WT (X-X’) and UAS-Tkv^ACT (Y-Y’). Scale bar in (A): 500 μm; (C): 100 μm; others: 50 μm.

Manipulating WNT and BMP signaling in differentiated EEs has a mild impact on the regional specification of class II and III EEs.
(A-L) Midguts stained with anti-Pros (red), anti-AstC (blue), anti-Tk (white) and anti-CCHa2 (green) in the following genotypes of flies driven by pros-Gal4: WT (A-E), UAS-dad (F-G), UAS-Tkv^ACT (H-J), U-nkd (K) and U-arm^S10 (L). Yellow arrow heads in (I and L) represent ectopic class II EEs in R4b region, and blue arrow heads in (J) represent ectopic class III EEs in R4c region. Scale bar represents 25 μm. (M-T) Quantification of the three major EE subtype percentage in different gut regions of UAS-nkd, UAS-arm^S10, WT, UAS-dad and UAS-Tkv^ACT flies. Error bars represent Mean ± SEM, **p <0.01; ***p <0.001; ****p <0.0001 (two-way ANOVA).

WNT and BMP signaling cooperatively regulate regional EE diversity
(A) Midguts stained with anti-AstC (blue), anti-Tk (white) and anti-CCHa2 (red) in esg-Gal4^ts>U-nkd, U-Tkv^ACT flies shift to 29 °C for 8 days since 48h APF. (B-F) Midguts stained with anti-AstC (blue), anti-Tk (white) and anti-CCHa2 (red) in the following genotypes of flies driven by esg-Gal4^ts for 8 days since 48h APF: WT (C-D) and UAS-arm^S10, UAS-Tkv^ACT (B and E-F). (G) Midguts stained with anti-AstC (blue), anti-Tk (white) and anti-CCHa2 (red) in esg-Gal4^ts>U-dad, U-arm^S10 flies shift to 29 °C for 8 days since 48h APF. (H-L) Midguts stained with anti-AstC (blue), anti-Tk (white) and anti-CCHa2 (red) in the following genotypes of flies driven by esg-Gal4^ts for 8 days since 48h APF: WT (L) and UAS-dad, UAS-nkd (H-K). (M) A schematic diagram describing the function of WNT and BMP gradients in regulating EEP division modes. (N) A schematic diagram on the relationships among EE subtype distribution pattern, WNT and BMP signaling activity and the EEP division modes along the fly midgut. Scale bar in (C) and (I): 50 μm; others: 300 μm.

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