Browsing by Author "Uribe, Rosa A."

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    A targeted CRISPR-Cas9 mediated F0 screen identifies genes involved in establishment of the enteric nervous system
    (Public Library of Science, 2024) Moreno-Campos, Rodrigo; Singleton, Eileen W.; Uribe, Rosa A.; Laboratory of Neural Crest and Enteric Nervous System Development
    The vertebrate enteric nervous system (ENS) is a crucial network of enteric neurons and glia resident within the entire gastrointestinal tract (GI). Overseeing essential GI functions such as gut motility and water balance, the ENS serves as a pivotal bidirectional link in the gut-brain axis. During early development, the ENS is primarily derived from enteric neural crest cells (ENCCs). Disruptions to ENCC development, as seen in conditions like Hirschsprung disease (HSCR), lead to the absence of ENS in the GI, particularly in the colon. In this study, using zebrafish, we devised an in vivo F0 CRISPR-based screen employing a robust, rapid pipeline integrating single-cell RNA sequencing, CRISPR reverse genetics, and high-content imaging. Our findings unveil various genes, including those encoding opioid receptors, as possible regulators of ENS establishment. In addition, we present evidence that suggests opioid receptor involvement in the neurochemical coding of the larval ENS. In summary, our work presents a novel, efficient CRISPR screen targeting ENS development, facilitating the discovery of previously unknown genes, and increasing knowledge of nervous system construction.
  • Thumbnail Image
    Item
    CHAF1A Blocks Neuronal Differentiation and Promotes Neuroblastoma Oncogenesis via Metabolic Reprogramming
    (Wiley, 2021) Tao, Ling; Moreno-Smith, Myrthala; Ibarra-García-Padilla, Rodrigo; Milazzo, Giorgio; Drolet, Nathan A.; Hernandez, Blanca E.; Oh, Young S.; Patel, Ivanshi; Kim, Jean J.; Zorman, Barry; Patel, Tajhal; Kamal, Abu Hena Mostafa; Zhao, Yanling; Hicks, John; Vasudevan, Sanjeev A.; Putluri, Nagireddy; Coarfa, Cristian; Sumazin, Pavel; Perini, Giovanni; Parchem, Ronald J.; Uribe, Rosa A.; Barbieri, Eveline
    Neuroblastoma (NB) arises from oncogenic disruption of neural crest (NC) differentiation. Treatment with retinoic acid (RA) to induce differentiation has improved survival in some NB patients, but not all patients respond, and most NBs eventually develop resistance to RA. Loss of the chromatin modifier chromatin assembly factor 1 subunit p150 (CHAF1A) promotes NB cell differentiation; however, the mechanism by which CHAF1A drives NB oncogenesis has remained unexplored. This study shows that CHAF1A gain-of-function supports cell malignancy, blocks neuronal differentiation in three models (zebrafish NC, human NC, and human NB), and promotes NB oncogenesis. Mechanistically, CHAF1A upregulates polyamine metabolism, which blocks neuronal differentiation and promotes cell cycle progression. Targeting polyamine synthesis promotes NB differentiation and enhances the anti-tumor activity of RA. The authors' results provide insight into the mechanisms that drive NB oncogenesis and suggest a rapidly translatable therapeutic approach (DFMO plus RA) to enhance the clinical efficacy of differentiation therapy in NB patients.
  • Thumbnail Image
    Item
    Elevated Hoxb5b Expands Vagal Neural Crest Pool and Blocks Enteric Neuronal Development in Zebrafish
    (Frontiers Media S.A., 2022) Howard, Aubrey G. A.; Nguyen, Aaron C.; Tworig, Joshua; Ravisankar, Priya; Singleton, Eileen W.; Li, Can; Kotzur, Grayson; Waxman, Joshua S.; Uribe, Rosa A.
    Neural crest cells (NCCs) are a migratory, transient, and multipotent stem cell population essential to vertebrate embryonic development, contributing to numerous cell lineages in the adult organism. While great strides have been made in elucidating molecular and cellular events that drive NCC specification, comprehensive knowledge of the genetic factors that orchestrate NCC developmental programs is still far from complete. We discovered that elevated Hoxb5b levels promoted an expansion of zebrafish NCCs, which persisted throughout multiple stages of development. Correspondingly, elevated Hoxb5b also specifically expanded expression domains of the vagal NCC markers foxd3 and phox2bb. Increases in NCCs were most apparent after pulsed ectopic Hoxb5b expression at early developmental stages, rather than later during differentiation stages, as determined using a novel transgenic zebrafish line. The increase in vagal NCCs early in development led to supernumerary Phox2b+ enteric neural progenitors, while leaving many other NCC-derived tissues without an overt phenotype. Surprisingly, these NCC-derived enteric progenitors failed to expand properly into sufficient quantities of enterically fated neurons and stalled in the gut tissue. These results suggest that while Hoxb5b participates in vagal NCC development as a driver of progenitor expansion, the supernumerary, ectopically localized NCC fail to initiate expansion programs in timely fashion in the gut. All together, these data point to a model in which Hoxb5b regulates NCCs both in a tissue specific and temporally restricted manner.
  • Thumbnail Image
    Item
    Expansion of a neural crest gene signature following ectopic MYCN expression in sympathoadrenal lineage cells in vivo
    (Public Library of Science, 2024) Ibarra-García-Padilla, Rodrigo; Nambiar, Annika; Hamre, Thomas A.; Singleton, Eileen W.; Uribe, Rosa A.
    Neural crest cells (NCC) are multipotent migratory stem cells that originate from the neural tube during early vertebrate embryogenesis. NCCs give rise to a variety of cell types within the developing organism, including neurons and glia of the sympathetic nervous system. It has been suggested that failure in correct NCC differentiation leads to several diseases, including neuroblastoma (NB). During normal NCC development, MYCN is transiently expressed to promote NCC migration, and its downregulation precedes neuronal differentiation. Overexpression of MYCN has been linked to high-risk and aggressive NB progression. For this reason, understanding the effect overexpression of this oncogene has on the development of NCC-derived sympathoadrenal progenitors (SAP), which later give rise to sympathetic nerves, will help elucidate the developmental mechanisms that may prime the onset of NB. Here, we found that overexpressing human EGFP-MYCN within SAP lineage cells in zebrafish led to the transient formation of an abnormal SAP population, which displayed expanded and elevated expression of NCC markers while paradoxically also co-expressing SAP and neuronal differentiation markers. The aberrant NCC signature was corroborated with in vivo time-lapse confocal imaging in zebrafish larvae, which revealed transient expansion of sox10 reporter expression in MYCN overexpressing SAPs during the early stages of SAP development. In these aberrant MYCN overexpressing SAP cells, we also found evidence of dampened BMP signaling activity, indicating that BMP signaling disruption occurs following elevated MYCN expression. Furthermore, we discovered that pharmacological inhibition of BMP signaling was sufficient to create an aberrant NCC gene signature in SAP cells, phenocopying MYCN overexpression. Together, our results suggest that MYCN overexpression in SAPs disrupts their differentiation by eliciting abnormal NCC gene expression programs, and dampening BMP signaling response, having developmental implications for the priming of NB in vivo.