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

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.

hyperglycemia and to potentiate insulin secretion 33 . However, the role of this pathway in the islet in vivo has not yet been demonstrated.
It recently was shown that a double deletion of Robo1 and Robo2 in lung pulmonary neuroendocrine cells (PNECs) results in the loss of PNECs' clustered architecture 34 . The delamination, migration and aggregation of the islets of Langerhans involve several Robo-related neuronal proteins such as Semaphorin, Ephrin/Eph and N-CAM [35][36][37][38][39][40] , as well as direct signals from the nervous system 41 . Moreover, Robo receptors themselves have been implicated in collective cell movement during organogenesis in various mammalian tissues 42,43 . We thus hypothesized that beyond their role in insulin secretion and β cell survival, Robo receptors may also be involved in the organogenesis of the islets of Langerhans, similar to their role in PNECs in the lung.
Here, we show that expression of Robo receptors in β cells is required for endocrine cell type sorting and mature islet architecture. Mice lacking Robo1 and Robo2 in all endocrine cells or selectively in β cells show complete loss of endocrine cell type sorting in the islets. Moreover, deletion of Robo receptors in mature β cells after islet formation has been completed also results in intermixing of endocrine cell types and loss of islet architecture. Finally, lineage-tracing experiments in β cell-selective Robo knockouts (Robo KO) provide evidence suggesting that disruption of islet architecture in Robo KO mice is not due to transdifferentiation, β cell death, or insufficient β cell differentiation or maturation.

Results
Robo receptors are required for endocrine cell type sorting and mature architecture of the islets of Langerhans. Current understanding of the formation of the mature architecture of the islets of Langerhans during development suggests that, beginning at E13.5, individual endocrine progenitors within the pancreatic duct independently turn on the transcription factor Neurogenin3 (Neurog3), and delaminate from the duct into the surrounding mesenchyme as single cells. These delaminated cells then migrate away from the duct and coalesce to form the mature islet architecture 35,37,44,45 . To test our hypothesis that Robo receptors are involved in the organogenesis of the islets of Langerhans, we generated an early endocrine progenitor knockout of Robo by crossing Robo1 Δ/Δ ,2 flx/flx mice 34 with Neurog3-Cre mice 46 . Robo1 Δ/Δ ,2 flx/flx mice harbor a linked Robo1 deletion allele (Robo1 Δ ) and conditional Robo2 deletion allele (Robo2 flx ). Neurog3 is expressed in all endocrine progenitors during their delamination from the duct, and its expression is subsequently turned off prior to endocrine cell aggregation to form proto-islet clusters 47,48 . The resulting Neurog3-Cre;Robo1 Δ/Δ ,2 flx/flx mice carry a whole-body deletion of Robo1, and a pancreatic endocrine-selective deletion of Robo2. This strategy was chosen to eliminate redundant Robo signaling and to avoid the homozygous lethality of Robo2 whole-body deletion 49 . Neurog3-Cre;Robo1 Δ/Δ ,2 flx/flx progeny are viable and appear normal.

Deletion of Robo receptors in β cells alone is sufficient to disrupt endocrine cell type sorting
and islet architecture. Because deletion of Robo receptors in Neurog3-Cre;Robo1 Δ/Δ ,2 flx/flx mice results in disruption of islet architecture, without apparent interference with endocrine cell aggregation into proto-islet clusters, we hypothesized that the deletion of Robo may affect later events in islet organogenesis. To test this hypothesis, we generated a β cell-selective deletion of Robo by crossing Robo1 Δ/Δ ,2 flx/flx mice with an Insulin-Cre line (Ins2-Cre) 50 .
SCientifiC REPORTS | (2018) 8:10876 | DOI:10.1038/s41598-018-29118-x Robo receptors are required for endocrine cell type sorting and islet architecture postnatally, and their expression is diminished in obesity. In some models of type-2 diabetes, islet architecture is distorted, with intermingling of α and β cells reminiscent of the Neurog3-Cre;Robo1 Δ/Δ ,2 flx/flx and Ins2-Cre;Robo1 Δ/Δ ,2 flx/flx phenotypes 12 . We therefore wondered whether Robo receptors may be involved in the loss of islet architecture in obesity and diabetes. To test for this possibility, we queried the Genomic Study of Parental Mice database developed by Keller and colleagues 51 , for the expression of Robo1 and Robo2. This database allows searching for gene expression in microarray experiments performed on islets from lean and obese (Lep ob/ob ) mice. We found that both Robo1 and Robo2 mRNAs are significantly downregulated in islets from obese mice compared to the lean controls as early as 4 weeks of age (Fig. 3A,B). Further literature search revealed that ROBO1 and ROBO2 are similarly downregulated in islets from human type-2 diabetics 28,29 . The diminished expression of Robo receptors in islets from obese, pre-diabetic mice and human type-2 diabetics suggests that these genes may be involved in maintaining islet architecture in the adult.
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, transdifferentiation or defects in differentiation or maturation. It has been shown that disrupting endocrine cell differentiation and maturation results in disordered islet architecture, reminiscent of our Robo KO phenotype [54][55][56][57][58][59][60][61] . To account for this possibility, we compared expression of the β cell maturation markers MafA and Ucn3 in Ucn3-Cre;Robo1 Δ/Δ ,2 flx/flx mice and controls by immunofluorescence (Fig. 4A,B). Surprisingly, we observed no change in expression of either MafA or Ucn3 in the β cells in Ucn3-Cre;Robo1 Δ/Δ ,2 flx/flx islets compared to controls, suggesting that deletion of Robo results in loss of islet architecture without apparent loss of β cell differentiation and maturation. It has previously been reported that reducing Slit-Robo signaling in islets promotes β cell death, while addition of recombinant Slit ligand improves β cell survival under diabetogenic stresses 33 . This suggests that both the intermixed endocrine cell phenotype and the increase in α cell ratio in Ucn3-Cre;Robo1 Δ/Δ ,2 flx/flx islets may be caused by β cell death, or replacement of β cells in the islet core by α cells through developmental reprogramming or transdifferentiation. To test the hypothesis that β cell death contributes to these phenotypes, we performed TUNEL analysis on Ucn3-Cre;Robo1 Δ/Δ ,2 flx/flx islets both at P13-16, when islets are still expanding, and at 8 weeks of age, when islet formation has been completed. We found no increase in α or β cell death in either of these stages (Fig. 5A). Analysis of β cell area in Ucn3-Cre;Robo1 Δ/Δ ,2 flx/flx islets showed a trend towards larger β cell area in the Robo KO when compared to controls though this did not reach statistical significance, further pointing against β cell death in Robo KO mice as an explanation for the islet architectural defects (Fig. 5B).
To test the hypothesis that β cell transdifferentiation to α cells is the cause of the observed disrupted islet architecture and increase in α cell ratio in Ucn3-Cre;Robo1 Δ/Δ ,2 flx/flx mice, we performed lineage tracing experiments.  (Fig. 6A). In the resulting progeny, any cell that has ever expressed the Insulin promoter in the former or the Ucn3 promoter in the latter is permanently marked with nuclear histone H2B-mCherry expression. The results obtained using this lineage tracing system showed no significant transdifferentiation of β cells to other cell types in the islet core in either Ucn3-Cre;Robo1 Δ/Δ ,2 flx/flx mice or Ins2-Cre;Robo1 Δ/Δ ,2 flx/flx mice (Fig. 6B, Supplemental Fig. 5). We thus concluded that the disrupted islet architecture in Robo KO lines and the increase in α cell ratio seen in the Ucn3-Cre;Robo1 Δ/Δ ,2 flx/flx mice are likely not caused by defects in β cell differentiation or maturation, accelerated β cell death, or transdifferentiation of β cells to α cells.

Discussion
In this study, we show that Robo receptors are essential for endocrine cell type sorting and mature architecture of the islets of Langerhans in mice. Conditional deletion of Robo2 in the islets on the background of whole-body deletion of Robo1 completely abolished the characteristic core-mantle organization of the islets. Instead of sorting to the periphery, α cells and δ cells became intermingled with β cells throughout the islet. The same phenotype was observed upon deletion of Robo in all endocrine cells, using a Neurog3-Cre;Robo1 Δ/Δ ,2 flx/flx model and upon selective deletion of Robo only in the β cell compartment, using the Ins2-Cre;Robo1 Δ/Δ ,2 flx/flx and Ucn3-Cre;Robo1 Δ/Δ ,2 flx/flx models. These results are in agreement with a recent study showing that α cells are dispensable for morphogenesis of the islets of Langerhans 63 , thus further highlighting the importance of β cells as the primary organizer of islet architecture. It should be noted that none of the three Cre lines used in this study are endocrine-specific. The Ucn3 promotor is inherently expressed in some cell types in the central nervous system 64 , and the Neurog3-Cre and Ins2-Cre lines have been shown to have leaky expression in the brain and other neuroendocrine tissues 65,66 . While this can influence behavior and glucose regulation, it is unlikely that leakiness expression of the transgenes in different tissues accounts for the remarkably similar defects in endocrine cell type sorting and islet architecture in all three models. We further show, using Ucn3-Cre;Robo1 Δ/Δ ,2 flx/flx mice, that deletion of Robo selectively in mature β cells, after initial islet formation has been completed, also results in loss of endocrine cell type sorting and improper islet architecture. One possible explanation for this observation is that Robo receptors are required for active maintenance of endocrine cell sorting. Ucn3 is a marker of functionally mature β cells in mice, which starts being expressed in β cells around P10 19,53 . While Ucn3-expressing β cells are functionally mature 52 , the islets of P13-P16 mice still undergo proliferative expansion 67 . Thus, a more plausible explanation to the phenotype observed in islets of Ucn3-Cre;Robo1 Δ/Δ ,2 flx/flx mice is that postnatal islets require Robo receptors to achieve mature islet architecture and endocrine cell type sorting during the expansion period. Future experiments, namely conditional deletion of Robo after weaning 68 , will resolve the question of active versus passive control of endocrine cell sorting and islet architecture in the adult. However, it would be interesting to see whether the observed reduction in Robo expression in islets from obese mice and type-2 diabetic humans accounts for the disruption of islet architecture during the islets' compensatory expansion under these conditions.
One notable difference between prenatal and postnatal deletion of Robo is the effect on α-to-β cell ratio. While prenatal deletion of Robo did not result in a significant difference in α-to-β cell ratio, postnatal Robo mutants showed increased α cell proportions. Our lineage-tracing experiments, as well as the trend towards larger β cell area in the mutant, strongly suggest that the increase in α cells in the islets of Ucn3-Cre;Robo1 Δ/Δ ,2 flx/flx mice is not the result of β cells transdifferentiating into α cells. One possible explanation for the difference in α-to-β cell ratio observed between deletion of Robo prenatally using Neurog3-cre and Ins2-Cre and that observed when Robo is deleted postnatally with Ucn3-Cre is that the former two models delete Robo in all β cells, while the latter model only deletes the gene only in mature β cells, thus potentially having a differential influence on the neogenic niche of virgin β cells at the islet periphery 52 .
Defects in endocrine cell type sorting and islet architecture have previously been associated with defects in β cell maturation and/or differentiation [54][55][56][57][58][59][60][61] . Using mature β cell-selective lineage tracing, we found no difference in the proportion of label-carrying non-β cells between islets from Robo KO mice and controls, suggesting that deletion of Robo does not cause β cells to lose their differentiated identity. This conclusion is supported by the apparently normal expression of the maturation markers MafA and Ucn3 in β cells of Robo KO mice. Furthermore, the similar or larger total β cell area in Robo KO and control islets, the lack of increase in cell death in Robo KO islets, and the similar or larger overall islet size between islets of Robo KO models and their respective controls strongly suggest that the defects seen in endocrine cell type sorting and islet architecture are not due to loss of β cells.
Robo receptors have been implicated in collective cell movement not only in migrating axons, but also in other mammalian tissues 42,43 . They can function together with their canonical ligands, Slit1-3, or by partnering with other cell surface molecules to regulate cell adhesion and cell motility 27 . Based on results shown here, we propose that the defects in endocrine cell type sorting and islet architecture seen upon deletion of Robo in β cells are caused either by the inability of the cells to sense positional cues, the inability of the cells to rearrange their own cytoskeleton in response to positional cues, or the inability of the cells to attach to neighboring cells or to the extracellular matrix to facilitate movement.
Taken together, our data shown here demonstrate that expression of Robo receptors in β cells is required for endocrine cell type sorting and mature architecture in the islets of Langerhans. These findings raise the possibility of using Robo to prevent and reverse the loss of correct islet architecture in type-2 diabetics, as well as for directing the architecture of islet-like clusters derived by stem cell differentiation to make bona fide islets in vitro for transplantation into type-1 diabetics.
Cell counting analysis. Cells were counted at maximum intensity projected images from 8-10, 10 µm thick confocal z-stacks using the ImageJ cell counter tool. Only cells with visible DAPI stained nuclei were counted. Cells were considered to be in the islet mantle if they fell within the 2 outermost cell layers of the islet, or core if they fell within any layers deeper than the outermost 2 cell layers. Only islets with at least 40 cells, with 10 or more cells being non β cells were counted for determining core/mantle localization. Islet size and shape analysis. Islet size and shape analyses were performed on the same images used for cell counting. All non-islet nuclei were erased using the ImageJ outline and clear outside functions. The subsequent images were then converted to 8 bit and set as threshold. Islet size and shape was determined using the Analyze Particles macro of ImageJ, which gives both a readout of area in µm 2 , and a Circularity Index that is calculated using the formula circularity = 4π(area/perimeter 2 ). β cell area analysis was performed on four whole pancreas sections per mouse, that were at least 100 µm apart. Whole sections were stained for insulin and DAPI, and then imaged as 10x tile scans on a Leica SP8 Scanning Confocal microscope. Using ImageJ, each channel was converted to threshold, background was erased, and the area was measured using the Analyze Particles function. To obtain the β cell area per section, the insulin area was divided by the DAPI area for each section and converted to a percentage. Statistical analysis. Data are presented as average ± SEM unless otherwise indicated. All P values were calculated using a Student's t-test using Prism 7 Graph Pad. P values of less than 0.05 were considered significant. Data availability. All data generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.