Figure 3. Phenotypic analysis of R26^T6B mice during homeostasis.

(A) Rosa26^+/+; Col1a1^T6B/T6B females were crossed with Rosa26rtTA/+; Col1a1^T6B/T6B males and doxycycline was administered by chow starting at 0.5 d.p.c. No viable pups positive for both the rtTA and T6B allele were observed (n = 15, p-value = 0.002, Fisher’s exact test). (B) Pregnant females were kept on doxycycline diet from E13.5 to E18.5 and the pups delivered on E18.5 by c-section. Note the significantly smaller size of Rosa26^rtTA/rtTA; Col1a1^T6B/T6B embryos relative to Rosa26^rtTA/rtTA;Col1a1^+/+ control littermates. Lower row: YFP detection by epifluorescence in E18.5 pups of the indicated genotypes. (C) Comparison of intestine architecture in H&E sections from R26^T6B and R26^CTL mice (n = 3 for each genotype) maintained on doxycycline for 2 months. (D) Immunofluorescence imaging of the small intestine of R26^T6B and R26^CTL mice (n = 3–5 for each genotype) kept on doxycycline diet for a month (upper row), showing a reduction in lysozyme expression in Paneth cells in the crypts. Lysozyme expression in R26^T6B mice returned to normal levels upon removal of doxycycline from the diet (lower row). (E) Peripheral blood analysis conducted in R26^T6B and R26^CTL mice (R26^CTL n = 4; R26^T6B n = 5). (F) Flow cytometric analysis of bone marrow of R26^T6B and R26^CTL mice kept on doxycycline diet for 3 weeks showing developmental block at the Pro-B to Pre-B. p-Values (from left to right): *p=0.0348, **p=0.0023, *p=0.0340, **p=0.0004, unpaired t-test. R26^CTL n = 4; R26^T6B n = 5. (G) Flow cytometry analysis of the bone marrow of control and R26^T6B mice kept on doxycycline diet for 3 weeks. p-Values (from left to right): p=0.0994, **p=0.0092, **p=0.0085, *p=0.0312, unpaired t-test. R26^CTL n = 4; R26^T6B n = 5.

Figure 3—figure supplement 1. Effects of FH-T6B-YFP fusion protein expression during development.

(A) Litter obtained by c-section from a pregnant Rosa26^rtTA/rtTA; Col1a1^T6B/+ female crossed to a Rosa26^rtTA/rtTA; Col1a1^T6B/+ male and maintained on doxycycline from d.p.c. 13.5 to d.p.c. 18.5. (B) Pups from (A) were weighted and genotyped and the results plotted. p-Value: two-tailed unpaired t-test.

Figure 3—figure supplement 2. Immunofluorescence imaging of the small and large intestine of R26^T6B and R26^CTL mice kept on doxycycline diet for a month.

An antibody against YFP was used to detect the T6B fusion protein.

Figure 3—figure supplement 3. Sections from the colon and small intestine sections of R26^T6B and control mice kept on doxycycline-containing diet for 2 months were probed by immunohistochemistry with an anti-Ki67 antibody.

Figure 3—figure supplement 4. Detection of goblet cells by staining of acidic and neutral mucins in intestine sections from R26^T6B and control mice kept on doxycycline diet for 2 months.

Neutral mucins are stained with periodic acid-Shiff, whereas acidic mucins are stained with Alcian blue.

Figure 3—figure supplement 5. Body weight of R26^T6B (n = 5) and control (n = 8) female mice was assessed after 2-month administration of doxycycline-containing chow.

ns, not significant (p=0. 6264, unpaired t-test).

Figure 3—figure supplement 6. Representative flow cytometry plots showing the gating strategy for the identification of hematopoietic stem and progenitor cells from whole bone marrow harvested from R26^T6B and R26^CTL mice maintained on doxycycline diet for 3 weeks.

LT-HSC: Lin- Kit+ Sca1+ CD150+ CD48-; ST-HSC: Lin- Kit+ Sca1+ CD150- CD48-; MPP2: Lin- Kit+ Sca1+ CD150+ CD48+; MPP3/4: Lin- Kit+ Sca1+ CD150 CD48+.

Figure 3—figure supplement 7. Representative flow cytometry plots showing the gating strategy for the identification of B cell lineage populations from whole bone marrow harvested from R26^T6B and R26^CTL mice maintained on doxycycline diet for 3 weeks.

Pro-B: B220+ CD19+ IgD-IgM-CD25-Kit+; Pre-B: B220+ CD19+ IgD-IgM-CD25+; Imm B: B220+ CD19+ IgD-IgM+; Mat B: B220+ CD19+ IgD + IgM+/lo.