Aldh1-Expressing Endocrine Progenitor Cells Regulate Secondary Islet Formation in Larval Zebrafish Pancreas

<div><p>Aldh1 expression is known to mark candidate progenitor populations in adult and embryonic mouse pancreas, and Aldh1 enzymatic activity has been identified as a potent regulator of pancreatic endocrine differentiation in zebrafish. However, the location and identity of Aldh1-expressing cells in zebrafish pancreas remain unknown. In this study we demonstrate that Aldh1-expressing cells are located immediately adjacent to 2F11-positive pancreatic ductal epithelial cells, and that their abundance dramatically increases during zebrafish secondary islet formation. These cells also express <i>neurod</i>, a marker of endocrine progenitor cells, but do not express markers of more mature endocrine cells such as <i>pax6b</i> or <i>insulin</i>. Using formal cre/lox-based lineage tracing, we further show that Aldh1-expressing pancreatic epithelial cells are the direct progeny of pancreatic notch-responsive progenitor cells, identifying them as a critical intermediate between multi-lineage progenitors and mature endocrine cells. Pharmacologic manipulation of Aldh1 enzymatic activity accelerates cell entry into the Aldh1-expressing endocrine progenitor pool, and also leads to the premature maturation of these cells, as evidenced by accelerated <i>pax6b</i> expression. Together, these findings suggest that Aldh1-expressing cells act as both participants and regulators of endocrine differentiation during zebrafish secondary islet formation.</p> </div

Cell proliferation among Aldh1- and 2F11-expressing cells.

<p>EdU signals (white arrows) were detected in subsets 2F11^pos cells at 15 (A), 20 (B) and 25 dpf (C). (D) Cells co-expressing Aldh1 and 2F11 (blue arrows) are not proliferative, but are localized in the adjacent to Aldh1-negative, EdU^pos, 2F11^pos cells (white arrows). (E and E’) Cells co-expressing NeuroD and 2F11 (yellow arrows) are not proliferative but are localized adjacent to NeuroD:eGFP-negative, EdU^pos, 2F11^pos cells (white arrows). (F and F’) Detailed geometry of 2F11, Aldh1, NeuroD:eGFP expression in association with EdU incorporation. White arrows indicate EdU ^pos/2F11 ^pos/Aldh1 ^neg/NeuroD:eGFP^neg cells. Blue arrows indicate EdU^neg /2F11 ^pos/Aldh1 ^pos/NeuroD:eGFP^pos cells. Yellow arrows indicate EdU ^neg/2F11 ^pos/Aldh1 ^neg/NeuroD:eGFP^pos cells. Green arrows indicate EdU ^neg/2F11^neg /Aldh1 ^neg/NeuroD:eGFP^pos cells.</p

Aldh1-expressing cells are derived from Notch-responsive progenitor cells.

<p>(A) Schematic timeline for 4OHT-induced lineage labeling. Tp1bglob:CreER ^T2/βactin:loxp-stop-loxp-hmgb-mCherry fish were treated with either 1% EtOH (B and D; negative controls) or with 4-hydroxy-tamoxifen (4OHT) between either 3-5 dpf (B and C) or 18-20 dpf (D and E), and then sacrificed for analysis at 25 dpf. Cells carrying the Tp1bglob:CreER^T2 lineage label display red nuclei. (C) 2F11 ^pos/Aldh1^pos cells arise from Notch-responsive cells labeled between 3-5 dpf (blue arrows). (E) Notch-responsive cells labeled between 18-20 dpf give rise to Aldh1^pos (blue arrows), Pax6:eGFP+ endocrine cells (green arrows) and ductal cells (white arrows). F, Schematic depiction of proposed lineage relationships between Notch-responsive progenitors (Notch-ON) and cells expressing 2F11, Aldh1, NeuroD and Pax6b during secondary islet formation. Notch-ON cells expressing 2F11 (red) give rise to Notch-OFF, 2F11^pos, NeuroD^pos, Aldh1^pos cells (blue), which then inactivate Aldh1 and activate Pax6 expression (green) prior to undergoing terminal endocrine differentiation (orange).</p

Aldh1-expressing cells increase in number and are spatially associated with endocrine cells during secondary islet formation.

<p>Expression of Aldh1 (red arrows) is compared with that of Insulin (white arrows), as assessed immunofluorescent labeling at 5 (A), 10 (B), 15 (C), 20 (D), 25 (E) and 30 dpf (F). (G) Quantification of the number of Aldh1-expressing cells located outside of the principle islet (black line), and their juxtaposition with Insulin-expressing cells (grey line). Values were derived from n=10 fish per developmental time point, with a minimum of 3 sections per fish. (*) and (**) indicate p<0.05 and p<0.01, respectively. Dotted white lines in (A) and (B) indicate region of principle islet; dotted white lines in (D) and (E) indicate region of pancreatic duct.</p

Inhibition of Aldh1 enzymatic activity increases the number of Aldh1- and NeuroD-expressing endocrine progenitor cells and induces their premature differentiation.

<p>Panels to the right of merged images represent corresponding single channel images. (A and B) NeuroD:eGFP/Tp1:hmgb-mCherry fish were treated with DMSO (A) or DEAB (B) at 72-120 hpf. DEAB treatment increased the number of Notch-responsive cells also expressing NeuroD:eGFP (red arrows). (C) Quantification of the number of eGFP^pos and/or mCherry^pos cells in 1000 µm³ of 2F11^pos tissue in NeuroD:eGFP/Tp1:hmgb-mCherry fish treated with DMSO or DEAB between 72-120 hpf. Inhibition of Aldh1a induced NeuroD:eGFP expression in Notch-responsive cells. Values indicate mean <u>+</u> SEM, with minimum of 20 larvae analyzed for each condition. (*) indicates p<0.05. (D) Quantification of the number of Aldh1-expresing cells in 100,000 µm³ of 2F11^pos tissue in NeuroD:eGFP/Tp1:hmgb-mCherry fish treated with DMSO or DEAB treatment between 72-120 hpf. Values indicate mean <u>+</u> SEM, with minimum of 20 larvae analyzed for each condition. (*) indicates p<0.05. (E and F) NeuroD:eGFP/Tp1:hmgb-mCherry fish were treated with DMSO (E) or DEAB (F) at 18-20 dpf. DEAB treatment increased the number of Notch-responsive cells also expressing NeuroD:eGFP (red arrows), and these cells were frequently adjacent to Aldh1-expressing cells (blue arrows). (G and H) Pax6b:eGFP/Tp1:hmgb-mCherry fish were treated with DMSO (G) or DEAB (H) between 18-20 dpf. DEAB treatment activated Pax6:eGFP expression in Aldh1-expressing cells. (I) Quantification of number of eGFP^pos and/or mCherry^pos cells in 1000 µm² of 2F11^pos tissue in NeuroD:eGFP/Tp1:hmgb-mCherry or Pax6b:eGFP/Tp1:hmgb-mCherry fish treated with DMSO or DEAB between 18-20 dpf. NeuroD:eGFP, but not Pax6:eGFP expression is activated within Notch responsive cells following DEAB treatment. In contrast, Pax6:eGFP but not NeuroD:eGFP expression is induced in cells not expressing Tp1:hmgb1-mCherry. (*) indicates p<0.05, with n=8 fish per condition and a minimum of 3 sections analyzed per fish. (J) Quantification of Aldh1-expressing cells also expressing either NeuroD:eGFP or Pax6:eGFP following treatment with either DMSO or DEAB between 18-20 dpf. DAEB treatment increases the number of cells Aldh1-expressing cells also expressing either NeuroD:eGFP or Pax6:eGFP. (*) and (**) indicates p<0.05 and p<0.01, respectively. n=10 fish per condition and a minimum of three sections analyzed per fish.</p

Proposed model for the role of Aldh1-expressing cells in zebrafish secondary islet formation.

<p>Notch-ON cells expressing 2F11 (red) give rise to Notch-OFF, 2F11^pos, NeuroD^pos Aldh1^pos cells (blue), which then inactivate Aldh1 and activate Pax6 expression (green) prior to undergoing terminal endocrine differentiation (orange). Aldh1 enzymatic activity and associated retinoic acid production inhibit the progression of Notch-ON cells into the NeuroD^pos, Aldh1^pos endocrine progenitor pool, as well as the progressive differentiation of NeuroD^pos, Aldh1^pos cells.</p

Stage- and tissue-dependent regulation of LGR5 expression during development.

<p>(A) Zygotic transcription of LGR5 begins in late embryogenesis during development in <i>X. laevis</i>. Total RNA was isolated from whole animals from stage 1 (fertilized egg) to stage 66 (the end of metamorphosis) and analyzed by qRT-PCR. The LGR5 expression shown in (C) was normalized against rpl8 expression determined at the same time. (B, C) Organ-specific temporal regulation of LGR5 during natural metamorphosis in <i>X. laevis</i>. Total RNA was isolated from tail, limb, and intestine of tadpoles at the indicated developmental stages and used for regular RT-PCR (B) or qRT-PCR (C) analysis of the LGR5 expression. In each RT-PCR, a primer set for the ribosomal gene rpl8 was also included in the same PCR reaction as an internal control, although the rpl8 result was shown only for the intestine in (B). The LGR5 expression shown in (C) was normalized against rpl8 expression determined at the same time.</p

Spatiotemporal expression of LGR5 mRNA in the small intestine during natural metamorphosis.

<p>Cross sections of the intestine at premetamorphic stage 54 (A, A′), prometamorphic stage 56/57 (B, B′), metamorphic climax stages 61 (C, C′, F) and 62 (D, D′, G), and the end of metamorphosis (stage 66) (E, E′) were hybridized with LGR5 antisense (A–E′) or sense probe (G). To compare the localization of LGR5 mRNA (C, C′) with that of adult epithelial progenitor cells, the serial sections at stage 60/61 were stained with methyl green-pyronin Y (MG-PY) (F). Arrows indicate the cells expressing LGR5 (A′–E′), while arrowheads indicate adult epithelial progenitor cells (F). Higher magnification of boxed areas in (A)–(E) are shown in (A′)–(E′). Sense probe did not produce any signal (G). Note that at metamorphic climax stage 61, LGR5 mRNA was localized in the islets between the larval epithelial cells and the connective tissue (C, C′). These islet cells were identified as the adult epithelial progenitor cells strongly stained red with pyronin Y (F) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013605#pone.0013605-IshizuyaOka5" target="_blank">[29]</a>. AE: adult epithelial cell including progenitor/stem cell, CT: connective tissue, LE: larval epithelial cell, Lu: lumen, M: muscle layer, Ty: typhlosole. Scale bars are 100 µm (A–E, G) and 20 µm (A′–E′, F), respectively.</p

T3 up-regulates LGR5 expression in premetamorphic tadpoles.

<p>Tadpoles at stage 54 were exposed to 5 nM T3 for 0–5 days before tissue isolation, RNA extraction, and qRT-PCR analyses as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013605#pone-0013605-g003" target="_blank">Fig. 3C</a>.</p

T3-dependent expression of LGR5 mRNA in the small intestine.

<p>Cross sections of the intestine from premetamorphic tadpoles (stage 54) treated with 10 nM T3 for 1 (A, A′), 3 (B, B′) and 5 days (C, C′) were hybridized with LGR5 antisense probe. AE: adult epithelial cell including the progenitor/stem cell, CT: connective tissue, LE: larval epithelial cell, Lu: lumen, M: muscle layer. Scale bars are 100 µm (A–C) and 20 µm (A′–C′), respectively.</p