SOX9 is required for maintenance of the pancreatic progenitor cell pool

Abstract

The factors necessary to maintain organ-specific progenitor cells are poorly understood and yet of extreme clinical importance. Here, we identify the transcription factor SOX9 as the first specific marker and maintenance factor of multipotential progenitors during pancreas organogenesis. In the developing pancreas, SOX9 expression is restricted to a mitotically active, Notch-responsive subset of PDX1(+) pluripotent progenitors and is absent from committed endocrine precursors or differentiated cells. Similar to Notch mutations, organ-specific Sox9 inactivation in mice causes severe pancreatic hypoplasia resulting from depletion of the progenitor cell pool. We show that Sox9 maintains pancreatic progenitors by stimulating their proliferation, survival, and persistence in an undifferentiated state. Our finding that SOX9 regulates the Notch-effector HES1 suggests a Notch-dependent mechanism and establishes a possible genetic link between SOX factors and Notch. These findings will be of major significance for the development of in vitro protocols for cell replacement therapies.

Fig. 1.

SOX9 expression is restricted to uncommitted pancreatic progenitor cells. At E9.0, SOX9 colocalizes with PDX1 in the prepancreatic endoderm (A), persists in the PDX1⁺ pancreatic progenitors at E12.5 (C), and by E15.5 becomes restricted to a core subset of PDX1⁺ epithelial cords (D). At E15.5, ≈40% of SOX9⁺ cells incorporate the mitotic marker BrdU (E, arrowheads) and coexpress HES1 (F, arrowheads). Committed NGN3⁺ endocrine progenitors are intercalated within the SOX9⁺ epithelial cords but rarely express SOX9 (G, arrowheads). SOX9 rarely colocalizes with the endocrine differentiation factors NKX2.2 (H, arrowhead), NKX6.1 (I, arrowheads), and MAFB (J, arrowhead), and it is similarly excluded from differentiated endocrine cells expressing ISL1 (K), insulin or glucagon (B and L), differentiated amylase⁺ acinar cells (M), and MUC1⁺ ductal cells (N). In the adult, SOX9 expression is restricted to a subset of ductal epithelial and centroacinar (arrows in O and P) cells in both mouse (O) and human (P). VP, ventral; DP, dorsal prepancreatic endoderm; BRDU, bromodeoxyuridine; GLU, glucagon; INS, insulin; AMY, amylase; MUC1, mucin-1; e, embryonic day. [Scale bar, 50 μm (A, B, D–P); 100 μm (C).]

Fig. 2.

Genetic regulation of SOX9 is consistent with a role as a pancreatic progenitor cell marker. The pattern of pancreatic SOX9 expression is identical in wild-type (A) and Ngn3-nullizygous embryos (B) at E15.5. Although SOX9 is restricted to a small subset of cells within the epithelial cords of E18.5 wild-type pancreas (C), SOX9 is maintained throughout the tubular network of undifferentiated PDX1⁺ epithelial cells in embryos that express an FGF10 transgene under the control of the Pdx1 promoter (Pdx1-FGF10) (D). In Sox9^flox/flox;Pdx1-Cre (Sox9^Δpan/Δpan) mice, SOX9 is robustly expressed throughout the PDX1⁺ prepancreatic endoderm at E9.0 (E and F). By E10.5, Pdx1-Cre has efficiently eliminated SOX9 from >95% of PDX1⁺ cells (G and H). PDX1 expression is maintained in the SOX9-deficient pancreatic epithelium (H). Likewise, SOX9 is expressed in pancreatic rudiments from PDX1-deficient embryos (J). DP, dorsal pancreas; VP, ventral pancreas; STOM, stomach; e, embryonic day. (Scale bar, 50 μm.)

Fig. 3.

Decreased contribution of SOX9-deficient progenitors to cell neogenesis results in pancreatic hypoplasia. Conditional Sox9 deletion with a Pdx1-Cre transgene results in pancreatic hypoplasia at E18.5 (A and B; pancreas outlined by a red dashed line). Using the ROSA26R allele, progeny of cells that underwent Pdx1-Cre-mediated recombination are identified by X-Gal staining (C and D). Uniform blue X-Gal staining in Sox9^flox/+;Pdx1-Cre;ROSA26R/+ (Sox9^+/Δpan;ROSA26R/+) embryos at E18.5 shows that all pancreatic cells have arisen from Pdx1-Cre-expressing progenitors (C). By contrast, the pancreatic remnant of Sox9^Δpan/Δpan;ROSA26R/+ littermates comprises a variable mosaic of β-gal⁺ recombined cells and β-gal⁻ unrecombined cells (D), indicating that cells that failed to undergo recombination have a selective advantage to contribute to the pancreas. Consistent with the presence of unrecombined cells in Sox9^Δpan/Δpan pancreas at E18.5, immunohistochemical staining reveals substantial numbers of SOX9-immunopositive pancreas cells in Sox9^Δpan/Δpan embryos bearing relatively large remnants (E and F). In concordance, PCR of genomic DNA from E18.5 pancreatic rudiments of Sox9^Δpan/Δpan embryos detects the Pdx1-Cre transgene, the unrecombined Sox9^flox allele, as well as the recombined, deleted Sox9 allele, revealing only partial Cre-mediated recombination (G). In Sox9^flox/+ controls that do not carry the Pdx1-Cre transgene, the wild-type Sox9 and floxed Sox9 alleles are amplified, but no deleted Sox9 allele is detected. e, embryonic day. [Scale bar, 200 μm (A and B); 100 μm (C–F).]

Fig. 4.

Pancreatic growth and cell differentiation require SOX9 activity. Hematoxylin/eosin (H&E) staining of pancreatic sections from E18.5 embryos (A and B) shows that pancreatic rudiments from Sox9^flox/flox;Pdx1-Cre (Sox9^Δpan/Δpan) embryos comprise predominantly fibrous tissue and epithelial cysts surrounding isolated clusters of acini, some of which show densely packed nuclei (B Inset). Sox9^Δpan/Δpan pancreatic rudiments display an almost complete absence of insulin⁺, glucagon⁺, somatostatin⁺, or pancreatic polypeptide⁺ endocrine cells (D and F) and scattered acinar cells that are weakly amylase⁺ (H). INS, insulin; GLU, glucagon; SOM, somatostatin; PP, pancreatic polypeptide; AMY, amylase; e, embryonic day. (Scale bar, 100 μm.)

Fig. 5.

Sox9 ensures maintenance of pancreatic progenitors through regulation of the Notch-effector HES1. At E11.5, Sox9^flox/flox;Pdx1-Cre (Sox9^Δpan/Δpan) embryos exhibit a significantly smaller mean sectional area of both dorsal and ventral pancreatic buds than wild-type littermates (A). The SOX9-deficient pancreatic epithelium shows reduced numbers of BrdU-incorporating cells among the PDX1⁺ pancreatic progenitor pool at E11.5 (B) and an increased number of TUNEL⁺ cells per total epithelial cells (C). At E11.5, the ratio of glucagon⁺ endocrine cells to the total number of PDX1⁺ progenitors is 4.4-fold increased in the pancreatic epithelium of Sox9^Δpan/Δpan compared with wild-type embryos (D–F). SOX9 deficiency is associated with decreased HES1 staining within the PDX1⁺ progenitor pool at E10.5 (G–I). (A–C) n = 3. (F and I) n = 4. DP, dorsal pancreas; VP, ventral pancreas; GLU, glucagon; E-CAD, E-cadherin; e, embryonic day. (Scale bar, 50 μm.)