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 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.

Genetic regulation of enteric nervous system development in zebrafish.

The enteric nervous system (ENS) is a complex series of interconnected neurons and glia that reside within and along the entire length of the gastrointestinal tract. ENS functions are vital to gut homeostasis and digestion, including local control of peristalsis, water balance, and intestinal cell barrier function. How the ENS develops during embryological development is a topic of great concern, as defects in ENS development can result in various diseases, the most common being Hirschsprung disease, in which variable regions of the infant gut lack ENS, with the distal colon most affected. Deciphering how the ENS forms from its progenitor cells, enteric neural crest cells, is an active area of research across various animal models. The vertebrate animal model, zebrafish, has been increasingly leveraged to understand early ENS formation, and over the past 20 years has contributed to our knowledge of the genetic regulation that underlies enteric development. In this review, I summarize our knowledge regarding the genetic regulation of zebrafish enteric neuronal development, and based on the most current literature, present a gene regulatory network inferred to underlie its construction. I also provide perspectives on areas for future zebrafish ENS research.

Systematic analysis of proximal midgut- and anorectal-originating contractions in larval zebrafish using event feature detection and supervised machine learning algorithms.

BACKGROUND: Zebrafish larvae are translucent, allowing in vivo analysis of gut development and physiology, including gut motility. While recent progress has been made in measuring gut motility in larvae, challenges remain which can influence results, such as how data are interpreted, opportunities for technical user error, and inconsistencies in methods. METHODS: To overcome these challenges, we noninvasively introduced Nile Red fluorescent dye to fill the intraluminal gut space in zebrafish larvae and collected serial confocal microscopic images of gut fluorescence. We automated the detection of fluorescent-contrasted contraction events against the median-subtracted signal and compared it to manually annotated gut contraction events across anatomically defined gut regions. Supervised machine learning (multiple logistic regression) was then used to discriminate between true contraction events and noise. To demonstrate, we analyzed motility in larvae under control and reserpine-treated conditions. We also used automated event detection analysis to compare unfed and fed larvae. KEY RESULTS: Automated analysis retained event features for proximal midgut-originating retrograde and anterograde contractions and anorectal-originating retrograde contractions. While manual annotation showed reserpine disrupted gut motility, machine learning only achieved equivalent contraction discrimination in controls and failed to accurately identify contractions after reserpine due to insufficient intraluminal fluorescence. Automated analysis also showed feeding had no effect on the frequency of anorectal-originating contractions. CONCLUSIONS & INFERENCES: Automated event detection analysis rapidly and accurately annotated contraction events, including the previously neglected phenomenon of anorectal contractions. However, challenges remain to discriminate contraction events based on intraluminal fluorescence under treatment conditions that disrupt functional motility.

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.

Hox proteins as regulators of extracellular matrix interactions during neural crest migration.

Emerging during embryogenesis, the neural crest are a migratory, transient population of multipotent stem cell that differentiates into various cell types in vertebrates. Neural crest cells arise along the anterior-posterior extent of the neural tube, delaminate and migrate along routes to their final destinations. The factors that orchestrate how neural crest cells undergo delamination and their subsequent sustained migration is not fully understood. This review provides a primer about neural crest epithelial-to-mesenchymal transition (EMT), with a special emphasis on the role of the Extracellular matrix (ECM), cellular effector proteins of EMT, and subsequent migration. We also summarize published findings that link the expression of Hox transcription factors to EMT and ECM modification, thereby implicating Hox factors in regulation of EMT and ECM remodeling during neural crest cell ontogenesis.

In toto imaging of early enteric nervous system development reveals that gut colonization is tied to proliferation downstream of Ret.

The enteric nervous system 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 these are pertinent subjects 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 wild type and a novel ret heterozygous background zebrafish (retwmr1/+) to gain insight into ENCC dynamics in vivo. We observed that retwmr1/+ zebrafish produced fewer ENCCs localized along the gut, and these ENCCs 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. These results suggest that Ret signaling may regulate maintenance of a stem state in ENCCs.

Expanding the eukaryotic genetic code with a biosynthesized 21st amino acid.

Genetic code expansion technology allows for the use of noncanonical amino acids (ncAAs) to create semisynthetic organisms for both biochemical and biomedical applications. However, exogenous feeding of chemically synthesized ncAAs at high concentrations is required to compensate for the inefficient cellular uptake and incorporation of these components into proteins, especially in the case of eukaryotic cells and multicellular organisms. To generate organisms capable of autonomously biosynthesizing an ncAA and incorporating it into proteins, we have engineered a metabolic pathway for the synthesis of O-methyltyrosine (OMeY). Specifically, we endowed organisms with a marformycins biosynthetic pathway-derived methyltransferase that efficiently converts tyrosine to OMeY in the presence of the co-factor S-adenosylmethionine. The resulting cells can produce and site-specifically incorporate OMeY into proteins at much higher levels than cells exogenously fed OMeY. To understand the structural basis for the substrate selectivity of the transferase, we solved the X-ray crystal structures of the ligand-free and tyrosine-bound enzymes. Most importantly, we have extended this OMeY biosynthetic system to both mammalian cells and the zebrafish model to enhance the utility of genetic code expansion. The creation of autonomous eukaryotes using a 21st amino acid will make genetic code expansion technology more applicable to multicellular organisms, providing valuable vertebrate models for biological and biomedical research.

CHAF1A Blocks Neuronal Differentiation and Promotes Neuroblastoma Oncogenesis via Metabolic Reprogramming.

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.

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 cells, 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.

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.

Immunohistochemical and ultrastructural analysis of the maturing larval zebrafish enteric nervous system reveals the formation of a neuropil pattern.

The gastrointestinal tract is constructed with an intrinsic series of interconnected ganglia that span its entire length, called the enteric nervous system (ENS). The ENS exerts critical local reflex control over many essential gut functions; including peristalsis, water balance, hormone secretions and intestinal barrier homeostasis. ENS ganglia exist as a collection of neurons and glia that are arranged in a series of plexuses throughout the gut: the myenteric plexus and submucosal plexus. While it is known that enteric ganglia are derived from a stem cell population called the neural crest, mechanisms that dictate final neuropil plexus organization remain obscure. Recently, the vertebrate animal, zebrafish, has emerged as a useful model to understand ENS development, however knowledge of its developing myenteric plexus architecture was unknown. Here, we examine myenteric plexus of the maturing zebrafish larval fish histologically over time and find that it consists of a series of tight axon layers and long glial cell processes that wrap the circumference of the gut tube to completely encapsulate it, along all levels of the gut. By late larval stages, complexity of the myenteric plexus increases such that a layer of axons is juxtaposed to concentric layers of glial cells. Ultrastructurally, glial cells contain glial filaments and make intimate contacts with one another in long, thread-like projections. Conserved indicators of vesicular axon profiles are readily abundant throughout the larval plexus neuropil. Together, these data extend our understanding of myenteric plexus architecture in maturing zebrafish, thereby enabling functional studies of its formation in the future.

Migration and diversification of the vagal neural crest.

Arising within the neural tube between the cranial and trunk regions of the body axis, the vagal neural crest shares interesting similarities in its migratory routes and derivatives with other neural crest populations. However, the vagal neural crest is also unique in its ability to contribute to diverse organs including the heart and enteric nervous system. This review highlights the migratory routes of the vagal neural crest and compares them across multiple vertebrates. We also summarize recent advances in understanding vagal neural crest ontogeny and discuss the contribution of this important neural crest population to the cardiovascular system and endoderm-derived organs, including the thymus, lungs and pancreas.

Tracking neural crest cell cycle progression in vivo.

Analysis of cell cycle entry/exit and progression can provide fundamental insights into stem cell propagation, maintenance, and differentiation. The neural crest is a unique stem cell population in vertebrate embryos that undergoes long-distance collective migration and differentiation into a wide variety of derivatives. Using traditional techniques such as immunohistochemistry to track cell cycle changes in such a dynamic population is challenging, as static time points provide an incomplete spatiotemporal picture. In contrast, the fluorescent, ubiquitination-based cell cycle indicator (Fucci) system provides in vivo readouts of cell cycle progression and has been previously adapted for use in zebrafish. The most commonly used Fucci systems are ubiquitously expressed, making tracking of a specific cell population challenging. Therefore, we generated a transgenic zebrafish line, Tg(-4.9sox10:mAG-gmnn(1/100)-2A-mCherry-cdt1(1/190)), in which the Fucci system is specifically expressed in delaminating and migrating neural crest cells. Here, we demonstrate validation of this new tool and its use in live high-resolution tracking of cell cycle progression in the neural crest and derivative populations.

Retinoic acid temporally orchestrates colonization of the gut by vagal neural crest cells.

The enteric nervous system arises from neural crest cells that migrate as chains into and along the primitive gut, subsequently differentiating into enteric neurons and glia. Little is known about the mechanisms governing neural crest migration en route to and along the gut in vivo. Here, we report that Retinoic Acid (RA) temporally controls zebrafish enteric neural crest cell chain migration. In vivo imaging reveals that RA loss severely compromises the integrity and migration of the chain of neural crest cells during the window of time window when they are moving along the foregut. After loss of RA, enteric progenitors accumulate in the foregut and differentiate into enteric neurons, but subsequently undergo apoptosis resulting in a striking neuronal deficit. Moreover, ectopic expression of the transcription factor meis3 and/or the receptor ret, partially rescues enteric neuron colonization after RA attenuation. Collectively, our findings suggest that retinoic acid plays a critical temporal role in promoting enteric neural crest chain migration and neuronal survival upstream of Meis3 and RET in vivo.

A novel subset of enteric neurons revealed by ptf1a:GFP in the developing zebrafish enteric nervous system.

The enteric nervous system, the largest division of the peripheral nervous system, is derived from vagal neural crest cells that invade and populate the entire length of the gut to form diverse neuronal subtypes. Here, we identify a novel population of neurons within the enteric nervous system of zebrafish larvae that express the transgenic marker ptf1a:GFP within the midgut. Genetic lineage analysis reveals that enteric ptf1a:GFP(+) cells are derived from the neural crest and that most ptf1a:GFP(+) neurons express the neurotransmitter 5HT, demonstrating that they are serotonergic. This transgenic line, Tg(ptf1a:GFP), provides a novel neuronal marker for a subpopulation of neurons within the enteric nervous system, and highlights the possibility that Ptf1a may act as an important transcription factor for enteric neuron development.

Histone demethylase KDM4B regulates otic vesicle invagination via epigenetic control of Dlx3 expression.

In vertebrates, the inner ear arises from the otic placode, a thickened swathe of ectoderm that invaginates to form the otic vesicle. We report that histone demethylase KDM4B is dynamically expressed during early stages of chick inner ear formation. A loss of KDM4B results in defective invagination and striking morphological changes in the otic epithelium, characterized by abnormal localization of adhesion and cytoskeletal molecules and reduced expression of several inner ear markers, including Dlx3. In vivo chromatin immunoprecipitation reveals direct and dynamic occupancy of KDM4B and its target, H3K9me3, at regulatory regions of the Dlx3 locus. Accordingly, coelectroporations of DLX3 or KDM4B encoding constructs, but not a catalytically dead mutant of KDM4B, rescue the ear invagination phenotype caused by KDM4B knockdown. Moreover, a loss of DLX3 phenocopies a loss of KDM4B. Collectively, our findings suggest that KDM4B play a critical role during inner ear invagination via modulating histone methylation of the direct target Dlx3.

Meis3 is required for neural crest invasion of the gut during zebrafish enteric nervous system development.

During development, vagal neural crest cells fated to contribute to the enteric nervous system migrate ventrally away from the neural tube toward and along the primitive gut. The molecular mechanisms that regulate their early migration en route to and entry into the gut remain elusive. Here we show that the transcription factor meis3 is expressed along vagal neural crest pathways. Meis3 loss of function results in a reduction in migration efficiency, cell number, and the mitotic activity of neural crest cells in the vicinity of the gut but has no effect on neural crest or gut specification. Later, during enteric nervous system differentiation, Meis3-depleted embryos exhibit colonic aganglionosis, a disorder in which the hindgut is devoid of neurons. Accordingly, the expression of Shh pathway components, previously shown to have a role in the etiology of Hirschsprung's disease, was misregulated within the gut after loss of Meis3. Taken together, these findings support a model in which Meis3 is required for neural crest proliferation, migration into, and colonization of the gut such that its loss leads to severe defects in enteric nervous system development.

Midkine-A functions upstream of Id2a to regulate cell cycle kinetics in the developing vertebrate retina.

BACKGROUND: Midkine is a small heparin binding growth factor expressed in numerous tissues during development. The unique midkine gene in mammals has two paralogs in zebrafish: midkine-a (mdka) and midkine-b (mdkb). In the zebrafish retina, during both larval development and adult photoreceptor regeneration, mdka is expressed in retinal stem and progenitor cells and functions as a molecular component of the retina's stem cell niche. In this study, loss-of-function and conditional overexpression were used to investigate the function of Mdka in the retina of the embryonic zebrafish. RESULTS: The results show that during early retinal development Mdka functions to regulate cell cycle kinetics. Following targeted knockdown of Mdka synthesis, retinal progenitors cycle more slowly, and this results in microphthalmia, a diminished rate of cell cycle exit and a temporal delay of cell cycle exit and neuronal differentiation. In contrast, Mdka overexpression results in acceleration of the cell cycle and retinal overgrowth. Mdka gain-of-function, however, does not temporally advance cell cycle exit. Experiments to identify a potential Mdka signaling pathway show that Mdka functions upstream of the HLH regulatory protein, Id2a. Gene expression analysis shows Mdka regulates id2a expression, and co-injection of Mdka morpholinos and id2a mRNA rescues the Mdka loss-of-function phenotype. CONCLUSIONS: These data show that in zebrafish, Mdka resides in a shared Id2a pathway to regulate cell cycle kinetics in retinal progenitors. This is the first study to demonstrate the function of Midkine during retinal development and adds Midkine to the list of growth factors that transcriptionally regulate Id proteins.

An ENU mutagenesis screen in zebrafish for visual system mutants identifies a novel splice-acceptor site mutation in patched2 that results in Colobomas.

PURPOSE: To identify recessive mutations affecting development and/or maintenance of the zebrafish visual system. METHODS: A three-generation ENU (N-Nitroso-N-ethylurea)-based forward genetic screen was performed. F3 embryos were screened visually from 1 to 5 days postfertilization (dpf) for ocular abnormalities, and 5 dpf embryos were fixed and processed for cryosectioning, after which eye sections were screened for defects in cellular organization within the retina, lens, and cornea. A combination of PCR and DNA sequencing, in situ hybridization, and pharmacological treatments were used to clone and characterize a coloboma mutant. RESULTS: A total of 126 F2 families were screened, and, from these, 18 recessive mutations were identified that affected eye development. Phenotypes included lens malformations and cataracts, photoreceptor defects, oculocutaneous albinism, microphthalmia, and colobomas. Analysis of one such coloboma mutant, uta(1), identified a splice-acceptor mutation in the patched2 gene that resulted in an in-frame deletion of 19 amino acids that are predicted to contribute to the first extracellular loop of Patched2. ptch2(uta1) mutants possessed elevated Hedgehog (Hh) pathway activity, and blocking the Hh pathway with cyclopamine prevented colobomas in ptch2(uta1) mutant embryos. CONCLUSIONS: We have identified 18 recessive mutations affecting development of the zebrafish visual system and we have characterized a novel splice-acceptor site mutation in patched2 that results in enhanced Hh pathway activity and colobomas.