**DL islet cells are found as isolated single cells at birth.** (**A**) Expected recombination events predicted by the Poisson distribution (left) and recombination events per islet observed at birth (right). White, gray and black areas represent the proportion of islets containing one, two or three DL cells per islet (*n*=4, 150 DL cells), respectively. (**B**) Proportion of homogenous clusters of DL cells, calculated per islet cell type with the knowledge that 22% of islets contain more than one cluster of DL cells. Expected and observed proportions of homogeneous clusters were compared using Fisher's exact test. No statistical difference was found. This indicates that the observed frequencies of homogeneous clusters correspond to those expected if labeled Ngn3^{+} cells were unipotent. *n*=4 mice per group, 100-200 islets per group; NS, not significant. (**C**) Mean cross-section diameter of homogeneous and heterogeneous clusters. Heterogeneous clusters are more dispersed (15 clusters per group, from four mice, ^{*}*P*<0.05). (**D**) Size (number of cells) of homogeneous and heterogeneous clusters at 2 and 10 months. Note the increased size of homogeneous clusters between 2 and 10 months of age. At 10 months, heterogeneous clusters are twice the size of homogeneous clusters (*n*=4-5 mice, greater than 50 clusters per group, ^{***}*P*<0.001). (**E**) Number of DL cells per cluster in adult islets. The prevailing size of DL cell clusters changes with age in 2- and 10-month-old transgenic mice. Diamonds indicate the median cluster size; *n*=4-5 mice per age group; 220 and 340 clusters of DL cells were scored in 800 and 1200 islets, respectively. (**F**) DL cluster size and islet size are not correlated. The average number of cells per cluster is the same in small and large islets (coefficient of determination: R^{2}=0.036; 223 labeled islets scored of all sizes, from five mice). (**G-I**) Paraffin sections of P0, 2- and 10-month-old islets from *Ngn3-GR/RG* mice stained for EGFP (DAB) and counterstained with Hemalun. At birth (G), one isolated DL cell is shown (arrowhead), whereas one cluster of two adjacent DL cells is seen at 2 months (H), and another of four DL cells at 10 months (I). Scale bars: 25 μm in G; 50 μm in H,I.

## PubMed Commons