Pancreas lineage allocation and specification are regulated by sphingosine-1-phosphate signalling

Abstract

During development, progenitor expansion, lineage allocation, and implementation of differentiation programs need to be tightly coordinated so that different cell types are generated in the correct numbers for appropriate tissue size and function. Pancreatic dysfunction results in some of the most debilitating and fatal diseases, including pancreatic cancer and diabetes. Several transcription factors regulating pancreas lineage specification have been identified, and Notch signalling has been implicated in lineage allocation, but it remains unclear how these processes are coordinated. Using a combination of genetic approaches, organotypic cultures of embryonic pancreata, and genomics, we found that sphingosine-1-phosphate (S1p), signalling through the G protein coupled receptor (GPCR) S1pr2, plays a key role in pancreas development linking lineage allocation and specification. S1pr2 signalling promotes progenitor survival as well as acinar and endocrine specification. S1pr2-mediated stabilisation of the yes-associated protein (YAP) is essential for endocrine specification, thus linking a regulator of progenitor growth with specification. YAP stabilisation and endocrine cell specification rely on Gαi subunits, revealing an unexpected specificity of selected GPCR intracellular signalling components. Finally, we found that S1pr2 signalling posttranscriptionally attenuates Notch signalling levels, thus regulating lineage allocation. Both S1pr2-mediated YAP stabilisation and Notch attenuation are necessary for the specification of the endocrine lineage. These findings identify S1p signalling as a novel key pathway coordinating cell survival, lineage allocation, and specification and linking these processes by regulating YAP levels and Notch signalling. Understanding lineage allocation and specification in the pancreas will shed light in the origins of pancreatic diseases and may suggest novel therapeutic approaches.

Fig 1. S1pr2 and Sphk expression peaks during pancreas secondary transition.

(A-C) β-galactosidase activity by X-gal staining and immunofluorescence on cryosections in heterozygous 14.5 dpc S1P2 ^tm1lacz embryos indicated that S1pr2 is expressed in E-cad⁺ (A) and Pdx1⁺ (B) cells in the developing epithelium and in the mesenchyme (A, B). qPCR analysis showed that S1pr2 expression peaks at 14.5 dpc and subsequently declines (C). (D-F) Immunofluorescence showed that Sphk localisation is predominantly epithelial, as indicated by the strong Sphk co-staining with E-cadherin (D) and Pdx1 (E). qPCR analysis showed that Sphk1 and Sphk2 expression peaks at 14.5 dpc (F). Scale bars, 50 μm; *p < 0.05, **p < 0.01, ***p < 0.001 (C, F) in reference to E14.5 samples; error bars show standard error of the mean (SEM). For raw data please refer to S1 Data.

Fig 2. S1pr2 null pancreata are developmentally delayed but up-regulate Lpar1 and S1pr3.

(A-D) PCA of RNA Seq gene expression profiles of 14.5 dpc S1pr2^tm1Rlp null pancreata (in red) and 13.5, 14.5, and 15.5 dpc wt pancreata (in shades of black). PC1 shows developmental time because it shows the highest variability among wt samples and there is only a small variance in PC2. S1pr2 null pancreata cluster away from their wt counterparts, suggesting that they were developmentally delayed (A). Analysis of these RNA Seq data revealed that endocrine- (B) and acinar- (C) specific genes were strongly repressed in S1pr2 null pancreata at 14.5 dpc as compared to 14.5 dpc wt controls, whereas expression of duct-specific genes was less affected (D). (E) qPCR analysis of Lpar1 and S1pr3 expression in the FACS-isolated epithelial and mesenchymal components of 14.5 dpc wt (black bars) and S1pr2 null (red bars) pancreata indicated that expression of both Lpar1 and S1pr3 was selectively up-regulated in the S1pr2 null epithelium by 7- and 5-fold, respectively. (F-J) air–liquid interface (ALI) cultures of 13.5 dpc wt embryonic pancreata for 3 d results in the generation of several C-pep⁺ and Gcg⁺ cells (F), and combined treatment with 20 μM and 50 μM of the specific Lpar1 and S1pr3 inhibitors, respectively, had no effect (G, J). In contrast, the already reduced number of endocrine cells in the S1pr2 nulls (H, J) is further reduced upon addition of the two antagonists in these concentrations (I, J). Padj for all genes <0.05 except Foxa2 where Padj <0.1 (B-D); **p < 0.01, ***p < 0.001, ns not significant in reference to corresponding epithelial or mesenchymal values (E, F); error bars in E show SEM; error bars in J show standard deviation (SD); scale bars are at 80 μm. For raw data, please refer to the S1 Data file.

Fig 3. S1pr2 signalling promotes commitment of pancreas progenitors.

(A-H) Immunofluorescence analysis of embryonic pancreata at 14.5 dpc and after 2 d in ALI cultures (14.5 dpc + 2 d) shows the progression of the developmental program. This was apparent by the reduction of Nkx6-1⁺ (A, E) and Ngn3⁺ (C, G) progenitor cells, the corresponding increase of strongly labelled Pdx1⁺ cells (B, F) and the appearance of Ptf1a⁺/Pdx^- cells (D, H; arrows in H). (I-L) Immunofluorescence analysis at 14.5 dpc + 2 d in the absence of S1pr2 signalling clearly showed that the Nkx6-1⁺ population was expanded (I), Ngn3⁺ cells (K) and strongly labelled Pdx1⁺ cells (J) were dramatically reduced, and Ptf1a⁺/Pdx1^- cells were lost (L). (M-P) Quantitation of Nkx6.1⁺and Ptf1a⁺ cells (M), Pdx1⁺ cells (N), Ngn3⁺ cells (O), and acinar progenitors (Ptf1a⁺ / Pdx1^-) (P) in 14.5 dpc pancreata, 14.5 dpc pancreata cultured in ALI for 2 d in the absence or presence of 15 uM of the S1pr2 specific inhibitor JTE013. Statistical significance is shown for the comparisons between 14.5 dpc and 14.5 dpc + 2 d stainings (14.5 dpc + 2 d bar) as well as between 14.5 dpc + 2 d with and without S1pr2 block (14.5 dpc + 2 d + S1pr2 block bar). (Q-S) RNA Seq gene expression analysis on 14.5 dpc pancreatic explants after 2 d in ALI culture with S1pr2 signalling blocked revealed a coordinated repression of transcription factors and terminal differentiation markers of the endocrine (Q) and acinar (R) lineages and, to a lesser extent, of the ductal lineage (S) compared to untreated control explants at 14.5 dpc + 2 d. Scale bars, 80 μm; **p < 0.01, ***p < 0.001, Padj for all genes <0.05; error bars M, N, P show SD; error bars in O show SEM. For raw data, please refer to the S1 Data file.

Fig 4. S1pr2 signalling promotes survival and lineage specification of pancreas progenitors.

(A-H, M, N) Immunofluorescence analysis showed that S1pr2 block with 15 μM JTE013 in ALI cultures of 14.5 dpc embryonic pancreata for 6 d (14.5 dpc + 6 d) lead to the elimination of C-peptide⁺ endocrine cells (A, E, N) and Amylase⁺ acinar cells (B, F, N) and to a greatly expanded number of CK19⁺ duct-like cells (B, F, N). Markers of mature β cells (Pdx1) and acinar cells (Ptf1a) were also eliminated upon S1pr2 block (C, G, M), and cell survival was severely compromised, as shown by TUNEL immunofluorescence analysis (D, H). (I-L, M, N) Immunofluorescence and TUNEL analysis showed that addition of CTGF in the ALI cultures along with the S1pr2 block virtually eliminated cell death (L), without reversing the loss of C-peptide⁺ endocrine (I, N), Amylase⁺ acinar cells (J, N). A large number of CK19⁺ duct-like cells (J, N) and Pdx1⁺ cells (K, M) persisted. Scale bars, 80 μm (A, B, D-F, H-J, L) and 70 μm (C, G, K); ***p < 0.001; error bars show SD. For raw data please refer to the S1 Data file.

Fig 5. G_αi subunits mediate endocrine pancreas specification.

(A-I) Activation of PTX expression from two alleles of the ROSA26 ^LSLPTX transgene using the Tg^Pdx1Cre driver resulted in a striking loss of C-pep⁺ and Gcg⁺ cells at P1, as shown by immunofluorescence analysis (A, B; Quantitations are provided in S6L Fig). ALI cultures of 14.5 dpc embryonic pancreata expressing PTX from two alleles in Pdx1⁺ progenitors also resulted in reduced numbers of C-pep⁺ and Gcg⁺ cells after 6 d in culture (C, D, E), preceded by an equivalent reduction in the number of Ngn3⁺ cells at 2 d (F, G). ALI cultures of 14.5 dpc wt pancreata in the presence of 10 μg/ml of PTX showed a dramatic reduction of Ngn3⁺ cells (I) and the total number of C-pep⁺ and Gcg⁺ cells (C, H) at 2 and 6 d, respectively. Neither acinar (Amy⁺ cells) nor ductal (CK19⁺ cells) specification was affected by PTX in either the genetic or the pharmacological approach (C). Scale bars, 80 μm (A, B) and 70 μm (D-I); ***p < 0.001 in reference to control ALI cultures (C); error bars show SD. For raw data please refer to the S1 Data file.

Fig 6. YAP mediates G_αi-dependent S1pr2 signalling and is essential for endocrine specification.

(A-B) Immunofluorescence analysis of embryonic pancreata at 14.5 dpc showed that YAP protein is specifically expressed in E-cadherin⁺ epithelial cells (A) and that it is dramatically reduced in S1pr2 nulls (B). (C-F) YAP expression is retained in the epithelium of 14.5 ALI embryonic pancreas explants cultured for 2 d (D) but dramatically reduced upon either S1pr2 block by 15 μM JTE013 (E) or G_αi inactivation by 10 μg/ml PTX (C). YAP expression levels are restored in JTE013-treated pancreata by 20 μM of S1p (F). (G) YAP protein levels detected by western blot were significantly reduced in S1pr2 null pancreata at 14.5 dpc and in 14.5 dpc ALI cultures subjected to S1pr2 blocking with 15 μM JTE013 for 2 d or subjected to G_αi inhibition by 10 μg/ml of PTX. YAP protein levels were restored in JTE013 treated pancreata by 20 μM of S1p or 1 uM of the proteasome inhibitor MG132. (H, I) Immunofluorescence analysis showed that C-pep⁺ and Gcg⁺ cells were restored in JTE013-treated 14.5 dpc + 6 d ALI cultures in the presence of 1 μM MG132 for the first 2 d (H). Amy⁺ cells, however, were not restored under these conditions (I) and CK19 expression remained high (I). Quantitations are provided in S7A Fig. (J–L) Inactivation of YAP in pancreas progenitors using the YAP^fl/fl conditional allele, the Tg^Pdx1CreERT2 driver, ip tamoxifen injections, and immunofluorescence analysis showed a nearly 40% reduction in the number of endocrine C-pep⁺ and Gcg⁺ cells compared to controls at P1. Scale bars, 80 μm (B-F, H, I) and 70 μm (K, L); ***p < 0.001, ns, not significant in reference to E14.5 samples (A) and wt embryonic pancreata; error bars show SEM with the exception of J, for which they show SD. For raw data, please refer to the S1 Data file.

Fig 7. S1pr2-mediated Notch attenuation triggers endocrine and acinar differentiation.

(A-C) Stimulation of Notch signalling with 10 μM DSL in 14.5 dpc + 2 d ALI cultures did not affect YAP protein levels, as shown by western blot analysis (A) but resulted in elimination of Ngn3⁺ cells (B, immunostaining in S8A Fig) and a significant reduction in the number of endocrine and acinar cells in favour of an expanded population of ductal cells (C, immunostainings in S8B and S8C Fig). (A-C, D-F) Inhibition of Notch signalling with 10 μM DAPT in 14.5 dpc + 2 d ALI cultures had no effect on YAP protein levels (A) but resulted in a large increase in the number of Ngn3⁺ (B, D), C-pep⁺, and Gcg⁺ (E, C) cells at the expense of Amy⁺ and CK19⁺ cells (F, C), as shown by immunofluorescence. (A-C, G-I). Simultaneous inhibition of Notch and S1pr2 signalling in 14.5 dpc + 2 d ALI cultures transiently resulted in a large increase in the number of Ngn3⁺ cells (B, G) shown by immunofluorescence. However, immunofluorescence analysis showed that C-pep⁺, Gcg⁺ (H, C), and Amy⁺ (I, C) cells were eventually eliminated in favour of an expanded population of CK19⁺ cells (I, C). Absence of terminally differentiated endocrine and acinar cells can be attributed to a significant reduction in YAP protein levels observed under these conditions (A). Scale bars, 80 μm (D-I); ***p < 0.001 in reference to control ALI cultures (B); error bars in B show SEM; error bars in C show SD. For raw data, please refer to the S1 Data file.

Fig 8. S1pr2 signalling attenuates Notch signalling.

(A-F) Immunofluorescence analysis showed that Sel1l is strongly expressed in 14.5 dpc pancreata maintained for 1 d (14.5 dpc + 1 d) in ALI cultures compared to 14.5 dpc pancreata (A, D), and this correlated with a drop in the expression levels of NICD (B, E) and Hes1 (C, F). (G-I) S1pr2 signalling block with 15 μM JTE013 in 14.5 dpc + 1 d ALI cultures resulted in loss of Sel1l expression (G), concomitant with maintenance of high NICD (H) and Hes1 (I) expression. (J-L) Sel1l expression was restored in the absence of S1pr2 signalling by 1 μM of the specific proteasome inhibitor MG132 in 14.5 dpc + 1 d ALI cultures (J), and this was sufficient to restore attenuation of both NICD (K) and Hes1 (L) expression. Scale bars, 50μm (A-L). For raw data, please refer to the S1 Data file.

Fig 9. S1pr2 signalling regulates pancreatic lineage allocation and specification.

S1pr2 signalling attenuates Notch signalling to promote the segregation of tip MPCs that will give rise to acinar progenitors and bipotent trunk progenitors and, in the latter, to promote the generation of endocrine progenitors via Ngn3 stabilisation. These are two temporally distinct steps shown here as one for simplicity. Ngn3 is stabilised post-translationally in Notch^low cells [19]. Additionally, S1p signalling stabilises YAP to enable progenitor survival. G_αi activation mediates the stabilisation of the YAP protein to enable endocrine specification. Both YAP stabilisation and Notch down-regulation are necessary for endocrine specification.