Fig 1: Sex differences in the developmental patterning of SCN Vip-tdT cell density plots aggregated across samples for each age illustrating total number of neighboring cells for each cell type in each sex. Blue lattice frames illustrate SCN boundaries at each age. Number in bottom right corner for each map indicates the number of aggregated samples. Additional orientations at select ages are shown in Supplementary Videos S1–S12.
Fig 2: Developmental appearance of SCN Avp-tdT+ and Vip-tdT+ neurons. (A) Timeline illustrating ages of tissue collection. Breeding and pup development data are in Supplementary Figure S9. (B) Labeled cells aggregated across SCN slice positions for a representative mouse from each age group illustrating progressive appearance of Avp-tdT+ and Vip-tdT+ cells during postnatal development. (C) Top: Total number of SCN Avp-tdT+ and Vip-tdT+ cells across development. Cre: F(1,99) = 89.6, p < 0.001, Age: F(7,99) = 411.9, p < 0.0001, Sex: F(1,99) = 0.7, p = 0.4, Cre*Age: F(7,99) = 11.3, p < 0.0001, Cre*Sex: F(1,99) = 0.01, p > 0.9, Age*Sex: F(7,99) = 5.2, p < 0.0001, Cre*Age*Sex: F(7,99) = 0.9, p > 0.5. Bottom: To compare developmental patterns across cell type, cell counts at each age were expressed as a percent relative to the number of labeled cells in adults. Cre: F(1,99) = 0.2, p > 0.6, Age: F(7,99) = 409.4, p < 0.0001, Sex: F(1,99) = 50.2, p < 0.0001, Cre*Age: F(7,99) = 2.9, p < 0.01, Cre*Sex: F(1,99) = 1.6, p > 0.2, Age*Sex: F(7,99) = 8.3, p < 0.0001, Cre*Age*Sex: F(7,99) = 1.1, p > 0.3. Total cell counts divided by sex, with magnification of E18-P01 data, are in Supplementary Figure S10A. (D) Percent labeled cells at each age divided by sex and cell type. Avp-tdT+: Age: F(7,47) = 204.3, p < 0.0001, Sex: F(1,47) = 16.4, p < 0.0005, Age*Sex: F(7,47) = 3.0.1, p < 0.01. Vip-tdT+: Age: F(7,52) = 222.5, p < 0.0001, Sex: F(1,52) = 36.0, p < 0.0001, Age*Sex: F(7,52) = 6.7, p < 0.0001. Contrasts comparing genotype or sex in each cell type, *p < 0.05.
Fig 3: Developmental appearance of Avp-tdT+ and Vip-tdT+ neurons across the anteroposterior SCN. (A) Representative samples illustrating spatial patterning of Avp-tdT+ development in the reconstructed horizontal plane. Coronal sections are illustrated in Supplementary Figure S10A. (B) Regional gradients in the developmental appearance of Avp-tdT+ neurons are influenced by sex. Age: F(7,47) = 190.4, p < 0.0001, Sex: F(1,47) = 18.5, p < 0.0001, Position: F(2,94) = 14.7, p < 0.001, Age*Sex: F(7,47) = 3.3, p < 0.01, Age*Position: F(14,94) = 3.3, p < 0.0005, Sex*Position: F(2,94) = 4.2, p < 0.02, Age*Sex*Position: F(14,94) = 0.8, p > 0.6. (C) Representative samples illustrating spatial patterning Vip-tdT+ development in the reconstructed horizontal plane. Coronal sections are illustrated in Supplementary Figure S10B. (D) Regional gradients in the developmental appearance of Vip-tdT+ neurons are influenced by sex. Age: F(7,53) = 97.5, p < 0.0001, Sex: F(1,53) = 22.7, p < 0.0001, Position: F(2,106) = 3.3, p < 0.05, Age*Sex: F(7,53) = 4.0, p < 0.005, Age*Position: F(14,106) = 2.9, p < 0.001, Sex*Position: F(2,106) = 1.8, p > 0.1, Age*Sex*Position: F(14,106) = 0.7, p > 0.7. Post hoc contrasts comparing male and female data for each slice position are indicated by color-coded asterisks below the x axis of each female graph in panels (B,D). *p < 0.05.
Fig 4: Spatial mapping of SCN neurons in each peptide class in adulthood. (A) A representative subset of SCN slices collected through the anteroposterior axis from a female Avp-tdT or Vip-tdT mouse. Each slice is color-coded by slice position, with cells in all SCN slices superimposed in rightmost panel. The full set of SCN slices from this mouse is illustrated in Supplementary Figure S1A. (B) Total number of SCN Avp-tdT+ and Vip-tdT+ neurons in each sex. Cre: F(1,9) = 18.1, p < 0.005, Sex: F(1,9) = 7.3, p < 0.05; Sex*Cre: F(1,9) = 1.1, p > 0.3. (C) Sex influences the number of Avp-tdT+ and Vip-tdT+ neurons in different anteroposterior SCN regions. Avp-tdT - Sex: F(1,5) = 5.6, p = 0.06; Position: F(2,10) = 25.1, p = 0.0001; Sex*Position: F(2,10) = 4.5, p < 0.05. Vip-tdT – Sex: F(1,4) = 3.6, p > 0.1; Position: F(2,8) = 63.3, p < 0.0001; Sex*Position: F(2,8) = 1.5, p > 0.2. aSCN, mSCN, and pSCN: Anterior, middle, and posterior SCN. Contrasts comparing male and female data for each cell type, *p < 0.05.
Fig 5: Genetic strategy to label spatial location of SCN peptide classes. (A) Schematic illustrating genetic approach to label Avp- and Vip-expressing SCN neurons with tdT. (B) tdT labeling is Cre-dependent in the SCN. Also see Supplementary Figure S4. Cre: F(2,22) = 24.1, p < 0.0001; Sex: F(1,22) = 0.3, p > 0.5; Cre*Sex: F(2,22) = 0.6, p > 0.5. (C) Representative SCN slices illustrating tdT and AVP/VIP expression in a male Avp-tdT or Vip-tdT SCN. Scale bars = 100 µm. (D) Total number of labeled neurons collapsed across three SCN slices. AVP/VIP IHC: Cre: F(1,15) = 175, p < 0.0001; Sex: F(1,15) = 0.2, p > 0.6; Cre*Sex: F(1,15) = 0.3, p > 0.5. tdT+: Cre: F(1,15) = 4.12, p = 0.06; Sex: F(1,15) = 2.3, p > 0.1; Cre*Sex: F(1,15) = 2.9, p > 0.1. (E) More Avp-tdT+ neurons express AVP compared to Vip-tdT+ neurons that express VIP. Cre: F(1,15) = 112.5, p < 0.0001; Sex: F(1,15) = 3.2, p = 0.09; Cre*Sex: F(1,15) = 0.1, p > 0.8. (F) More Vip-tdT+ express tdT compared to Avp-tdT+ neurons. Cre: F(1,15) = 105.8, p < 0.0001; Sex: F(1,15) = 1.6, p > 0.2; Cre*Sex: F(1,15) = 0.2, p > 0.6. Numbers below x-axis in panels (B,D) indicate sample sizes for each group. Contrasts comparing genotype, *p < 0.05.
Supplier Page from MilliporeSigma for Anti-VIP antibody produced in rabbit