ALDH1B1 is a potential stem/progenitor marker for multiple pancreas progenitor pools

Abstract

Aldehyde dehydrogenase (ALDH) genes are increasingly associated with stem/progenitor cell status but their role in the maintenance of pluripotency remains uncertain. In a screen conducted for downstream Ngn3 target genes using ES derived pancreas progenitors we identified Aldh1b1, encoding a mitochondrial enzyme, as one of the genes strongly up regulated in response to Ngn3 expression. We found both by in situ hybridization and immunofluorescence using a specific antibody that ALDH1B1 is exclusively expressed in the emerging pancreatic buds of the early embryo (9.5 dpc) in a Pdx1 dependent manner. Around the time of secondary transition, ALDH1B1 expression was restricted in the tip tripotent progenitors of the branching epithelium and in a subset of the trunk epithelium. Expression in the latter was Ngn3 dependent. Subsequently, ALDH1B1 expression persisted only in the tip cells that become restricted to the exocrine lineage and declined rapidly as these cells mature. In the adult pancreas we identified rare ALDH1B1(+) cells that become abundant following pancreas injury in either the caerulein or streptozotocin paradigms. Blocking ALDH catalytic activity in pancreas embryonic explants resulted in reduced size of the explants and accelerated differentiation suggesting for the first time that ALDH activity may be necessary in the developing pancreas for the maintenance and expansion of progenitor pools.

Figure 1. Aldh1b1 is expressed in the pancreatic buds in a Pdx1 dependent manner

(A, B) In situ whole mount hybridization shows that Aldh1b1 is expressed in both the dorsal and ventral pancreatic buds (dp and vp, respectively) at 9.5 dpc (arrows in A) marked at the same time point by whole mount immunostaining for PDX1 (arrows in B). Combined in situ hybridization for Aldh1b1 expression and PDX1 immunostaining showed co-localisation (insert in A). (C–F) Triple immunofluorescence at 9.5 dpc for ALDH1B1, E-CADHERIN and PDX1 or ISL1. Both ALDH1B1 and PDX1 proteins are present in the same cells of wt embryos (compare C and D). ALDH1B1 expression was completely lost in 9.5 dpc Pdx1 null embryos (E, F). In E, F mesenchymal cells are marked by ISL1 expression. (G–J) Triple immunofluorescence at 10.5 dpc for ALDH1B1, E-CADHERIN and PDX1 or ISL1. ALDH1B1 and PDX1 proteins continue to be co-expressed in wt embryos (compare G and H). ALDH1B1 expression was completely lost in 10.5 dpc Pdx1 null embryos (I, J). In I, J mesenchymal cells are marked by Isl1 expression. Scale bars: A (inlet), C–F 50 um; G–J 100 um.

Figure 2. Aldh1b1 is expressed in tip and trunk pancreas progenitors and expression in the latter is Ngn3 dependent

(A, B) Double immunofluorescence for ALDH1B1 and E-CADHERIN showed that ALDH1B1 is expressed exclusively in the 12.5 dpc pancreatic epithelium in both the tips and the trunks. (C, D) Double immunofluorescence for ALDH1B1 and NKX6.1 at 14.5 dpc wt embryonic pancreata showed that most (arrows in D) but not all (arrowheads in D) NKX6.1⁺ trunk cells were also ALDH1B1⁺. (E–G) Triple immunofluorescence experiments for ALDH1B1, E-CADHERIN and NGN3 14.5 dpc wt embryonic pancreata showed that all trunk ALDH1B1⁺ cells were also NGN3⁺ (arrows in G). (H–J) Triple immunofluorescence experiments for ALDH1B1, E-CADHERIN and NGN3 at NGN3 null embryonic pancreata showed that expression of ALDH1B1 in the trunk is NGN3 dependent (arrowheads in H–J). Scale bars: A–B, C–D, F–G, I–J 50 um; E, H 100 um.

Figure 3. Aldh1b1 is strongly expressed in the developing pancreas compared to other Aldh genes

Real Time PCR in 12.5 and 14.5 dpc wt and 14.5 Ngn3 null pancreata was performed for all known Raldh genes as well as for Aldh1b1 and the closely related Aldh2. Aldh1b1 appears to be expressed in higher levels compared to Aldh1a1 and Aldh1a2. Aldh1b1 and Aldh1a1 expression levels drop in 14.5 dpc Ngn3 null pancreata in a statistically significant manner (*, p < 0.05).

Figure 4. Blocking of ALDH activity in mouse embryonic pancreas explants reduces mitosis in the epithelium

(A–D) Double immunofluorescence in untreated (A, C) and DEAB treated (B, D) pancreata for E-Cadherin and BrdU showed that BrdU incorporation apparent in the trunk epithelium of control pancreata (arrows in C) was greatly reduced in the trunk epithelium of DEAB treated pancreata. Areas marked by squares in A, B are represented in high magnification in C, D respectively. (E, F) Immunofluorescence in untreated (E) and DEAB treated (F) pancreata showed a drop in SOX9 expression following DEAB exposure. (G, H) Immunofluorescence in untreated (G) and DEAB treated (H) pancreata showed a drop in NGN3 expression following DEAB exposure. (I) Epithelial BrdU incoroporation was reduced in DEAB treated pancreata. Inclusion of RA further reduced incorporation to levels similar by the inclusion of RA alone (p<0.02). Scale bars: A–D, E–F, G–J 100 um; C–D 25 um.

Figure 5. Blocking of Aldh activity in mouse embryonic pancreas explants accelerates differentiation and changes the relative linage distribution

(A, B) Double immunofluorescence for AMYLASE and INSULIN in untreated (A) and DEAB treated (B) pancreata showed an increase in the number of INSULIN⁺ cells (depicted graphically in E). (C, D) Double immunofluorescence for CK19 and glucagon in untreated (C) and DEAB treated (D) pancreata showed a strong increase in the number of GLUCAGON⁺ cells (depicted graphically in E). Scale bars: A–D 100 um.

Figure 6. Very rare ALDH1B1⁺ cells persist in the adult pancreas but their number expands vastly following either caerulein or streptozotocin insults

(A–C) Triple immunofluorescence of wt, untreated pancreata showed the presence of rare elongated cells expressing ALDH1B1 (A–C, arrows in A), E-CADHERIN (A, B) and PNA (C). (D–F) The number of ALDH1B1⁺ cells expanded by 20 – fold in response to acute pancreatitis as shown by triple immunofluorescence for ALDH1B1 (D–F, arrows in D), E-CADHERIN (D, E) and PNA (F). Most ALDH1B1⁺ cells were PNA⁺ (F, arrows) but some were PNA⁻ (F, arrowheads). (G–I) The number of ALDH1B1⁺ cells expanded by 10 – fold in response to β cell ablation as shown by triple immunofuorescence for ALDH1B1 (G–I, arrows in G), E-CADHERIN (G, H) and PNA (I). (J–L) ALDH1B1⁺ cells occasionally surrounded β cell depleted islets (J, arrows). Occasionally, ALDH1B1⁺/DBA⁺ (K, arrow) or ALDH1B1⁺/DBA⁺/PNA⁺ (L, arrow) cells were observed. Scale bars: A, D, G 100 um; B–C, E–F, H–I, J–L 25 um.