Figure 2. SOX2 is essential for establishing SOX10+ acini during organogenesis.

(A–D) SMG+SLG from Krt14^CreERT2; Sox2^fl/fl and wild-type (WT) embryos in which recombination was induced before gland ontogenesis at E10.5 and 11.5. (A) Representative brightfield images of Krt14^CreERT2; Sox2^fl/fl and wild-type (WT) SMG+SLG at E16.5. Scale bar is 1 mm. (B) Representative images of SMG+SLG immunostained for nerves (TUBB3) and epithelium (Ecadherin, ECAD). Scale bar is 1 mm. SMG = submandibular gland, SLG = sublingual gland. Dashed white line denotes SLG. (C) qPCR analysis of E16.5 Krt14^CreERT2; Sox2^fl/fl and wild-type (WT) SMG+SLG for genes involved in acinar differentiation, ductal differentiation and innervation, with expression normalized to Rsp29. Red dashed line = WT. n = 3 embryos per genotype. Data are means+s.d. and were analyzed using a one-way analysis of variance with post-hoc Dunnett’s test. *p<0.05, **p<0.01. (D) Quantification of cells expressing acinar or ductal markers, cleaved caspase-3 or Ki67 in acini of E16.5 in Krt14^CreERT2; Sox2^fl/fl and wild-type (WT). n = 2–4 glands/genotype and cells were counted in 3–4 acini/gland. Data are means+s.d. and were analyzed using a Student’s t-test, ***p<0.001. (E and F) SMG+SLG from Krt14^CreERT2; Rosa26^mTmG and Krt14^CreERT2; Rosa26^mTmG; Sox2^fl/fl in which recombination was induced at E10.5 and 11.5 were immunostained for SOX10 and AQP5 (E) and GFP+ cells expressing SOX10 and AQP5 were quantified (F). n = 3 glands/genotype and cells were counted in 3–4 acini/gland. Data were subjected to a Student’s t-test, ***p<0.001. Scale bar in E is 20 µm. Arrowheads indicate double positive cells. (G) qPCR for enrichment of Sox10 in SOX2 ChIP. n = 20 pooled SLG, average three experiments, *p<0.05. Additional data for this figure in Figure 2—figure supplement 1.

DOI: http://dx.doi.org/10.7554/eLife.26620.004

Figure 2—figure supplement 1. Acini are depleted in the absence of Sox2.

Representative images of Krt14^CreERT2; Sox2^fl/fl and wild-type (WT) SMG+SLG at E13 (A; scale bar is 100 μm), E13.5 (B; top and bottom panels; scale bars are 100 and 50 μm, respectively) and E16.5 (C; scale bar is 200 µm (top panels), 50 µm (second and fourth panels) and 10 µm (third panels)). SMG = submandibular gland, SLG = sublingual gland. E13.5 SGs (B) were immunostained for SOX2 and E-cadherin (ECAD) and E16.5 SMG+SLG (C) for KRT19, MIST1, SOX10, AQP5, KRT5, Ecadherin (ECAD) and nuclei. Dashed white lines outline acini. Lower panel in B highlights the oral epithelium of the E13.5 SG. (D–E) Quantification of acini at E13.5 and E16.5 is shown in D (n = 3–7) and E (n = 3), with WT set to 100%. Data in D and E were analyzed using a Student’s t-test, *p<0.05, **p<0.01. (F and G) qPCR analysis of gene expression of SG from Krt14^CreERT2; Sox2^fl/fl versus wild-type littermate (WT) at E13.5 (F) and E16.5 (G). Red dashed line = WT. Data are means+s.d. of 3–4 SMG+SLG per genotype. Data in F and G were analyzed using a one-way analysis of variance with a post-hoc Dunnett’s test. *p<0.05, **p<0.01.

DOI: http://dx.doi.org/10.7554/eLife.26620.009