Gas6 is dispensable for pubertal mammary gland development

Abstract

Mammary gland development is a complex and dynamic process that occurs mainly postnatally. Ductal elongation and branching morphogenesis are regulated by a plethora of factors, including cytokines, hormones, growth factors and the extracellular matrix. Gas6 is a secreted gamma-carboxylated protein that binds to a family of receptors tyrosine kinase receptors known as the TAMR family (Tyro3, Axl, Mer). Gas6 function in developmental processes has been shown in nervous, reproductive and immune systems. In this study, we found that Gas6 is highly expressed in virgin adult mammary glands but declines during pregnancy and lactation. Specifically, Gas6 is highly expressed in luminal and basal mammary epithelial cells during puberty and adulthood, while TAMR expression is low. Mammary whole mount analysis revealed that Gas6 germline deletion does not impact ductal elongation, branching morphogenesis or terminal end bud formation. Masson's trichrome staining showed that collagen deposition is similar in Gas6-/- mice as compared to wildtype mice. Gas6-/- mammary glands presented an organized luminal and myoepithelial bilayer of cells, and the proportion of mammary stem cells was unchanged in Gas6-/- mammary glands as compared to wildtype. Finally, proliferation of epithelial cells and macrophage number were similar in both groups. These studies suggest that Gas6 is not essential for pubertal mammary gland development in nulliparous mice.

Fig 1. TAMR expression and their ligands in mammary epithelial cells during mammary gland development.

2-D t-SNE projection plots of scRNA-seq data from mammary epithelial cells isolated from pubertal and adult mouse mammary gland. Tyro3, Axl, Mertk, Gas6 and Pros1 expression in different epithelial cell clusters [20]. Each cell was colored by normalized expression of the genes in the cell. Color scale represents the normalized UMI (unique molecular identifier) counts for each gene under a log10 scale.

Fig 2. TAMR protein expression and their ligands in normal mammary gland.

(A) Mammary glands (n = 3 per time point) were harvested in diestrus from 5 and 12 week old animals and stained with an antibody to Gas6. Representative images of mouse mammary tissue showing Gas6 expression in mammary epithelial cells and mammary ductal lumen. Scale bar = 20 μm. (B) Western blot analysis of TAMR (n = 3) and Pros1 (n = 2) in epithelial organoids isolated from adult animals. Spleen and brain were used as positive controls. Uncropped blots are depicted in S2 Fig.

Fig 3. Ductal morphogenesis is normal in Gas6^-/- mammary glands.

Mammary glands (5–7 mice per timepoint, 2 mammary glands per mouse) were harvested in diestrus from 5, 8 and 12 week old animals. (A) Carmine alum stained whole mounts of mammary glands from wildtype and Gas6^-/- animals. Scale bar = 1 mm. (B) Ductal elongation was quantified by measuring the distance from lymph node to the most distal tip of a duct or TEB. (5 wk p = 0.08; 8 wk p = 0.06; 12 wk p = 0.86). (C) Quantification of TEB number in wildtype and Gas6^-/- glands (p = 0.49). (D) Branching morphogenesis was quantified by counting the branch points in the entire gland (5 wk p = 0.48; 8 wk p = 0.48; 12 wk p = 0.11).

Fig 4. Histological characterization of Gas6^-/- mammary ducts.

(A) Representative images of mouse mammary tissue stained with H&E and Masson’s trichrome (3 glands from 3 mice). Scale bar = 20 μm. (B) Representative images of mouse mammary tissue from wildtype and Gas6^-/- animals stained with an antibody for F4/80. Graph depicts quantification of the average number of F4/80⁺ cells in wild type and Gas6^-/- mammary sections. For each gland, a minimum of 4 ducts and 4 TEBs were counted from 3 glands (3 mice) for each group (5 wk TEB p = 0.81; 5 wk duct p = 0.25; 12 wk duct p = 0.56). Scale bar = 20 μm. (C) Representative immunofluorescence images of mammary sections stained with antibodies for Ki-67 (red) and panCK (green) are depicted. Graph shows quantification of the average number of Ki-67⁺ cells in WT (5 week, n = 7; 12 week, n = 5) and Gas6^-/- (5 week, n = 5; 12 week, n = 4) mammary glands, where n is the number of animals. For each gland, a minimum of 4 ducts and 4 TEBs were counted per animal (5 week TEB p = 0.41; 5 week duct p = 0.46; 12 week duct p = 0.64). Scale bar = 20 μm.

Fig 5. Gas6^-/- mammary glands are comprised of normal luminal and basal cell layers.

(A) Representative images of mammary glands from wild type and Gas6^-/- mice stained with antibodies to CK14 (red) and CK8 (green) (3 glands from 3 mice). Scale bar = 20 μm. (n = 3) (B) Dot plots depict the percentages of LIN⁻CD24^hiCD29^lo (luminal) or LIN⁻CD24⁺CD29^hi (basal) cell population in mammary epithelial cells from wild type and Gas6^-/- animals (n = 3). Graph depicts the percentages of luminal and basal cells (mean and SEM) from wildtype and Gas6^-/- mammary glands from 3 independent experiments (*p< 0.05).