Biology subjects

Uribe, R. A.

Publications and source records attributed to Uribe, R. A..

8 recordsLinked to original sources

bioRxiv2025

Whole-gut spatial genomic analysis reveals molecular regionalization of the differentiating zebrafish enteric nervous system

The enteric nervous system (ENS) is the intrinsic nervous system of the gut and controls essential functions, such as gut motility, intestinal barrier function, and water balance. The ENS displays a complex 3D architecture within the context of the gut and specific transcriptional states needed to control gut homeostasis. During development, the ENS develops from enteric neural progenitor cells (ENPs) that migrate into the gut and differentiate into functionally diverse neuron types. Incorrect ENS development can disrupt ENS function and induce various gut disorders, including the congenital disease Hirschsprung disease, or various other functional gut neurological disorders, such as esophageal achalasia. In this study, we used the zebrafish larval model and performed whole gut spatial genomic analysis (SGA) of the differentiating ENS at cellular resolution. To that end, a pipeline was developed that integrated early and late developmental ENS stages by linking various spatial and transcriptional dimensions to discover regionalized cellular groups and their co-expression similarity. We identified 3D networks of intact ENS surrounding the gut and predicted cellular connectivity properties based on the stage. Spatial variable genes, such as hoxb5b, hoxa4a, etv1, and ret, were regionalized along gut axes, suggesting they may have a precise spatiotemporal control of ENS development. The application of SGA to ENS development provides new insights into its cellular transcriptional networks and interactions, and provides a baseline data set to further advance our understanding of gut neurodevelopmental disorders such as Hirschsprung disease and congenital enteric neuropathies.

neuroscience↗

bioRxiv2024

Expansion of a neural crest gene signature following ectopic MYCN expression in sympathoadrenal lineage cells in vivo

Neural crest cells (NCC) are multipotent migratory stem cells which originate from the neural tube during early vertebrate embryogenesis. NCC 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 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, and suggesting BMP is functionally important for the NCC to SAP differentiation transition. In agreement, 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.

developmental biology↗

bioRxiv2023

A targeted CRISPR-Cas9 mediated F0 screen identifies genes involved in establishment of the enteric nervous system

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 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 for opioid receptors, as possible regulators of ENS establishment. In addition, we present evidence that suggests opioid receptor involvement in 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.

neuroscience↗

bioRxiv2023

BMP signaling pathway member expression is enriched in enteric neural progenitors and required for zebrafish enteric nervous system development

The vertebrate enteric nervous system (ENS) consists of a series of interconnected ganglia within the gastrointestinal (GI) tract, formed during development following the migration of enteric neural crest cells (ENCCs) into the primitive gut tube. Much work has been done to unravel the complex nature of extrinsic and intrinsic factors that regulate processes that direct the migration, proliferation, and differentiation of ENCCs. However, ENS development is a complex process and we still have much to learn regarding the signaling factors that regulate ENCC development. Here in zebrafish, through transcriptomic, in situ transcript expression, immunohistochemical analysis, and chemical attenuation, we identified a time-dependent role for bone morphogenetic protein (BMP) in the maintenance of Phox2bb+ enteric progenitor numbers and/or time of differentiation of the progenitor pool. In support of our in silico transcriptomic analysis, we identified expression of a novel ENS ligand-encoding transcript, bmp5, within developmental regions of ENCCs. Through generation of a novel mutant bmp5wmr2, we identified a functional role for BMP5 in proper GI tract colonization, whereby phox2bb+ enteric progenitor numbers were reduced. Altogether, this work has identified time-dependent roles for BMP signaling and identification of a novel extrinsic factor, BMP5, that is necessary for proper formation of vertebrate ENS.

developmental biology↗

bioRxiv2022

In Toto imaging of early Enteric Nervous System Development reveals that gut colonization is tied to proliferation downstream of Ret

The enteric nervous system (ENS) is a vast intrinsic network of neurons and glia within the gastrointestinal tract and is largely derived from enteric neural crest cells (ENCCs) that emigrate into the gut during vertebrate embryonic development. Study of ENCC migration dynamics and their genetic regulators provides great insights into fundamentals of collective cell migration and nervous system formation, and are a pertinent subject for study due to their relevance to the human congenital disease, Hirschsprung disease (HSCR). For the first time, we performed in toto gut imaging and single-cell generation tracing of ENCC migration in WT and a novel ret heterozygous background zebrafish (retwmr1/+) to gain insight into ENCC dynamics in vivo. We observed that retwmr1/+ zebrafish produced fewer ENCCs while localized along the gut, which failed to reach the hindgut, resulting in HSCR-like phenotypes. Specifically, we observed a proliferation dependent migration mechanism, where cell divisions were associated with inter-cell distances and migration speed. Lastly, we detected a premature neuronal differentiation gene expression signature in retwmr1/+ ENCCs, collectively suggesting that Ret signaling may function to regulate maintenance of a stem-state in ENCCs.

developmental biology↗

bioRxiv2021

Elevated Hoxb5b expands vagal neural crest pool and blocks enteric neuronal development in zebrafish

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.

developmental biology↗

bioRxiv2020

An atlas of neural crest lineages along the posterior developing zebrafish at single-cell resolution

Neural crest cells (NCCs) are vertebrate stem cells that give rise to various cell types throughout the developing body in early life. Here, we utilized single-cell transcriptomic analyses to delineate NCC-derivatives along the posterior developing vertebrate, zebrafish, during the late embryonic to early larval stage, a period when NCCs are actively differentiating into distinct cellular lineages. We identified several major NCC/NCC-derived cell-types including mesenchyme, neural crest, neural, neuronal, glial, and pigment, from which we resolved over three dozen cellular subtypes. We dissected gene expression signatures of pigment progenitors delineating into chromatophore lineages, mesenchyme subtypes, and enteric NCCs transforming into enteric neurons. Global analysis of NCC derivatives revealed they were demarcated by combinatorial hox gene codes, with distinct profiles within neuronal cells. From these analyses, we present a comprehensive cell-type atlas that can be utilized as a valuable resource for further mechanistic and evolutionary investigations of NCC differentiation.

developmental biology↗

bioRxiv2020

Frequency-independent biological signal identification (FIBSI): A free program that simplifies intensive analysis of non-stationary time series data

Extracting biological signals from non-linear, dynamic and stochastic experimental data can be challenging, especially when the signal is non-stationary. Many currently available methods make assumptions about the data structure (e.g., signal is periodic, sufficient recording time) and modify the raw data in pre-processing using filters and/or transformations. With an agnostic approach to biological data analysis as a goal, we implemented a signal detection algorithm in Python that quantifies the dimensional properties of waveform deviations from baseline via a running fit function. We call the resulting free program frequency-independent biological signal identification (FIBSI). We demonstrate the utility of FIBSI on two disparate types of experimental data: in vitro whole-cell current-clamp electrophysiological recordings of rodent sensory neurons (i.e., nociceptors) and in vivo fluorescence image time-lapse movies capturing gastrointestinal motility in larval zebrafish. In rodent nociceptors, depolarizing fluctuations in membrane potential are irregular in shape and difficult to distinguish from noise. Using FIBSI, we determined that nociceptors from naïve mice generate larger, more frequent fluctuations compared to naïve rats, suggesting species-specific specializations in rodent nociceptors. In zebrafish, measuring gut motility is a useful tool for addressing developmental and disease-related mechanisms associated with gut function. However, available methods are laborious, technically complex, and/or not cost-effective. We developed and tested a novel assay that can characterize intestinal peristalsis using imaging time series datasets. We used FIBSI to identify muscle contractions in the fluorescence signals and compared their frequencies in unfed and fed larvae. Additionally, FIBSI allowed us to discriminate between peristalsis and oscillatory sphincter-like movements in functionally distinct gut segments (foregut, midgut, and cloaca). We conclude that FIBSI, which is freely available via GitHub, is widely useful for the unbiased analysis of non-stationary signals and extraction of biologically meaningful information from experimental time series data and can be employed for both descriptive and hypothesis-driven investigations.Author Summary Biologists increasingly work with large, complex experimental datasets. Those datasets often encode biologically meaningful signals along with background noise that is recorded along with the biological data during experiments. Background noise masks the real signal but originates from other sources, for example from the equipment used to perform the measurements or environmental disturbances. When it comes to analyzing the data, distinguishing between the real biological signals and the background noise can be very challenging. Many existing programs designed to help scientists with this problem are either difficult to use, not freely available, or only appropriate to use on very specific types of datasets. The research presented here embodies our goal of helping others to analyze their data by employing a powerful but novice-friendly program that describes multiple features of biological activity in its raw form without abstract transformations. We show the program’s applicability using two different kinds of biological activity measured in our labs. It is our hope that this will help others to analyze complex datasets more easily, thoroughly, and rigorously.Competing Interest StatementThe authors have declared no competing interest.View Full Text

physiology↗