PhD Graduate: May Yin LEE
Thesis Title: The roles of Gli3 in murine embryonic mammary gland development
The five pairs of mouse mammary glands are ectoderm-derived appendages that are formed in the embryo. The cellular and molecular mechanisms that regulate embryonic mammary gland development are largely unknown. To understand these mechanisms, I study the involvement and roles of GLI Kruppel family member 3 (Gli3) in embryonic mammary gland development using the Gli3Xt-J/Xt-J (null) mouse model. Previous studies have shown that Gli3Xt-J/Xt-J embryos fail to induce mammary rudiment (MR) pairs 3 and 5. Here, I show that, in addition, Gli3Xt-J/Xt-J MR2 and MR4 are severely hypoplastic and protrude outwardly. In contrast, MR1 is mildly hypoplastic but develops fairly normally.
In the first part of this study, I sought to determine the cellular mechanisms that normally regulate embryonic mammary gland development and the involvement of Gli3 in regulating these mechanisms. I show that cell migration and cell hypertrophy are the primary mechanisms that regulate induction and growth of the MRs during the first two days of mammogenesis. These cellular processes are compromised with the loss of Gli3, resulting in the lack of induction or hypoplasia of Gli3Xt-J/Xt-J MRs.
In the second part of the study, I sought to determine the molecular mechanisms regulated by Gli3 in embryonic mammary gland development. Building on previous observations that Wnt10b is downregulated in the mammary line of Gli3Xt-J/Xt-J embryos, I show that WNT/ß-catenin signalling is downregulated during the first day of mammogenesis in Gli3Xt-J/Xt-J embryos. Reconstitution of WNT/ß-catenin signalling using an ectoderm specific stabilized ß-catenin transgene rescues the induction of MR3, but not MR5, and attenuates the hypoplasia of MR1, MR2, and MR4 in Gli3Xt-J/Xt-J embryos. However, proper morphogenesis was not restored in Gli3Xt-J/Xt-J MR2 and MR3. In addition, stabilized ß-catenin did not rescue the MR phenotype of null mutants for the genes encoding fibroblast growth factor 10 (FGF10), or its main receptor FGFR2B which have lower basal levels of WNT/β-catenin signalling compared to Gli3Xt-J/Xt-J embryos during the first day of mammogenesis. We propose that sufficiently high levels of WNT/β-catenin signalling are crucial in mediating MR induction and growth, but not morphogenesis.
In the third part of the study, I perform a meta-analysis of reported mouse genetic models that have an embryonic MR phenotype and observed that different genes are required for the induction and morphogenesis of each MR pair. This analysis revealed that the mammogenic potential along the mammary line is different – MR4 is the most resistant to compromised induction caused by single and double gene deletions, while the induction of MR3 is frequently compromised by the loss of gene function. The differential mammogenic potential could, in part, explain the diversity of mammary gland numbers and positions among the mammalian species.
My MR-specific analyses in this thesis uncovered position-specific roles for Gli3, leading to the unexpected notion that different mechanisms are involved in the early development of these functionally-identical organs. The utilization of different molecular mechanisms represents an ingenious way of safeguarding the mammary glands in the event of an inhibitory mutation, and highlights the importance of using mammary gland-specific comparisons during research.
Figure Legend: Relationship between Gli3 expression and MR growth in mouse embryos.
(A-H) Hematoxylin/eosin stained transversal histological sections through the center of each MR in E13.5 Gli3Xt-J/+ and Gli3Xt-J/Xt-J embryos. Black contours in (B) exemplify the boundary of the MR (solid) and mammary mesenchyme (dotted). Scale bar in A H: 100 µm. (I-J) Scanning electron micrographs of the external view on epidermis and MR2, showing the outlet of the prospective milk-canal in a wt, and the outwardly protruding mammary rudiment in a Gli3Xt-J/Xt-J embryo at E15.5. (K-N) Carmine red-stained skins with MR2 and MR4 at E18.5. White arrowheads indicate some end buds of the branched mammary rudiment in wt and Gli3Xt-J/Xt-J embryos. Black arrowheads indicate the nipple. (O) Volumetric growth of each MR in wt and Gli3Xt J/Xt-J embryos between E11.75 and E13.5. Error bars represent (one-sided) standard deviations. (P-U’) Radioactive section in situ hybridization of wt MRs with a Gli3 mRNA probe. (P-U) Brightfield images visualizing the MRs. (P’-U’) Darkfield images of the same sections, with white grains indicating hybridization of the probe. (V) Quantification of Gli3 expression in micro-array data of each of the individual MRs, ectoderm and mesenchyme at E12.5 (n=5, solid error bars indicate standard deviation) and E13.5 (n=2, dashed error bars extend between the two measured values). Asterisks indicate a significantly higher Gli3 expression in MR5 than in MR3 and MR4 at E12.5 (p<0.05, Student’s t-test).
Part of this thesis has recently been published, and is accessible here:
For more information on Jacqueline VELTMAAT’s lab, please click here.