Goldberg-Shprintzen syndrome (GOSHS) is a rare clinical disorder characterized by central and enteric nervous system defects. This syndrome is caused by inactivating mutations in the Kinesin Binding Protein (KBP) gene, which encodes a protein of which the precise function is largely unclear. We show that KBP expression is upregulated during neuronal development in mouse cortical neurons. Moreover, KBP-depleted PC12 cells were defective in nerve growth factor-induced differentiation and neurite outgrowth, suggesting that KBP is required for cell differentiation and neurite development. To identify KBP interacting proteins, we performed a yeast twohybrid screen and found that KBP binds almost exclusively to microtubule associated or related proteins, specifically SCG10 and several kinesins. We confirmed these results by validating KBP interaction with one of these proteins: SCG10, a microtubule destabilizing protein. Zebrafish studies further demonstrated an epistatic interaction between KBP and SCG10 in vivo. To investigate the possibility of direct interaction between KBP and microtubules, we undertook co-localization and in vitro binding assays, but found no evidence of direct binding. Thus, our data indicate that KBP is involved in neuronal differentiation and that the central and enteric nervous system defects seen in GOSHS are likely caused by microtubule-related defects.

Background Hirschsprung disease (HSCR), which is congenital obstruction of the bowel, results from a failure of enteric nervous system (ENS) progenitors to migrate, proliferate, differentiate, or survive within the distal intestine. Previous studies that have searched for genes underlying HSCR have focused on ENS-related pathways and genes not fitting the current knowledge have thus often been ignored. We identify and validate novel HSCR genes using whole exome sequencing (WES), burden tests, in silico prediction, unbiased in vivo analyses of the mutated genes in zebrafish, and expression analyses in zebrafish, mouse, and human. Results We performed de novo mutation (DNM) screening on 24 HSCR trios. We identify 28 DNMs in 21 different genes. Eight of the DNMs we identified occur in RET, the main HSCR gene, and the remaining 20 DNMs reside in genes not reported in the ENS. Knockdown of all 12 genes with missense or loss-of-function DNMs showed that the orthologs of four genes (DENND3, NCLN, NUP98, and TBATA) are indispensable for ENS development in zebrafish, and these results were confirmed by CRISPR knockout. These genes are also expressed in human and mouse gut and/or ENS progenitors. Importantly, the encoded proteins are linked to neuronal processes shared by the central nervous system and the ENS. Conclusions Our data open new fields of investigation into HSCR pathology and provide novel insights into the development of the ENS. Moreover, the study demonstrates that functional analyses of genes carrying DNMs are warranted to delineate the full genetic architecture of rare complex diseases.

The guidance molecule Netrin and its receptor DCC (deleted in colorectal cancer) attract commissural axons toward the midline en route to their final destination. To test whether these molecules can also guide dendrites, we studied the contralateral dendrites of zebrafish octavolateralis efferent (OLe) neurons, which are unusual in that they navigate toward and cross the midline. We found that, at the time of dendrite outgrowth, OLe neurons express dcc, and the hindbrain midline expresses netrin1. Knocking down dcc or netrin1 function by injecting antisense morpholino oligonucleotides prevented OLe contralateral dendrites from crossing the midline, showing that dcc and netrin1 are necessary for dendrite guidance or formation. Furthermore, by transplanting cells from dcc morphants into wild-type embryos and vice versa, we demonstrated that dcc acts cell autonomously in OLe dendrites. This work is the first evidence that Netrin/DCC signaling acts in dendrites in a vertebrate system.

Background The enteric nervous system (ENS) is the largest subdivision of the peripheral nervous system and forms a complex circuit of neurons and glia that controls the function of the gastrointestinal (GI) tract. Within this circuit, there are multiple subtypes of neurons and glia. Appropriate differentiation of these various cell subtypes is vital for normal ENS and GI function. Studies of the pediatric disorder Hirschprung's Disease (HSCR) have provided a number of important insights into the mechanisms and molecules involved in ENS development; however, there are numerous other GI disorders that potentially may result from defects in development/differentiation of only a subset of ENS neurons or glia. Purpose Our understanding of the mechanisms and molecules involved in enteric nervous system differentiation is far from complete. Critically, it remains unclear at what point the fates of enteric neural crest cells (ENCCs) become committed to a specific subtype cell fate and how these cell fate choices are made. We will review our current understanding of ENS differentiation and highlight key questions that need to be addressed to gain a more complete understanding of this biological process.

The enteric nervous system (ENS) regulates many gastrointestinal functions including peristalsis, immune regulation and uptake of nutrients. Defects in the ENS can lead to severe enteric neuropathies such as Hirschsprung disease (HSCR). Zebrafish have proven to be fruitful in the identification of genes involved in ENS development and HSCR pathogenesis. However, composition and specification of enteric neurons and glial subtypes at larval stages, remains mainly unexplored. Here, we performed single cell RNA sequencing of zebrafish ENS at 5 days post-fertilization. We identified vagal neural crest progenitors, Schwann cell precursors, and four clusters of differentiated neurons. In addition, a previously unrecognized elavl3+/phox2bb-population of neurons and cx43+/phox2bb-enteric glia was found. Pseudotime analysis supported binary neurogenic branching of ENS differentiation, driven by a notch-responsive state. Taken together, we provide new insights on ENS development and specification, proving that the zebrafish is a valuable model for the study of congenital enteric neuropathies.

The zebrafish enteric nervous system (ENS), like those of all other vertebrate species, is principally derived from the vagal neural crest cells (NCC). The developmental controls that govern the migration, proliferation and patterning of the ENS precursors are not well understood. We have investigated the roles of endoderm and Sonic Hedgehog (SHH) in the development of the ENS. We show that endoderm is required for the migration of ENS NCC from the vagal region to the anterior end of the intestine. We show that the expression of shh and its receptor ptc-1 correlate with the development of the ENS and demonstrate that hedgehog (HH) signaling is required in two phases, a pre-enteric and an enteric phase, for normal ENS development. We show that HH signaling regulates the proliferation of vagal NCC and ENS precursors in vivo. We also show the zebrafish hand2 is required for the normal development of the intestinal smooth muscle and the ENS. Furthermore we show that endoderm and HH signaling, but not hand2, regulate gdnf expression in the intestine, highlighting a central role of endoderm and SHH in patterning the intestine and the ENS.

Smad-interacting protein-1 (SIP1) has been implicated in the development of Mowat-Wilson syndrome whose patients exhibit Hirschsprung disease, an aganglionosis of the large intestine, as well as other phenotypes. We have identified and cloned two sip1 orthologues in zebrafish. Both sip1 orthologues are expressed maternally and have dynamic zygotic expression patterns that are temporally and spatially distinct. We have investigated the function of both orthologues using translation and splice-blocking morpholino antisense oligonucleotides. Knockdown of the orthologues causes axial and neural patterning defects consistent with the previously described function of SIP1 as an inhibitor of BMP signaling. In addition, knockdown of both genes leads to a significant reduction/loss of the post-otic cranial neural crest. This results in a subsequent absence of neural crest precursors in the posterior pharyngeal arches and a loss of enteric precursors in the intestine.

The enteric nervous system is composed of neurons and glia that modulate many aspects of intestinal function. The ability to use both forward and reverse genetic approaches and to visualize development in living embryos and larvae has made zebrafish an attractive model in which to study mechanisms underlying enteric nervous system development. Here we review recent work describing the development and organization of the zebrafish enteric nervous system and how this relates to intestinal motility. We also discuss the cellular, molecular and genetic mechanisms that have been revealed by these studies and how they are providing new insights into human enteric nervous system diseases.

We have taken advantage of the strengths of the zebrafish model system to introduce developmental biology and genetics to undergraduates in their second semester of the Introductory Biology course at Emory. We designed a 6-week laboratory module based on research being undertaken by faculty in the department, and incorporated experiments that used current research methods including bioinformatics. Students undertook a range of experiments including direct observation of live wild-type zebrafish at different stages of embryogenesis, whole-mount in situ hybridization of mutant and wild-type embryos, vital dye staining of mutant and wild-type embryos, and pharmacological treatments to perturb normal development. These laboratories engaged the students by providing a hands-on, research-centered experience, while also enhancing their written (worksheets and laboratory reports) and oral (group presentation) communication skills. We describe the proceedings of each lab and the logistics of preparing and running these labs for 400–500 students (120 students taking lab each day), and provide a preliminary assessment of the success of the laboratories data based on student evaluations.

The phox2b gene encodes a transcription factor that is expressed in the developing enteric nervous system (ENS). An enhancer element has been identified in the zebrafish phox2b locus that can drive tissue specific expression of reporter genes in enteric neuron precursor cells. We have generated a transgenic zebrafish line in which the Kaede fluorescent protein is under the control of this phox2b enhancer. This line has stable expression of the Kaede protein in enteric neuron precursor cells over 3 generations. To demonstrate the utility of this line we compared the migration and division rates of enteric neuron precursor cells in wild type and the zebrafish ENS mutant lessen.