Multiscale Regulation of Cellular Differentiation During Organogenesis

During Vertebrate organogenesis, stem cells from multiple embryonic germ layers differentiate and give rise to intricate tissue morphologies. However, the molecular and cellular mechanisms governing the differentiation of diverse cell types and the subsequent emergence of complex functional organs remain poorly understood. The zebrafish embryo is an excellent vertebrate model system to study this problem, due to its rapid external development, optical transparency, ease of genetic/chemical perturbation, and genomic similarity to other vertebrates. In my thesis work, I use high-resolution microscopy and targeted knockdown of key genes and signaling pathways to study cell fate regulation during the differentiation of multiple stem cell populations in the developing zebrafish embryo. At the border between the neural plate and non-neural ectoderm, a group of multipotent stem cells called neural crest cells (NCCs) are specified. NCCs proliferate and migrate across the entire embryo, participate in cell-cell signaling, and activate distinct gene regulatory programs to produce diverse cell types. Previous and ongoing studies have focused primarily on characterizing the genetic interactions, expression of transcription factors, and signaling pathways necessary for multivariate NCC differentiation. However, the importance of cell cycle in the modulation of NCC cell fate during differentiation has been unclear. This gap in our knowledge has been primarily due to a shortage of tools needed to visualize and capture cell cycle dynamics of NCCs in vivo while they are undergoing active migration and differentiation. In Chapter 2, I describe the development of an NCC-specific fluorescent, ubiquitination-based cell cycle indicator (Fucci) system which provides in vivo readouts of cell cycle progression. This transgenic Fucci system is expressed in delaminating and migrating NCCs as they undergo differentiation. I demonstrated the validation of this tool and its use in live tracking cell cycle progression in NCCs and their derivative cell types. NCCs are known to intermix and interact with cells at the anterior edge of the neural plate where an ectodermal thickening called the olfactory placode is formed. The olfactory placode grows and develops into the olfactory epithelium (OE), i.e., the vertebrate nose. The mature zebrafish OE houses a diverse repertoire of olfactory sensory neurons (OSNs) that can detect odors and other signals. The OE also contains olfactory multiciliated cells (OMCCs) with motile cilia that are known to direct fluid flow toward OSNs for odor detection. The mechanisms that drive the formation and integration of new OSNs and OMCCs into the developing OE are not well understood. Specifically, the intercellular interactions and gene expression patterns within progenitors and stem cells that generate new OSNs and OMCCs remain unclear. In the work for Chapter 3, I tracked single cells undergoing stochastic Notch signaling activation in the developing OE and performed targeted inhibition of multiple signaling pathways to assay for changes in OSN differentiation. I found that transient groups of progenitor cells self-organize into neighborhoods with reciprocal expression patterns for Notch signaling and the transcription factor Insulinoma-associated 1a to facilitate the formation of new OSNs. Using computational modeling followed by experimental validation, and tracking of basal to apical cell movements, I have uncovered mechanisms operating from molecular to tissue-wide scales that coordinate olfactory neurogenesis in vivo. In Chapter 4, using live imaging and cell tracking, I identified the earliest time window of OMCC differentiation and demonstrated these cells’ derivation from peridermal cells. In addition, targeted cell ablation, and temporally-specific signaling inhibition revealed how Notch signaling represses differentiation/ciliogenesis-associated genes foxj1a and foxj1b to regulate OMCC specification in a region-specific manner. In Chapter 5, I describe preliminary data showing heterogeneity amongst olfactory progenitors and their differential sensitivity to Notch signaling. I also discuss how the broad implications of this work may provide new insights into the role of stochasticity in cell-cell signaling during organogenesis.