Endocrine cell type sorting and mature architecture in the islets of Langerhans require expression of Roundabout receptors in β cells

Abstract

Pancreatic islets of Langerhans display characteristic spatial architecture of their endocrine cell types. This architecture is critical for cell-cell communication and coordinated hormone secretion. Islet architecture is disrupted in type-2 diabetes. Moreover, the generation of architecturally correct islets in vitro remains a challenge in regenerative approaches to type-1 diabetes. Although the characteristic islet architecture is well documented, the mechanisms controlling its formation remain obscure. Here, we report that correct endocrine cell type sorting and the formation of mature islet architecture require the expression of Roundabout (Robo) receptors in β cells. Mice with whole-body deletion of Robo1 and conditional deletion of Robo2 either in all endocrine cells or selectively in β cells show complete loss of endocrine cell type sorting, highlighting the importance of β cells as the primary organizer of islet architecture. Conditional deletion of Robo in mature β cells subsequent to islet formation results in a similar phenotype. Finally, we provide evidence to suggest that the loss of islet architecture in Robo KO mice is not due to β cell transdifferentiation, cell death or loss of β cell differentiation or maturation.

Figure 1

Robo receptors are required for endocrine cell type sorting and mature architecture of the islets of Langerhans. (A) Immunofluorescence staining for β cells (Insulin, red) and α cells (glucagon, green) of control (Robo1^+/Δ,2^+/flx and Neurog3-Cre;Robo1^+/+,2^+/+) and Neurog3-Cre;Robo1^Δ/Δ,2^flx/flx islets from 2 month old mice. (B) Average Circularity Index of control (Robo1^+/Δ,2^+/flx and Neurog3-Cre;Robo1^+/+,2^+/+) and Neurog3-Cre;Robo1^Δ/Δ,2^flx/flx islets. (C) Percentage of total α cells found in periphery of the islet in control (Robo1^+/Δ,2^+/flx and Neurog3-Cre;Robo1^+/+,2^+/+) vs. Neurog3-Cre;Robo1^Δ/Δ,2^flx/flx mice. (D) Average size of control (Robo1^+/Δ,2^+/flx and Neurog3-Cre;Robo1^+/+,2^+/+) and Neurog3-Cre;Robo1^Δ/Δ,2^flx/flx. (E) Percent of α cells out of total cells in control (Robo1^+/Δ,2^+/flx and Neurog3-Cre;Robo1^+/+,2^+/+) and Neurog3-Cre;Robo1^Δ/Δ,2^flx/flx.

Figure 2

Deletion of Robo receptors in β cells alone is sufficient to disrupt endocrine cell type sorting and islet architecture. (A) Immunofluorescence staining for β cells (Insulin, red) and α cells (glucagon, green) of control (Robo1^+/+,2^+/+ and Ins2-Cre;Robo1^+/+,2^+/+) and Ins2-Cre;Robo1^Δ/Δ,2^flx/flx islets from 2 month old mice. (B) Average Circularity Index of control (Robo1^+/+,2^+/+ and Ins2-Cre;Robo1^+/+,2^+/+) and Ins2-Cre;Robo1^Δ/Δ,2^flx/flx islets. (C) Percentage of total α cells found in periphery in control (Robo1^+/+,2^+/+ and Ins2-Cre;Robo1^+/+,2^+/+) vs. Ins2-Cre;Robo1^Δ/Δ,2^flx/flx islets. (D) Average size of control (Robo1^+/+,2^+/+ and Ins2-Cre;Robo1^+/+,2^+/+) and Ins2-Cre;Robo1^Δ/Δ,2^flx/flx islets. (E) Percent of α cells out of total cells in control (Robo1^+/+,2^+/+ and Ins2-Cre;Robo1^+/+,2^+/+) and Ins2-Cre;Robo1^Δ/Δ,2^flx/flx.

Figure 3

Robo receptors maintain endocrine cell type sorting and islet architecture in the adult, and their expression is diminished in obesity. (A,B) Microarray data adapted from Keller and colleagues (Keller et al.). Relative expression of Robo1 (A) and Robo2 (B) in lean and obese C57BL6 mice at 4 and 10 weeks. Relative expression is defined here as the log₁₀ of the ratio of (C) Immunofluorescence staining for β cells (Insulin, red) and α cells (glucagon, green) of control (Robo1^+/+,2^+/+ and Ucn3-Cre;Robo1^+/+,2^+/+) and Ucn3-Cre;Robo1^Δ/Δ,2^flx/flx islets from 2 month old mice. (D) Average Circularity Index of control (Robo1^+/+,2^+/+ and Ucn3-Cre;Robo1^+/+,2^+/+) and Ucn3-Cre;Robo1^Δ/Δ,2^flx/flx islets. (E) Percentage of peripheral α cells in control (Robo1^+/+,2^+/+ and Ucn3-Cre;Robo1^+/+,2^+/+) vs. Ucn3-Cre;Robo1^Δ/Δ,2^flx/flx islets. (F) Average size of control (Robo1^+/+,2^+/+ and Ucn3-Cre;Robo1^+/+,2^+/+) and Ucn3-Cre;Robo1^Δ/Δ,2^flx/flx islets. (G) Percent of α cells out of total cells in control (Robo1^+/+,2^+/+ and Ucn3-Cre;Robo1^+/+,2^+/+) and Ucn3-Cre;Robo1^Δ/Δ,2^flx/flx islets from 2 month old mice.

Figure 4

The disrupted islet architecture and increase in α cell ratio in Robo mβKO islets is not due to defects in differentiation or maturation of β cells. (A) Immunofluorescent staining for Insulin (red) and Ucn3 (green) of control Ucn3-Cre;Robo1^+/+,2^+/+ and Ucn3-Cre;Robo1^Δ/Δ,2^flx/flx islets from 1.5–3 month old mice. (B) Immunofluorescent staining for Insulin (red) and MafA (green) of control Ucn3-Cre;Robo1^+/+,2^+/+ and Ucn3-Cre;Robo1^Δ/Δ,2^flx/flx islets from 1.5–3 months old mice. Disorganized islets do not show loss of β cell maturation.

Figure 5

The disrupted islet architecture and increase in α cell ratio in Ucn3-Cre;Robo1^Δ/Δ,2^flx/flx islets is not due to β cell death. (A) TUNEL staining for apoptotic cells (white), counterstained with Insulin (red), and DAPI (blue), showing no cell death in adult and p13 control Ucn3-Cre;Robo1^+/+,2^+/+ and Ucn3-Cre;Robo1^Δ/Δ,2^flx/flx islets. (B) Percent β cell area of pancreatic sections in control Ucn3-Cre;Robo1^+/+,2^+/+ and Ucn3-Cre;Robo1^Δ/Δ,2^flx/flx showing a trend towards higher β cell area in Ucn3-Cre;Robo1^Δ/Δ,2^flx/flx.

Figure 6

The disrupted islet architecture and increase in α cell ratio in Ucn3-Cre;Robo1^Δ/Δ,2^flx/flx islets is not due to transdifferentiation of β to α cells. (A) Schematic of constructs used for lineage tracing Ucn3-Cre expressing cells. (B) Lineage tracing of mature β cells in control Ucn3-Cre;Robo1^+/+,2^+/+ and Ucn3-Cre;Robo1^Δ/Δ,2^flx/flx islets. Lineage traced β cells express nuclear mCherry (white), and counterstained for Insulin (red), Glucagon (green), and DAPI (blue), showing no transdifferentiation of α to β cells.

Figure 7

Robo receptors are important for glucose tolerance in male mice. (A) Glucose Tolerance Test on male control Ucn3-Cre;Robo1^+/+,2^+/+ and Ucn3-Cre;Robo1^Δ/Δ,2^flx/flx mice fasted overnight showing mild glucose intolerance. (B) Glucose Tolerance Test on female control Ucn3-Cre;Robo1^+/+,2^+/+ and Ucn3-Cre;Robo1^Δ/Δ,2^flx/flx mice fasted overnight showing no glucose intolerance.