Browsing by Author "Howard, Aubrey Gaylon Adam"
Now showing 1 - 2 of 2
Results Per Page
Sort Options
Item A protocol for whole-mount immuno-coupled hybridization chain reaction (WICHCR) in zebrafish embryos and larvae(Cell Press, 2021) Ibarra-García-Padilla, Rodrigo; Howard, Aubrey Gaylon Adam; Singleton, Eileen Willey; Uribe, Rosa AnnaCharacterizing mRNA and protein expression with temporal and spatial resolution is a valuable component of nearly every developmental study. Here, we describe a protocol that combines in situ hybridization chain reaction (HCR) and immunofluorescence, allowing for the detection of mRNAs and proteins simultaneously, in zebrafish embryos and larvae. This protocol expands the flexibility of multiplexed HCR by coupling it with traditional immunofluorescence detection. For complete details on the use and execution of this protocol, please refer to Choi et al. (2010, 2016, 2018) and Howard et al. (2021).Item Posterior Hox Transcription Factors Drive Multiple Phases of Neural Crest Development(2022-12-02) Howard, Aubrey Gaylon Adam; Uribe, Rosa AIn vertebrate embryos, neural crest cells (NCC) as a stem cell population demonstrate a remarkable capacity for both migratory potential as well as a high degree of plasticity. From early embryonic stages, these multipotent cells delaminate from the neural tube, migrate throughout the embryonic body, and differentiate in a wide host of cells lineages, including pigment lineages, corneal endothelium, bone, mesenchyme, glia, and various populations of neurons, such as the enteric nervous system. NCC differentiate into terminal tissues collinear with the anterior-posterior axis, dividing them into four groups: cranial, vagal, trunk, and sacral. Notably, the developmental span of these NCC subpopulations is coincident with the complex and overlapping expression of hox genes, which encode an ancient family of conserved transcription factors. Throughout each of the NCC developmental phases (specification, epithelial-to-mesenchymal transition (EMT), migration, and differentiation), the development of NCC is regulated by a complex and dynamic gene regulatory network, which remains to be full characterized, particularly among the vagal and trunk NCC. Further, the role for more posterior hox transcription factors are here unto unknown within the posterior NCC populations, unlike the cranial NCC. To this end, I have utilized the vertebrate model zebrafish (Danio rerio) to investigate the transcriptional landscape of NCC. I led a team to build and identify an atlas of sox10 cell lineages which includes thousands of transcriptomes from single cells representing stages spanning the lifetime of the fish. Using this transcriptional atlas, I have found a dynamic and diverse set of hox expression codes which define specific NCC and non-NCC lineages in the posterior embryo. Among these codes, hoxb5b was highly enriched and was selected for deeper characterization. Overexpression of hoxb5b revealed that it was sufficient to expand NCC localization domains, but was restricted in its function to early developmental spans. Further, suppression of single and overlapping members of the hox codes identified in the transcriptional atlas demonstrated a requirement for specific hox codes in NCC EMT. Cumulatively these findings support a model in which hox genes participate as potent and required drivers of vagal NCC patterning, expanding our fundamental knowledge of vertebrate development.