Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Developmental, Regenerative and Stem Cell Biology

Language

English (en)

Date of Award

Spring 4-22-2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Raphael Kopan

Abstract

Notch is a membrane-bound transcription factor that mediates signals between adjacent cells. It is the central member of a highly conserved pathway that is important for proliferation, cell fate decisions, and apoptosis not only during development, but also in adulthood acting either in stem cell maintenance or differentiation. In the skin, Notch is essential for epidermal differentiation, maintenance of hair follicle morphology and terminal differentiation of the inner and outer root sheath cells. Ligand-dependent activation, gamma-secretase-dependent cleavage, and RBPjk-mediated downstream transcriptional activities of Notch receptors constitute the "canonical" Notch signaling pathway. This implies that loss of gamma-secretase (no Notch Intracellular Domain (NICD) release), Notch receptors (no NICD), or RBPjk (no activation of downstream targets) should generate similar phenotypes based on the canonical model. Unexpectedly, keratinocyte-specific deletion of Rbpj gene (RBPjcKO) produced a significantly milder phenotype than the keratinocyte-specific deletion of both Notch1 and Notch2 genes (N1N2dcKO) or Presenilin1 and Presenilin2 genes (PSdcKO). Based on these observations, we sought to determine the underlying mechanisms and investigated the potential role of non-canonical Notch signaling in generating these phenotypic differences.

First, we characterized N1N2dcKO, PSdcKO, and RBPjcKO embryonic skin. We found that all these mutants are similar at birth with no significant differences in the gross morphology of their epidermis and hair follicles as well as mRNA and protein expression patterns.

Secondly, we genetically dissected the Notch signaling pathway to define the elements ameliorating the phenotype. We showed that even a limiting amount of cleaved Notch1ICD is sufficient for the milder phenotype observed in RBPjcKO animals.

Lastly, we showed that the loss of Notch signaling in the hair follicles drives the severe phenotypes in PSdcKO and N1N2dcKO animals. Although the levels of epidermal TSLP expression are similar in all Notch mutants, RBPjcKO hair follicles are less distorted and produce less TSLP leading to lower serum TSLP levels. The reduced TSLP in serum results in less aggressive B cell expansion allowing RBPjcKO animals to live significantly longer than N1N2dcKO and PSdcKO animals. In conclusion, gamma-secretase cleaved Notch1ICD likely stimulate RBPjk-independent signals in the Rbpj-/- hair follicle keratinocytes causing a milder phenotype in RBPjcKO animals. Future experiments will interrogate whether RBPjk-independent NICD activity directly generates a different transcriptional response or affects other signaling pathways.

Comments

Permanent URL: http://dx.doi.org/10.7936/K7GB222H

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