The long Filamin-A isoform is required for intestinal development and motility: implications for chronic intestinal pseudo-obstruction.

Filamin A (FLNA) is a cytoplasmic actin binding protein, recently shown to be expressed as a long and short isoform. Mutations in FLNA are associated with a wide spectrum of disorders, including an X-linked form of chronic intestinal pseudo-obstruction (CIPO). However, the role of FLNA in intestinal development and function is largely unknown. In this study, we show that FLNA is expressed in the muscle layer of the small intestine from early human fetal stages. Expression of FLNA variants associated with CIPO, blocked expression of the long flna isoform and led to an overall reduction of RNA and protein levels. As a consequence, contractility of human intestinal smooth muscle cells was affected. Lastly, our transgenic zebrafish line showed that the flna long isoform is required for intestinal elongation and peristalsis. Histological analysis revealed structural and architectural changes in the intestinal smooth muscle of homozygous fish, likely triggered by the abnormal expression of intestinal smooth muscle markers. No defect in the localization or numbers of enteric neurons was observed. Taken together, our study demonstrates that the long FLNA isoform contributes to intestinal development and function. Since loss of the long FLNA isoform does not seem to affect the enteric nervous system, it likely results in a myopathic form of CIPO, bringing new insights to disease pathogenesis.

Identification of Variants in RET and IHH Pathway Members in a Large Family With History of Hirschsprung Disease.

BACKGROUND & AIMS: Hirschsprung disease (HSCR) is an inherited congenital disorder characterized by absence of enteric ganglia in the distal part of the gut. Variants in ret proto-oncogene (RET) have been associated with up to 50% of familial and 35% of sporadic cases. We searched for variants that affect disease risk in a large, multigenerational family with history of HSCR in a linkage region previously associated with the disease (4q31.3-q32.3) and exome wide. METHODS: We performed exome sequencing analyses of a family in the Netherlands with 5 members diagnosed with HSCR and 2 members diagnosed with functional constipation. We initially focused on variants in genes located in 4q31.3-q32.3; however, we also performed an exome-wide analysis in which known HSCR or HSCR-associated gene variants predicted to be deleterious were prioritized for further analysis. Candidate genes were expressed in HEK293, COS-7, and Neuro-2a cells and analyzed by luciferase and immunoblot assays. Morpholinos were designed to target exons of candidate genes and injected into 1-cell stage zebrafish embryos. Embryos were allowed to develop and stained for enteric neurons. RESULTS: Within the linkage region, we identified 1 putative splice variant in the lipopolysaccharide responsive beige-like anchor protein gene (LRBA). Functional assays could not confirm its predicted effect on messenger RNA splicing or on expression of the mab-21 like 2 gene (MAB21L2), which is embedded in LRBA. Zebrafish that developed following injection of the lrba morpholino had a shortened body axis and subtle gut morphological defects, but no significant reduction in number of enteric neurons compared with controls. Outside the linkage region, members of 1 branch of the family carried a previously unidentified RET variant or an in-frame deletion in the glial cell line derived neurotrophic factor gene (GDNF), which encodes a ligand of RET. This deletion was located 6 base pairs before the last codon. We also found variants in the Indian hedgehog gene (IHH) and its mediator, the transcription factor GLI family zinc finger 3 (GLI3). When expressed in cells, the RET-P399L variant disrupted protein glycosylation and had altered phosphorylation following activation by GDNF. The deletion in GDNF prevented secretion of its gene product, reducing RET activation, and the IHH-Q51K variant reduced expression of the transcription factor GLI1. Injection of morpholinos that target ihh reduced the number of enteric neurons to 13% ± 1.4% of control zebrafish. CONCLUSIONS: In a study of a large family with history of HSCR, we identified variants in LRBA, RET, the gene encoding the RET ligand (GDNF), IHH, and a gene encoding a mediator of IHH signaling (GLI3). These variants altered functions of the gene products when expressed in cells and knockout of ihh reduced the number of enteric neurons in the zebrafish gut.

Enteric nervous system development in avian and zebrafish models.

Our current understanding of the developmental biology of the enteric nervous system (ENS) and the genesis of ENS diseases is founded almost entirely on studies using model systems. Although genetic studies in the mouse have been at the forefront of this field over the last 20 years or so, historically it was the easy accessibility of the chick embryo for experimental manipulations that allowed the first descriptions of the neural crest origins of the ENS in the 1950s. More recently, studies in the chick and other non-mammalian model systems, notably zebrafish, have continued to advance our understanding of the basic biology of ENS development, with each animal model providing unique experimental advantages. Here we review the basic biology of ENS development in chick and zebrafish, highlighting conserved and unique features, and emphasising novel contributions to our general understanding of ENS development due to technical or biological features.

In vivo visualization of the development of the enteric nervous system using a Tg(-8.3bphox2b:Kaede) transgenic zebrafish.

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 three 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.

Pericentrosomal targeting of Rab6 secretory vesicles by Bicaudal-D-related protein 1 (BICDR-1) regulates neuritogenesis.

Membrane and secretory trafficking are essential for proper neuronal development. However, the molecular mechanisms that organize secretory trafficking are poorly understood. Here, we identify Bicaudal-D-related protein 1 (BICDR-1) as an effector of the small GTPase Rab6 and key component of the molecular machinery that controls secretory vesicle transport in developing neurons. BICDR-1 interacts with kinesin motor Kif1C, the dynein/dynactin retrograde motor complex, regulates the pericentrosomal localization of Rab6-positive secretory vesicles and is required for neural development in zebrafish. BICDR-1 expression is high during early neuronal development and strongly declines during neurite outgrowth. In young neurons, BICDR-1 accumulates Rab6 secretory vesicles around the centrosome, restricts anterograde secretory transport and inhibits neuritogenesis. Later during development, BICDR-1 expression is strongly reduced, which permits anterograde secretory transport required for neurite outgrowth. These results indicate an important role for BICDR-1 as temporal regulator of secretory trafficking during the early phase of neuronal differentiation.

CLMP is required for intestinal development, and loss-of-function mutations cause congenital short-bowel syndrome.

BACKGROUND & AIMS: Short-bowel syndrome usually results from surgical resection of the small intestine for diseases such as intestinal atresias, volvulus, and necrotizing enterocolitis. Patients with congenital short-bowel syndrome (CSBS) are born with a substantial shortening of the small intestine, to a mean length of 50 cm, compared with a normal length at birth of 190-280 cm. They also are born with intestinal malrotation. Because CSBS occurs in many consanguineous families, it is considered to be an autosomal-recessive disorder. We aimed to identify and characterize the genetic factor causing CSBS. METHODS: We performed homozygosity mapping using 610,000 K single-nucleotide polymorphism arrays to analyze the genomes of 5 patients with CSBS. After identifying a gene causing the disease, we determined its expression pattern in human embryos. We also overexpressed forms of the gene product that were and were not associated with CSBS in Chinese Hamster Ovary and T84 cells and generated a zebrafish model of the disease. RESULTS: We identified loss-of-function mutations in Coxsackie- and adenovirus receptor-like membrane protein (CLMP) in CSBS patients. CLMP is a tight-junction-associated protein that is expressed in the intestine of human embryos throughout development. Mutations in CLMP prevented its normal localization to the cell membrane. Knock-down experiments in zebrafish resulted in general developmental defects, including shortening of the intestine and the absence of goblet cells. Because goblet cells are characteristic for the midintestine in zebrafish, which resembles the small intestine in human beings, the zebrafish model mimics CSBS. CONCLUSIONS: Loss-of-function mutations in CLMP cause CSBS in human beings, likely by interfering with tight-junction formation, which disrupts intestinal development. Furthermore, we developed a zebrafish model of CSBS.

Expression of neuropeptides and anoctamin 1 in the embryonic and adult zebrafish intestine, revealing neuronal subpopulations and ICC-like cells.

This immunohistochemical study in zebrafish aims to extend the neurochemical characterization of enteric neuronal subpopulations and to validate a marker for identification of interstitial cells of Cajal (ICC). The expression of neuropeptides and anoctamin 1 (Ano1), a selective ICC marker in mammals, was analyzed in both embryonic and adult intestine. Neuropeptides were present from 3 days postfertilization (dpf). At 3 dpf, galanin-positive nerve fibers were found in the proximal intestine, while calcitonin gene-related peptide (CGRP)- and substance P-expressing fibers appeared in the distal intestine. At 5 dpf, immunoreactive fibers were present along the entire intestinal length, indicating a well-developed peptidergic innervation at the onset of feeding. In the adult intestine, vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), galanin, CGRP and substance P were detected in nerve fibers. Colchicine pretreatment enhanced only VIP and PACAP immunoreactivity. VIP and PACAP were coexpressed in enteric neurons. Colocalization stainings revealed three neuronal subpopulations expressing VIP and PACAP: a nitrergic noncholinergic subpopulation, a serotonergic subpopulation and a subpopulation expressing no other markers. Ano1-immunostaining revealed a 3-dimensional network in the adult intestine containing multipolar cells at the myenteric plexus and bipolar cells interspersed between circular smooth muscle cells. Ano1 immunoreactivity first appeared at 3 dpf, indicative of the onset of proliferation of ICC-like cells. It is shown that the Ano1 antiserum is a selective marker of ICC-like cells in the zebrafish intestine. Finally, it is hypothesized that ICC-like cells mediate the spontaneous regular activity of the embryonic intestine.

Unbiased characterization of the larval zebrafish enteric nervous system at a single cell transcriptomic level.

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.

KBP interacts with SCG10, linking Goldberg-Shprintzen syndrome to microtubule dynamics and neuronal differentiation.

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 up-regulated 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 two-hybrid 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.

Churchill and Sip1a repress fibroblast growth factor signaling during zebrafish somitogenesis.

Cell-type specific regulation of a small number of growth factor signal transduction pathways generates diverse developmental outcomes. The zinc finger protein Churchill (ChCh) is a key effector of fibroblast growth factor (FGF) signaling during gastrulation. ChCh is largely thought to act by inducing expression of the multifunctional Sip1 (Smad Interacting Protein 1). We investigated the function of ChCh and Sip1a during zebrafish somitogenesis. Knockdown of ChCh or Sip1a results in misshapen somites that are short and narrow. As in wild-type embryos, cycling gene expression occurs in the developing somites in ChCh and Sip1a compromised embryos, but expression of her1 and her7 is maintained in formed somites. In addition, tail bud fgf8 expression is expanded anteriorly in these embryos. Finally, we found that blocking FGF8 restores somite morphology in ChCh and Sip1a compromised embryos. These results demonstrate a novel role for ChCh and Sip1a in repression of FGF activity.

Abnormal nuclear pore formation triggers apoptosis in the intestinal epithelium of elys-deficient zebrafish.

BACKGROUND & AIMS: Zebrafish mutants generated by ethylnitrosourea-mutagenesis provide a powerful tool for dissecting the genetic regulation of developmental processes, including organogenesis. One zebrafish mutant, "flotte lotte" (flo), displays striking defects in intestinal, liver, pancreas, and eye formation at 78 hours postfertilization (hpf). In this study, we sought to identify the underlying mutated gene in flo and link the genetic lesion to its phenotype. METHODS: Positional cloning was employed to map the flo mutation. Subcellular characterization of flo embryos was achieved using histology, immunocytochemistry, bromodeoxyuridine incorporation analysis, and confocal and electron microscopy. RESULTS: The molecular lesion in flo is a nonsense mutation in the elys (embryonic large molecule derived from yolk sac) gene, which encodes a severely truncated protein lacking the Elys C-terminal AT-hook DNA binding domain. Recently, the human ELYS protein has been shown to play a critical, and hitherto unsuspected, role in nuclear pore assembly. Although elys messenger RNA (mRNA) is expressed broadly during early zebrafish development, widespread early defects in flo are circumvented by the persistence of maternally expressed elys mRNA until 24 hpf. From 72 hpf, elys mRNA expression is restricted to proliferating tissues, including the intestinal epithelium, pancreas, liver, and eye. Cells in these tissues display disrupted nuclear pore formation; ultimately, intestinal epithelial cells undergo apoptosis. CONCLUSIONS: Our results demonstrate that Elys regulates digestive organ formation.

Endoderm-derived Sonic hedgehog and mesoderm Hand2 expression are required for enteric nervous system development in zebrafish.

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.

Conditional deletion of Hand2 reveals critical functions in neurogenesis and cell type-specific gene expression for development of neural crest-derived noradrenergic sympathetic ganglion neurons.

Neural crest-derived structures that depend critically upon expression of the basic helix-loop-helix DNA binding protein Hand2 for normal development include craniofacial cartilage and bone, the outflow tract of the heart, cardiac cushion, and noradrenergic sympathetic ganglion neurons. Loss of Hand2 is embryonic lethal by E9.5, obviating a genetic analysis of its in-vivo function. We have overcome this difficulty by specific deletion of Hand2 in neural crest-derived cells by crossing our line of floxed Hand2 mice with Wnt1-Cre transgenic mice. Our analysis of Hand2 knock-out in neural crest-derived cells reveals effects on development in all neural crest-derived structures where Hand2 is expressed. In the autonomic nervous system, conditional disruption of Hand2 results in a significant and progressive loss of neurons as well as a significant loss of TH expression. Hand2 affects generation of the neural precursor pool of cells by affecting both the proliferative capacity of the progenitors as well as affecting expression of Phox2a and Gata3, DNA binding proteins important for the cell autonomous development of noradrenergic neurons. Our data suggest that Hand2 is a multifunctional DNA binding protein affecting differentiation and cell type-specific gene expression in neural crest-derived noradrenergic sympathetic ganglion neurons. Hand2 has a pivotal function in a non-linear cross-regulatory network of DNA binding proteins that affect cell autonomous control of differentiation and cell type-specific gene expression.

Characterization of spatial and temporal expression pattern of SCG10 during zebrafish development.

SCG10 (Superior Cervical Ganglia 10, STMN2) is a member of the stathmin family of proteins. Stathmins regulate microtubule dynamics by inhibiting polymerization and promoting their depolymerization. SCG10 is believed to be a neuronal-specific stathmin that is enriched in the growth cones of developing neurons and plays a role in regulating neurite outgrowth. In all species examined so far, SCG10 is expressed in both the CNS and PNS. We have cloned two zebrafish SCG10 homologues and have determined the temporal and spatial expression pattern of both of these genes by RT-PCR and in situ hybridization. RT-PCR shows that both transcripts are expressed maternally and zygotically through at least 5 days. In situ hybridization analysis reveals that both SCG10 orthologues have dynamic, spatial expression patterns that are nearly identical to each other. Initially, these orthologues are expressed in discrete areas of the forebrain, midbrain, and hindbrain, as well as in the anterior and posterior lateral line ganglia and transiently in the spinal cord Rohon-Beard neurons. From 48hpf onwards, the level of expression of both genes increases and becomes mainly restricted to the anterior CNS (the forebrain region, retina, optic tectum, and hindbrain), and to the cranial ganglia. From 72 to 96hpf, SCG10 genes are also expressed in the developing neurons in the gut and in the surrounding intestinal mesenchyme. Our results provide a starting point for future studies that will investigate the in vivo function of SCG10 orthologues in zebrafish neural development.

Netrin/DCC signaling controls contralateral dendrites of octavolateralis efferent neurons.

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.

Differential roles of transcriptional mediator complex subunits Crsp34/Med27, Crsp150/Med14 and Trap100/Med24 during zebrafish retinal development.

The transcriptional mediator complex has emerged as an important component of transcriptional regulation, yet it is largely unknown whether its subunits have differential functions in development. We demonstrate that the zebrafish mutation m885 disrupts a subunit of the mediator complex, Crsp34/Med27. To explore the role of the mediator in the control of retinal differentiation, we employed two additional mutations disrupting the mediator subunits Trap100/Med24 and Crsp150/Med14. Our analysis shows that loss of Crsp34/Med27 decreases amacrine cell number, but increases the number of rod photoreceptor cells. In contrast, loss of Trap100/Med24 decreases rod photoreceptor cells. Loss of Crsp150/Med14, on the other hand, only slightly reduces dopaminergic amacrine cells, which are absent from both crsp34(m885) and trap100(lessen) mutant embryos. Our data provide evidence for differential requirements for Crsp34/Med27 in developmental processes. In addition, our data point to divergent functions of the mediator subunits Crsp34/Med27, Trap100/Med24, and Crsp150/Med14 and, thus, suggest that subunit composition of the mediator contributes to the control of differentiation in the vertebrate CNS.

Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes.

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.

Development of the zebrafish enteric nervous system.

The enteric nervous system (ENS) 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 ENS development. In this chapter, we review the recent work describing the development and organization of the zebrafish ENS 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 ENS diseases.

Neurochemical coding of enteric neurons in adult and embryonic zebrafish (Danio rerio).

Although the morphology and development of the zebrafish enteric nervous system have been extensively studied, the precise neurochemical coding of enteric neurons and their proportional enteric distribution are currently not known. By using immunohistochemistry, we determined the proportional expression and coexpression of neurochemical markers in the embryonic and adult zebrafish intestine. Tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), and pituitary adenylate cyclase-activating peptide (PACAP) were observed only in nerve fibers, whereas other markers were also detected in neuronal cell bodies. Calretinin and calbindin had similar distributions. In embryos, all markers, except for choline acetyltransferase (ChAT) and TH, were present from 72 hours postfertilization. Nitrergic neurons, evenly distributed and remaining constant in time, constituted the major neuronal subpopulation. The neuronal proportions of the other markers increased during development and were characterized by regional differences. In the adult, all markers examined were expressed in the enteric nervous system. A large percentage of enteric neurons displayed calbindin and calretinin, and serotonin was the only marker showing significant distribution differences in the three intestinal regions. Colocalization studies showed that serotonin was not coexpressed with any of the other markers. At least five neuronal subpopulations were determined: a serotonergic, a nitrergic noncholinergic, two cholinergic nonnitrergic subpopulations along with one subpopulation expressing both ChAT and neuronal nitric oxide synthase. Analysis of nerve fibers revealed that nitrergic neurons coexpress VIP and PACAP, and that nitrergic neurons innervate the tunica muscularis, whereas serotonergic and cholinergic nonnitrergic neurons innervate the lamina propria and the tunica muscularis.

Zebrafish development and genetics: introducing undergraduates to developmental biology and genetics in a large introductory laboratory class.

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.