Michael J. F. Barresi

Somite development in zebrafish

A full understanding of somite development requires knowledge of the molecular genetic pathways for cell determination as well as the cellular behaviors that underlie segmentation, somite epithelialization, and somite patterning. The zebrafish has long been recognized as an ideal organism for cellular and histological studies of somite patterning. In recent years, genetics has proven to be a very powerful complementary approach to these embryological studies, as genetic screens for zebrafish mutants defective in somitogenesis have identified over 50 genes that are necessary for normal somite development. Zebrafish is thus an ideal system in which to analyze the role of specific gene products in regulating the cell behaviors that underlie somite development. We review what is currently known about zebrafish somite development and compare it where appropriate to somite development in chick and mouse. We discuss the processes of segmentation and somite epithelialization, and then review the patterning of cell types within the somite. We show directly, for the first time, that muscle cell and sclerotome migrations occur at the same time. We end with a look at the many questions about somitogenesis that are still unanswered.

Generality of vertebrate developmental patterns: evidence for a dermomyotome in fish

SUMMARY The somitic compartment that gives rise to trunk muscle and dermis in amniotes is an epithelial sheet on the external surface of the somite, and is known as the dermomyotome. However, despite its central role in the development of the trunk and limbs, the evolutionary history of the dermomyotome and its role in nonamniotes is poorly understood. We have tested whether a tissue with the morphological and molecular characteristics of a dermomyotome exists in nonamniotes. We show that representatives of the agnathans and of all major clades of gnathostomes each have a layer of cells on the surface of the somite, external to the embryonic myotome. These external cells do not show any signs of terminal myogenic or dermogenic differentiation. Moreover, in the embryos of bony fishes as diverse as sturgeons (Chondrostei) and zebrafish (Teleostei) this layer of cells expresses the pax3 and pax7 genes that mark myogenic precursors. Some of the pax7‐expressing cells also express the differentiation‐promoting myogenic regulatory factor Myogenin and appear to enter into the myotome. We therefore suggest that the dermomyotome is an ancient and conserved structure that evolved prior to the last common ancestor of all vertebrates. The identification of a dermomyotome in fish makes it possible to apply the powerful cellular and genetic approaches available in zebrafish to the understanding of this key developmental structure.

Multiple roles for Hedgehog signaling in zebrafish pituitary development

The endocrine-secreting lobe of the pituitary gland, or adenohypophysis, forms from cells at the anterior margin of the neural plate through inductive interactions involving secreted morphogens of the Hedgehog (Hh), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) families. To better understand when and where Hh signaling influences pituitary development, we have analyzed the effects of blocking Hh signaling both pharmacologically (cyclopamine treatments) and genetically (zebrafish Hh pathway mutants). While current models state that Shh signaling from the oral ectoderm patterns the pituitary after placode induction, our data suggest that Shh plays a direct early role in both pituitary induction and patterning, and that early Hh signals comes from adjacent neural ectoderm. We report that Hh signaling is necessary between 10 and 15 h of development for induction of the zebrafish adenohypophysis, a time when shh is expressed only in neural tissue. We show that the Hh responsive genes ptc1 and nk2.2 are expressed in preplacodal cells at the anterior margin of the neural tube at this time, indicating that these cells are directly receiving Hh signals. Later (15-20 h) cyclopamine treatments disrupt anterior expression of nk2.2 and Prolactin, showing that early functional patterning requires Hh signals. Consistent with a direct role for Hh signaling in pituitary induction and patterning, overexpression of Shh results in expanded adenohypophyseal expression of lim3, expansion of nk2.2 into the posterior adenohypophysis, and an increase in Prolactin- and Somatolactin-secreting cells. We also use the zebrafish Hh pathway mutants to document the range of pituitary defects that occur when different elements of the Hh signaling pathway are mutated. These defects, ranging from a complete loss of the adenohypophysis (smu/smo and yot/gli2 mutants) to more subtle patterning defects (dtr/gli1 mutants), may correlate to human Hh signaling mutant phenotypes seen in Holoprosencephaly and other congenital disorders. Our results reveal multiple and distinct roles for Hh signaling in the formation of the vertebrate pituitary gland, and suggest that Hh signaling from neural ectoderm is necessary for induction and functional patterning of the vertebrate pituitary gland.

Macondo crude oil from the Deepwater Horizon oil spill disrupts specific developmental processes during zebrafish embryogenesis

BackgroundThe Deepwater Horizon disaster was the largest marine oil spill in history, and total vertical exposure of oil to the water column suggests it could impact an enormous diversity of ecosystems. The most vulnerable organisms are those encountering these pollutants during their early life stages. Water-soluble components of crude oil and specific polycyclic aromatic hydrocarbons have been shown to cause defects in cardiovascular and craniofacial development in a variety of teleost species, but the developmental origins of these defects have yet to be determined. We have adopted zebrafish, Danio rerio, as a model to test whether water accumulated fractions (WAF) of the Deepwater Horizon oil could impact specific embryonic developmental processes. While not a native species to the Gulf waters, the developmental biology of zebrafish has been well characterized and makes it a powerful model system to reveal the cellular and molecular mechanisms behind Macondo crude toxicity.ResultsWAF of Macondo crude oil sampled during the oil spill was used to treat zebrafish throughout embryonic and larval development. Our results indicate that the Macondo crude oil causes a variety of significant defects in zebrafish embryogenesis, but these defects have specific developmental origins. WAF treatments caused defects in craniofacial development and circulatory function similar to previous reports, but we extend these results to show they are likely derived from an earlier defect in neural crest cell development. Moreover, we demonstrate that exposure to WAFs causes a variety of novel deformations in specific developmental processes, including programmed cell death, locomotor behavior, sensory and motor axon pathfinding, somitogenesis and muscle patterning. Interestingly, the severity of cell death and muscle phenotypes decreased over several months of repeated analysis, which was correlated with a rapid drop-off in the aromatic and alkane hydrocarbon components of the oil.ConclusionsWhether these teratogenic effects are unique to the oil from the Deepwater Horizon oil spill or generalizable for most crude oil types remains to be determined. This work establishes a model for further investigation into the molecular mechanisms behind crude oil mediated deformations. In addition, due to the high conservation of genetic and cellular processes between zebrafish and other vertebrates, our work also provides a platform for more focused assessment of the impact that the Deepwater Horizon oil spill has had on the early life stages of native fish species in the Gulf of Mexico and the Atlantic Ocean.

Estrogens Suppress a Behavioral Phenotype in Zebrafish Mutants of the Autism Risk Gene, CNTNAP2

Autism spectrum disorders (ASDs) are a group of devastating neurodevelopmental syndromes that affect up to 1 in 68 children. Despite advances in the identification of ASD risk genes, the mechanisms underlying ASDs remain unknown. Homozygous loss-of-function mutations in Contactin Associated Protein-like 2 (CNTNAP2) are strongly linked to ASDs. Here we investigate the function of Cntnap2 and undertake pharmacological screens to identify phenotypic suppressors. We find that zebrafish cntnap2 mutants display GABAergic deficits, particularly in the forebrain, and sensitivity to drug-induced seizures. High-throughput behavioral profiling identifies nighttime hyperactivity in cntnap2 mutants, while pharmacological testing reveals dysregulation of GABAergic and glutamatergic systems. Finally, we find that estrogen receptor agonists elicit a behavioral fingerprint anti-correlative to that of cntnap2 mutants and show that the phytoestrogen biochanin A specifically reverses the mutant behavioral phenotype. These results identify estrogenic compounds as phenotypic suppressors and illuminate novel pharmacological pathways with relevance to autism.

Functional Morphology and Developmental Biology of Zebrafish: Reciprocal Illumination from an Unlikely Couple'

Abstract Functional morphology has benefited greatly from the input of techniques and thinking from other disciplines. This has been especially productive in situations where each discipline has made significant contributions to a particular research topic. A combination of methodologies from functional morphology and developmental biology has allowed us to characterize feeding mechanics of first-feeding larval zebrafish (Danio rerio). Contrary to kinematic patterns commonly seen in adult teleosts, larval zebrafish showed no lateral abduction during the expansive phase of a suction-feeding event. Instead, dorsoventral expansion of the buccal chamber, more typical of patterns seen in primitive fishes, characterized the expansive phase. Moreover, a pronounced preparatory phase during which the buccal chamber is constricted by the protractor hyoideus was consistently seen in first-feeding larval kinematics. Key kinematic variables associated with first feeding correlated significantly with the hydrodynamic regime as measured by the Reynolds number. Using the tools of both functional morphology and developmental biology we have not only determined which cranial muscles are important for successful feeding but also uncovered important physiological differences in muscle structure. Muscles necessary for the rapid dorsoventral expansion of the head are composed primarily of fast-twitch fibers while those involved in more tonic contractions such as hyoid protraction have more slow-twitch muscle fibers. While most evolutionary developmental studies have examined mechanisms responsible for large evolutionary changes in morphology, we propose that the type of data uncovered in functional studies can lead to the generation of hypotheses concerning the developmental mechanisms responsible for smaller intra- and/or interspecific changes.

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered. Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions. Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes. Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras. We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another. We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures. In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo. These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Gfap-positive radial glial cells are an essential progenitor population for later-born neurons and glia in the zebrafish spinal cord.

Radial glial cells are presumptive neural stem cells (NSCs) in the developing nervous system. The direct requirement of radial glia for the generation of a diverse array of neuronal and glial subtypes, however, has not been tested. We employed two novel transgenic zebrafish lines and endogenous markers of NSCs and radial glia to show for the first time that radial glia are essential for neurogenesis during development. By using the gfap promoter to drive expression of nuclear localized mCherry we discerned two distinct radial glial‐derived cell types: a major nestin+/Sox2+ subtype with strong gfap promoter activity and a minor Sox2+ subtype lacking this activity. Fate mapping studies in this line indicate that gfap+ radial glia generate later‐born CoSA interneurons, secondary motorneurons, and oligodendroglia. In another transgenic line using the gfap promoter‐driven expression of the nitroreductase enzyme, we induced cell autonomous ablation of gfap+ radial glia and observed a reduction in their specific derived lineages, but not Blbp+ and Sox2+/gfap‐negative NSCs, which were retained and expanded at later larval stages. Moreover, we provide evidence supporting classical roles of radial glial in axon patterning, blood–brain barrier formation, and locomotion. Our results suggest that gfap+ radial glia represent the major NSC during late neurogenesis for specific lineages, and possess diverse roles to sustain the structure and function of the spinal cord. These new tools will both corroborate the predicted roles of astroglia and reveal novel roles related to development, physiology, and regeneration in the vertebrate nervous system. GLIA 2016;64:1170–1189

Vincristine and bortezomib cause axon outgrowth and behavioral defects in larval zebrafish.

Peripheral neuropathy is a common side effect of a number of pharmaceutical compounds, including several chemotherapy drugs. Among these are vincristine sulfate, a mitotic inhibitor used to treat a variety of leukemias, lymphomas, and other cancers, and bortezomib, a 26S proteasome inhibitor used primarily to treat relapsed multiple myeloma and mantle cell lymphoma. To gain insight into the mechanisms by which these compounds act, we tested their effects in zebrafish. Vincristine or bortezomib given during late embryonic development caused significant defects at both behavioral and cellular levels. Intriguingly, the effects of the two drugs appear to be distinct. Vincristine causes uncoordinated swimming behavior, which is coupled with a reduction in the density of sensory innervation and overall size of motor axon arbors. Bortezomib, in contrast, increases the duration and amplitude of muscle contractions associated with escape swimming, which is coupled with a preferential reduction in fine processes and branches of sensory and motor axons. These results demonstrate that zebrafish is a convenient in vivo assay system for screening potential pharmaceutical compounds for neurotoxic side effects, and they provide an important step toward understanding how vincristine and bortezomib cause peripheral neuropathy.

Radial glia inhibit peripheral glial infiltration into the spinal cord at motor exit point transition zones

In the mature vertebrate nervous system, central and peripheral nervous system (CNS and PNS, respectively) GLIA myelinate distinct motor axon domains at the motor exit point transition zone (MEP TZ). How these cells preferentially associate with and myelinate discrete, non‐overlapping CNS versus PNS axonal segments, is unknown. Using in vivo imaging and genetic cell ablation in zebrafish, we demonstrate that radial glia restrict migration of PNS glia into the spinal cord during development. Prior to development of radial glial endfeet, peripheral cells freely migrate back and forth across the MEP TZ. However, upon maturation, peripherally located cells never enter the CNS. When we ablate radial glia, peripheral glia ectopically migrate into the spinal cord during developmental stages when they would normally be restricted. These findings demonstrate that radial glia contribute to both CNS and PNS development and control the unidirectional movement of glial cell types across the MEP TZ early in development. GLIA 2016. GLIA 2016;64:1138–1153