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. 2017 Jul 3;36(13):1928-1945.
doi: 10.15252/embj.201695622. Epub 2017 May 22.

Intrinsic regulation of enteroendocrine fate by Numb

Jérémy Sallé  1   2 , Louis Gervais  1   2 , Benjamin Boumard  1   2   3 , Marine Stefanutti  1   2 , Katarzyna Siudeja  1   2 , Allison J Bardin  4   2
Affiliations

Intrinsic regulation of enteroendocrine fate by Numb

Jérémy Sallé et al. EMBO J. .

Abstract

How terminal cell fates are specified in dynamically renewing adult tissues is not well understood. Here we explore terminal cell fate establishment during homeostasis using the enteroendocrine cells (EEs) of the adult Drosophila midgut as a paradigm. Our data argue against the existence of local feedback signals, and we identify Numb as an intrinsic regulator of EE fate. Our data further indicate that Numb, with alpha-adaptin, acts upstream or in parallel of known regulators of EE fate to limit Notch signaling, thereby facilitating EE fate acquisition. We find that Numb is regulated in part through its asymmetric and symmetric distribution during stem cell divisions; however, its de novo synthesis is also required during the differentiation of the EE cell. Thus, this work identifies Numb as a crucial factor for cell fate choice in the adult Drosophila intestine. Furthermore, our findings demonstrate that cell-intrinsic control mechanisms of terminal cell fate acquisition can result in a balanced tissue-wide production of terminally differentiated cell types.

Keywords: Drosophila midgut; Numb; adult intestinal stem cell; enteroendocrine cells; fate homeostasis.

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Figures

Figure 1
Figure 1. Enteroendocrine fate choice does not relies on a negative feedback loop from the differentiated enteroendocrine cells
  1. A–F

    (A, C, E) Adult posterior midguts containing control clones, those lacking EEs, or with extra EEs (clones outlined), observed 10 days after heat shock (AHS). Images are compiled tile scans stitched together in Zen software. In (A, C), we used a modified MARCM technique in which ProsGal4 was clonally activated when GAL80 was clonally lost through FLP/FRT‐mediated mitotic recombination. Clones were detected by the loss of RFP baring Gal80 chromosome. Scale bar = 100 μm. (A) Control wild‐type clones marked by lack of RFP expression, DNA in blue, and EE cells marked by Pros in green. (B) Schematic view of (A) with EE cells represented in red and clone delineated in black and shown in white. (C) EE depletion by clonal expression of rpr under the control of prosGal4 using the GAL80 system in negatively marked clones (lacking RFP expression and GAL80 expression). (D) Scheme of (C). (E) EE accumulation in MARCM clones (green) expressing RNAi against Notch. DAPI in blue, EE cells marked through Pros, in red. (F) Scheme of (E).

  2. G

    Global EE density measured in clonal and non‐clonal compartments in control wild‐type clones, upon EE depletion and ectopic EE cells. The bars represent mean values ± SD.

  3. H, I

    Number of EE cells located in a 30‐μm‐wide band around each individual clone normalized by clonal area (H) and clonal perimeter (I). The bars represent mean values ± SEM.

Data information: Results were compared using a two‐tailed Mann–Whitney statistical test (ns = non‐significant, P‐values indicated).
Figure EV1
Figure EV1. Local EE fate imbalance does not trigger proliferative response
  1. A–C

    Adult posterior midguts containing control clones, those lacking EEs, or with extra EEs (clones outlined), observed 10 days AHS. Images are compiled tile scans stitched together in Zen software. In (A, C), we used a modified MARCM technique in which ProsGal4 was clonally activated when GAL80 was clonally lost through FLP/FRT‐mediated mitotic recombination. Clones were detected by the loss of RFP baring Gal80 chromosome. (A, A′) Control wild‐type clones marked by lack of RFP expression, DNA in blue, dividing cells marked by PH3 in green and EE cells marked by Pros in red (right panel). (B, B′) EE depletion by clonal expression of rpr under the control of prosGal4 using the GAL80 system in negatively marked clones (lacking RFP expression and GAL80 expression). (C, C′) EE accumulation in MARCM clones (green) expressing an RNAi against Notch. DAPI in blue, EE cells marked through Pros, in red and dividing cells marked by PH3 in green and red as indicated. Scale bar = 100 μm.

  2. D

    Global dividing cells (PH3) density measured in clonal and non‐clonal compartments in control wild‐type clones, upon EE depletion and ectopic EE cells . The bars represent mean values ± SD.

  3. E, F

    Number of dividing cells (PH3) cells located in a 30‐μm‐wide band around each individual clone normalized by clonal area (E) and clonal perimeter (F). Results were compared using a two‐tailed Mann–Whitney statistical test (ns = non‐significant, P‐value indicated). The bars represent mean values ± SEM.

Figure 2
Figure 2. Slit/Robo pathway is not involved on local EE cell fate regulation
  1. A–D

    Posterior midguts containing 10 days AHS MARCM clones outlined with dotted lines (GFP, green; EE cells, Pros, red; DAPI, blue). Scale bar = 30 μm. No variation of EE density was observed in the clonal periphery or within the clones when comparing (A, A′) control MARCM clones with (B–C′) clones expressing two different RNAis against slit or (D, D′) clones expressing UAS‐slit.

  2. E, F

    Number of EE cells located in 30 μm around each clone normalized by clonal area (E) and clonal perimeter (F).

  3. G–I

    Quantification of (G) the average number of EE cells per clone, (H) the percent of EE cells per clone, and (I) the global EE density measured in clonal and non‐clonal compartments in the different clonal condition.

Data information: Results were compared using a two‐tailed Mann–Whitney statistical test (ns = non‐significant). (E–H) The bars represent mean values ± SEM. (I) The bars represent mean values ± SD.
Figure EV2
Figure EV2. Numb is required for EE fate
  1. A

    Lineage tracing from Notch signaling‐activated cells using “Flp‐out” strategy: (A) UAS‐flippase under the control of a Notch response element (NRE)‐Gal4 triggered stable GFP expression from the “Flp‐out” cassette in cells where Notch pathway was active. After 5 days at 29°C, we observed that EE cells were never labeled by GFP suggesting that EE cells do not derive from a Notch active cell (GFP, green; EE cells marked by Pros, in red; DAPI, blue). (A′, A′′) Detail of a GFP‐negative dividing ISC. (A′′′, A′′′′) Detail of GFP‐negative EEs and GFP‐positive ECs. Scale bars = 20 μm (A), 10 μm (A′, A′′′).

  2. B, C

    (B, B′) Tk‐positive cells were present in wild‐type clones at 10 days AHS MARCM (GFP, green; Tachykinin (Tk) staining, red; DAPI, blue, whereas (C, C′) Tk‐positive cells were absent in numb 15 mutant clones. Scale bar = 30 μm.

  3. D, E

    (D, D′) EE cells were present in control clones at 5 days AHS, whereas (E, E′) EE cells are lost in numb 1 mutant clones (GFP, green; EE cells marked by Pros; DAPI, blue). Scale bar = 30 μm.

  4. F–H

    Quantification of (F) the average clone size, (G) the average number of EE cells per clone, and (H) the percentage of clones that contained at least one EE cell. Results were compared using two‐tailed Mann–Whitney test for (F, G); and the Fisher's exact test for (H) (ns = non‐significant (P‐value indicated). The bars represent mean values ± SEM.

  5. I, J

    Control (I) and numb RNAi (J) expressing clone made via heat‐shock‐induced excision of a stop cassette allowing Act‐Gal4‐driven RNAi expression (outlined GFP, green; EE cells marked by Pros; DAPI, blue). Scale bar = 30 μm.

  6. K

    Average number of EE cells per clone in wild‐type clones and clones expressing a numb RNAi. Results were compared using two‐tailed Mann–Whitney test (P‐value indicated). The bars represent mean values ± SEM.

  7. L, M

    (L, L′) EE cells were present in control clones at 10 days AHS, whereas (M, M′) EE cells are lacking in numb 124 mutant clones (GFP, green; EE cells marked by Pros; DAPI, blue). Scale bar = 30 μm.

  8. N

    Average number of EE cells per clone in wild‐type clones and numb 124. Results were compared using a two‐tailed Mann–Whitney test. The bars represent mean values ± SEM.

Figure EV3
Figure EV3. ISCs and EC are present in numb mutant clones
  1. A, B

    ISCs are present in control clones (A, A′) and numb 15 mutant clones (B, B′) at 10 days AHS (GFP, green; ISCs marked by Delta; DAPI, blue). Scale bar = 30 μm.

  2. C–F

    Quantification of (C) the average number of ISCs per clone, (D) the percentage of ISCs per clone, (E) the average number of ECs per clone, and (F) the percentage of ECs per clone. Results were compared using a two‐tailed Mann–Whitney test [ns = non‐significant (P > 0.05)]. The bars represent mean values ± SEM.

  3. G, H

    ISCs are present in control clones (G, G′) and UAS‐numb expressing clones (H, H′) at 10 days AHS (GFP, green; ISCs marked by Sanpodo; DAPI, blue). Scale bar = 30 μm.

  4. I–L

    Quantification of (I) the average number of ISCs per clone, (J) the percentage of ISCs per clone, (K) the average number of ECs per clone, and (L) the percentage of ECs per clone. Results were compared using a two‐tailed Mann–Whitney test (ns = non‐significant; P > 0.05). The bars represent mean values ± SEM.

Figure 3
Figure 3. Numb is required for EE fate determination in adult midgut
  1. A–D

    10 days AHS MARCM clones (GFP, in green; Pros, red or white as indicated; DAPI, blue) are outlined with dotted lines. (A, A′) Wild‐type clones contained EE cells. (B, B′) EE cells were lost in numb 15 mutant clones. (C, C′) EE cells were present upon expression of UAS‐numb. (D, D′) EE cells were present when UAS‐numb was expressed in numb 15.

  2. E–H

    Quantification of (E) the average clone size, (F) the average number of EE cells per clone, and (G) the percentage of EE cells per clone, and (H) the percentage of clones that contained at least one EE cell. Results were compared using two‐tailed Mann–Whitney test for (E, F, G); and the Fisher's exact test for (H) (ns = non‐significant, P‐values indicated). The bars represent mean values ± SEM.

Data information: Scale bar for D–G = 30 μm.
Figure 4
Figure 4. Numb segregates both asymmetrically and symmetric in dividing cells in the intestine
  1. A–C

    Numb‐GFP (green) was observed in all cells and showed a cytoplasmic and membrane localization [Numb‐GFP, green; PH3, red in (A); Dl, red in (A′); DAPI, blue]. In ISC early mitosis however, Numb‐GFP accumulated on the basal side of the cell [arrow in basal optical section in (A′′′)]. Scale bar = 5 μm. (B, C) Reconstructed lateral view of ISCs (Numb‐GFP, green; Dl, red; DAPI, blue). Scale bar = 5 μm. (B) In interphase, Numb‐GFP localized at the membrane and cytoplasm. (C) In early mitosis, Numb accumulated at the basal cortex (see arrows; see also Movie EV1).

  2. D, E

    Numb‐GFP localized asymmetrically in 78% of the divisions, and symmetrically in 22% of the divisions (n = 102; Numb‐GFP, green; PH3, red; DAPI, blue; see also Movies EV2 and EV3).

  3. F

    ISC divisions with symmetric segregation of Numb were preferentially parallel to the epithelial plane, P = 0.0021, a χ2 test was used.

Figure EV4
Figure EV4. Numb segregation and Prospero segregation
  1. A, B

    (A–A′′) Prospero was absent in most divisions (94.6%) showing an asymmetric distribution of Numb‐GFP. (B–B′′) However in some divisions (5.4%), co‐localization with Numb‐GFP was observed (n = 74; Numb‐GFP, green in A and B, white in A′ and B′; Pros, red in A and B, white in A′′ and B′′; PH3, white; DAPI, blue). Scale bar = 2 μm.

  2. C

    Upon expression of esgGal4, tub‐Gal80ts‐driven UAS‐ttk69‐RNAi, Numb‐GFP showed an asymmetric distribution in 77.7% of late anaphases (n = 9; PH3, red; Numb‐GFP, green in C, white in C′; DAPI, blue).

  3. D

    Upon expression of esgGal4, tub‐Gal80ts‐driven UAS‐aPKC‐CAAX, Numb‐GFP showed symmetric distribution in 92.9% of late anaphases (n = 17; PH3, red; Numb‐GFP, green in D, white in D′; DAPI, blue).

  4. E

    In lgl 4 mutant clones, Numb‐GFP showed asymmetric distribution in 100% of late anaphases (n = 4; PH3, red; Numb‐GFP, green in E, white in E′; DAPI, blue).

Figure 5
Figure 5. The Notch receptor is not activated in EE precursors

  1. ISCs (marked by Dl, red) showed NiGFP (green) localized in cytoplasmic vesicles, while in the adjacent presumptive EB cell, active cleaved NiGFP relocalized in the nucleus. NiGFP was not detected in ECs (polyploid Pdm1+, white, n = 68).

  2. A majority of EE cells (79.7% of Pros+ cells, n = 179) marked by Pros (red) did not express NiGFP (green).

  3. 20.3% of Pros+ cells co‐expressed NiGFP, though NiGFP remained cortical and did not show nuclear localization indicative of receptor activation. Of these, 59% (22/179 Pros+ cells) co‐expressed Dl (red). EE cells (Pros, white).

Data information: Scale bars = 5 μm.
Figure 6
Figure 6. Numb controls EE fate through Notch signaling inhibition
  1. A–K

    (A–B′; F–G′) 10 days AHS MARCM clones outlined with dotted lines (GFP, green; EE cells marked by Pros, in red or white as indicated; DAPI, blue). (A, A′) Notch RNAi resulted in extra EE cells. (B, B′) Similarly, a numb 15 mutant clone expressing Notch RNAi contained extra EE cells. Quantification of: (C) clone size, (D) the average number of EE cells per clone, and (E) the percentage of EE cells per clone. (F, F′) EE cells were greatly reduced in ada ear4 mutant clones. (G, G′) Expression of UAS‐numb did not promote EE cell production in ada ear4 mutant clones. Quantification of: (H) clone size, (I) the average number of EE cells per clone, (J) the percentage of EE cells per clone, and (K) and percentage of clones containing at least one EE cell.

Data information: Scale bars = 30 μm. Results were compared using two‐tailed Mann–Whitney statistical test for (C, D) and the Fisher's exact test for (E) (P‐values indicated). The bars represent mean values ± SEM.
Figure 7
Figure 7. Numb is an upstream regulator of EE fate determination
  1. A–K

    (A–F′, J–K′) 10 days AHS MARCM clones (GFP, in green) outlined with dotted lines; Pros, red or white as indicated or otherwise specified; DAPI, blue). (A, A′) EE cells were formed upon UAS‐scute expression and (B, B′) when UAS‐scute was expressed in numb 15 mutant clones. (C, C′) Similarly, UAS‐asense promoted EE cell formation in wild‐type or (D, D′) in UAS‐asense, numb 15 mutant clones. (E, E′) Ttk69 RNAi produced ectopic EE cells alone as well as (F, F′) in combination with numb 15 mutant. Quantification of (G) cells per clone, (H) average number of EE cells per clone, and (I) percentage of EE cells per clone. EE cells (marked by Tk in red or white) were lost in pros 17 mutant clones, (J, J′) The expression of UAS‐numb failed to promote EE cells in pros 17 mutant clones (Tk, red or white in F′). (K, K′) Extra EE cells were formed upon ttk69 RNAi expression. (F, F′) Likewise, ttk69 RNAi expression promoted EE fate in numb 15 mutant clones.

Data information: (G–I) Results were compared using a two‐tailed Mann–Whitney statistical test (ns = non‐significant, P‐values indicated). Scale bar = 30 μm. The bars represent mean values ± SEM.
Figure 8
Figure 8. Modification of cell polarity factors alters EE fate decisions
  1. A–F

    MARCM clones (GFP, green, outlined; Pros, red; DAPI, blue) observed at 10 days AHS. (A, A′) Wild‐type control clones. (B, B′) Expression of UAS‐aPKC‐CAAX showed a slight increase in EE cells [quantified in (H) and (I)] and a significant increase in clusters of EE cells [quantified in (J)]. A large clone is shown for illustration. (C, C′) EE cells were diminished in lgl 4 mutant clones. (D, D′) Overexpression of UAS‐Numb5A promoted EE formation. (E, E′) Control clones for F, F′. (F, F′) aPKC 06403 mutant clones showed reduced EE cells. Scale bars = 30 μm.

  2. G–J

    Quantification of: (G) the average clone size, (H) the average number of EE cells per clone, (I) the percentage of EE cells per clone, and (J) the percentage of EE clusters. The bars represent mean values ± SEM.

  3. K–N

    3 days AHS single‐cell MARCM clones (GFP, green; Pros, red; DAPI, blue). Arrows show single‐cell clones. Scale bar = 20 μm. (K) In wild type, single‐cell EE clones were 20%, (L) whereas single‐cell clones in numb 15 mutant were only 2.6% EEs. (M) Quantification of the percentage of differentiated cells EEs in single‐cell clones. (N) Quantification of the total single‐cell clones.

Data information: (G–J) Results were compared using two‐tailed Mann–Whitney statistical test for (G, H), Fisher's exact test for (I), and χ2 test for (J) (ns = non‐significant, P‐values indicated). (M, N) Results were compared using a Fisher's exact test (P‐value indicated).
Figure 9
Figure 9. A model for Numb promoting fate plasticity and controlling EE fate acquisition
Based on our data presented here and previously published observations (Biteau & Jasper, 2014; Wang et al, 2015; Zeng & Hou, 2015), we suggest the following model of Numb function and regulation of EE fate acquisition.

  1. In 80% of ISC divisions, Numb was found to be asymmetrically distributed. We hypothesize that the Notch pathway is activated in the cell that does not inherit Numb and which differentiates into an enterocyte. Furthermore, we propose that the cell inheriting Numb does not activate the Notch pathway and likely remains an ISC. Of note, we previously demonstrated that numb is not essential for ISC fate acquisition or maintenance or EC fate (Fig EV3 and Bardin et al, 2010).

  2. In 20% of ISC divisions, Numb is segregated symmetrically in the two daughter cells. We hypothesize that Notch activity is inhibited by Numb in the two daughter cells maintaining them in a plastic state capable of ISC and EE fate. The symmetrically segregated pool of Numb likely biases fate decisions by lowering Notch activity, but Numb expression must also be maintained to for EE terminal differentiation. Our genetic data suggest that Numb inhibits Notch through its interaction with the AP‐2 complex. Inactivation of Notch drives expression of the proneural genes Scute and Asense, which, in turn, promote Pros expression and EE fate acquisition. Parallel action of these genes cannot be ruled out. Ttk69 is also required to block EE fate (Wang et al, 2015).

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