A transgene targeted to the zebrafish nkx2.4b locus drives specific green fluorescent protein expression and disrupts thyroid development.

BACKGROUND: With the goal of labeling and manipulating the zebrafish hypothalamus, we sought to target a green fluorescent protein (gfp) transgene to the expression domains of nkx2.4b, a gene expressed during hypothalamic and thyroid development. We combined transcription activator-like effector nucleases (TALENs)-mediated mutagenesis with a targeting construct to enable insertion of a gfp transgene into the endogenous nkx2.4b genomic locus. RESULTS: Injection of TALENs targeted to the first exon of nkx2.4b created a predicted null allele, and homozygous mutant embryos displayed loss of thyroid markers. From embryos injected with both TALENs and a targeting construct carrying a gfp transgene, we recovered a line in which GFP was expressed specifically in the hypothalamus and thyroid. Fish homozygous for this allele lacked exon 1 of nkx2.4b and exhibited hypothyroid phenotypes. CONCLUSIONS: By combining TALENs injections with a targeting construct that contained a gfp transgene, we were able to recover an allele in which GFP is expressed in the nkx2.4b expression domains, with homozygous phenotypes suggesting the creation of a loss-of-function transgenic line. These results demonstrate the creation of a useful tool for studying hypothalamus and thyroid development.

Development of the hypothalamus: conservation, modification and innovation.

The hypothalamus, which regulates fundamental aspects of physiological homeostasis and behavior, is a brain region that exhibits highly conserved anatomy across vertebrate species. Its development involves conserved basic mechanisms of induction and patterning, combined with a more plastic process of neuronal fate specification, to produce brain circuits that mediate physiology and behavior according to the needs of each species. Here, we review the factors involved in the induction, patterning and neuronal differentiation of the hypothalamus, highlighting recent evidence that illustrates how changes in Wnt/ß-catenin signaling during development may lead to species-specific form and function of this important brain structure.

Lef1-dependent hypothalamic neurogenesis inhibits anxiety.

While innate behaviors are conserved throughout the animal kingdom, it is unknown whether common signaling pathways regulate the development of neuronal populations mediating these behaviors in diverse organisms. Here, we demonstrate that the Wnt/ß-catenin effector Lef1 is required for the differentiation of anxiolytic hypothalamic neurons in zebrafish and mice, although the identity of Lef1-dependent genes and neurons differ between these 2 species. We further show that zebrafish and Drosophila have common Lef1-dependent gene expression in their respective neuroendocrine organs, consistent with a conserved pathway that has diverged in the mouse. Finally, orthologs of Lef1-dependent genes from both zebrafish and mouse show highly correlated hypothalamic expression in marmosets and humans, suggesting co-regulation of 2 parallel anxiolytic pathways in primates. These findings demonstrate that during evolution, a transcription factor can act through multiple mechanisms to generate a common behavioral output, and that Lef1 regulates circuit development that is fundamentally important for mediating anxiety in a wide variety of animal species.

A toolbox to study epidermal cell types in zebrafish.

Epithelia provide a crucial protective barrier for our organs and are also the sites where the majority of carcinomas form. Most studies on epithelia and carcinomas use cell culture or organisms where high-resolution live imaging is inaccessible without invasive techniques. Here, we introduce the developing zebrafish epidermis as an excellent in vivo model system for studying a living epithelium. We developed tools to fluorescently tag specific epithelial cell types and express genes in a mosaic fashion using five Gal4 lines identified from an enhancer trap screen. When crossed to a variety of UAS effector lines, we can now track, ablate or monitor single cells at sub-cellular resolution. Using photo-cleavable morpholino oligonucleotides that target gal4, we can also express genes in a mosaic fashion at specific times during development. Together, this system provides an excellent in vivo alternative to tissue culture cells, without the intrinsic concerns of culture conditions or transformation, and enables the investigation of distinct cell types within living epithelial tissues.

Hypothalamic radial glia function as self-renewing neural progenitors in the absence of Wnt/ß-catenin signaling.

The vertebrate hypothalamus contains persistent radial glia that have been proposed to function as neural progenitors. In zebrafish, a high level of postembryonic hypothalamic neurogenesis has been observed, but the role of radial glia in generating these new neurons is unclear. We have used inducible Cre-mediated lineage labeling to show that a population of hypothalamic radial glia undergoes self-renewal and generates multiple neuronal subtypes at larval stages. Whereas Wnt/ß-catenin signaling has been demonstrated to promote the expansion of other stem and progenitor cell populations, we find that Wnt/ß-catenin pathway activity inhibits this process in hypothalamic radial glia and is not required for their self-renewal. By contrast, Wnt/ß-catenin signaling is required for the differentiation of a specific subset of radial glial neuronal progeny residing along the ventricular surface. We also show that partial genetic ablation of hypothalamic radial glia or their progeny causes a net increase in their proliferation, which is also independent of Wnt/ß-catenin signaling. Hypothalamic radial glia in the zebrafish larva thus exhibit several key characteristics of a neural stem cell population, and our data support the idea that Wnt pathway function may not be homogeneous in all stem or progenitor cells.

ZC4H2, an XLID gene, is required for the generation of a specific subset of CNS interneurons.

Miles-Carpenter syndrome (MCS) was described in 1991 as an XLID syndrome with fingertip arches and contractures and mapped to proximal Xq. Patients had microcephaly, short stature, mild spasticity, thoracic scoliosis, hyperextendable MCP joints, rocker-bottom feet, hyperextended elbows and knees. A mutation, p.L66H, in ZC4H2, was identified in a XLID re-sequencing project. Additional screening of linked families and next generation sequencing of XLID families identified three ZC4H2 mutations: p.R18K, p.R213W and p.V75in15aa. The families shared some relevant clinical features. In silico modeling of the mutant proteins indicated all alterations would destabilize the protein. Knockout mutations in zc4h2 were created in zebrafish and homozygous mutant larvae exhibited abnormal swimming, increased twitching, defective eye movement and pectoral fin contractures. Because several of the behavioral defects were consistent with hyperactivity, we examined the underlying neuronal defects and found that sensory neurons and motoneurons appeared normal. However, we observed a striking reduction in GABAergic interneurons. Analysis of cell-type-specific markers showed a specific loss of V2 interneurons in the brain and spinal cord, likely arising from mis-specification of neural progenitors. Injected human wt ZC4H2 rescued the mutant phenotype. Mutant zebrafish injected with human p.L66H or p.R213W mRNA failed to be rescued, while the p.R18K mRNA was able to rescue the interneuron defect. Our findings clearly support ZC4H2 as a novel XLID gene with a required function in interneuron development. Loss of function of ZC4H2 thus likely results in altered connectivity of many brain and spinal circuits.

Wnt/ß-catenin signaling is required for radial glial neurogenesis following spinal cord injury.

Spinal cord injury results in permanent sensorimotor loss in mammals, in part due to a lack of injury-induced neurogenesis. The regeneration of neurons depends upon resident neural progenitors, which in zebrafish persist throughout the central nervous system as radial glia. However the molecular mechanisms regulating spinal cord progenitors remain uncharacterized. Wnt/ß-catenin signaling is necessary for the regenerative response of multiple tissues in zebrafish as well as other vertebrates, but it is not known whether the pathway has a role in spinal cord regeneration. Here we show that spinal radial glia exhibit Wnt/ß-catenin activity as they undergo neurogenesis following transection. We then use Cre-mediated lineage tracing to label the progeny of radial glia and show that Wnt/ß-catenin signaling is required for progenitors to differentiate into neurons. Finally, we show that axonal regrowth after injury also requires Wnt/ß-catenin signaling, suggesting coordinated roles for the pathway in functional recovery. Our data thus establish Wnt/ß-catenin pathway activation as a necessary step in spinal cord regeneration.

High-resolution analysis of central nervous system expression patterns in zebrafish Gal4 enhancer-trap lines.

BACKGROUND: The application of the Gal4/UAS system to enhancer and gene trapping screens in zebrafish has greatly increased the ability to label and manipulate cell populations in multiple tissues, including the central nervous system (CNS). However the ability to select existing lines for specific applications has been limited by the lack of detailed expression analysis. RESULTS: We describe a Gal4 enhancer trap screen in which we used advanced image analysis, including three-dimensional confocal reconstructions and documentation of expression patterns at multiple developmental time points. In all, we have created and annotated 98 lines exhibiting a wide range of expression patterns, most of which include CNS expression. Expression was also observed in nonneural tissues such as muscle, skin epithelium, vasculature, and neural crest derivatives. All lines and data are publicly available from the Zebrafish International Research Center (ZIRC) from the Zebrafish Model Organism Database (ZFIN). CONCLUSIONS: Our detailed documentation of expression patterns, combined with the public availability of images and fish lines, provides a valuable resource for researchers wishing to study CNS development and function in zebrafish. Our data also suggest that many existing enhancer trap lines may have previously uncharacterized expression in multiple tissues and cell types.

Extraocular ectoderm triggers dorsal retinal fate during optic vesicle evagination in zebrafish.

Dorsal retinal fate is established early in eye development, via expression of spatially restricted dorsal-specific transcription factors in the optic vesicle; yet the events leading to initiation of dorsal fate are not clear. We hypothesized that induction of dorsal fate would require an extraocular signal arising from a neighboring tissue to pattern the prospective dorsal retina, however no such signal has been identified. We used the zebrafish embryo to determine the source, timing, and identity of the dorsal retina-inducing signal. Extensive cell movements occur during zebrafish optic vesicle morphogenesis, however the location of prospective dorsal cells within the early optic vesicle and their spatial relationship to early dorsal markers is currently unknown. Our mRNA expression and fate mapping analyses demonstrate that the dorsolateral optic vesicle is the earliest region to express dorsal specific markers, and cells from this domain contribute to the dorsal retinal pole at 24 hpf. We show that three bmp genes marking dorsal retina at 25 hpf are also expressed extraocularly before retinal patterning begins. We identified gdf6a as a dorsal initiation signal acting from the extraocular non-neural ectoderm during optic vesicle evagination. We find that bmp2b is involved in dorsal retina initiation, acting upstream of gdf6a. Together, this work has identified the nature and source of extraocular signals required to pattern the dorsal retina.

otpb functions in a Lef1-dependent transcriptional network required for expression of the stress response inhibitor crhbp in the zebrafish hypothalamus.

The vertebrate hypothalamus regulates physiological and behavioral responses to environmental stimuli through the function of evolutionarily-conserved neuronal subpopulations. Our previous work found that mutation of zebrafish lef1 , which encodes a transcriptional mediator of the Wnt signaling pathway, leads to the loss of hypothalamic neurons and behavioral phenotypes that are both associated with stress-related human mood disorders However, the specific Lef1 target genes that link neurogenesis to behavior remain unknown. One candidate is otpb , which encodes a transcription factor with known roles in hypothalamic development. Here we show that otpb expression in the posterior hypothalamus is Lef1-dependent, and that like lef1 , its function is required for the generation of crhbp + neurons in this region. Transgenic reporter analysis of a crhbp conserved noncoding element suggests that otpb participates in a transcriptional regulatory network with other Lef1 targets. Finally, consistent with a role for crhbp in inhibiting the stress response, zebrafish otpb mutants exhibit decreased exploration in a novel tank diving assay. Together our findings suggest a potential evolutionarily-conserved mechanism for the regulation of innate stress response behaviors through Lef1-mediated hypothalamic neurogenesis.

Tcf7l1 is required for spinal cord progenitor maintenance.

Neural progenitor cells must be maintained during development in order to produce the full complement of neuronal and glial derivatives. While molecular pathways have been identified that inhibit progenitor differentiation, it is unclear whether the progenitor state itself is actively maintained. In this study, we have investigated the role of Tcf7l1 (formerly named Tcf3) in maintaining spinal progenitor characteristics and allowing the continued production of neurons and glia following primary neurogenesis. We find that spinal cord progenitor markers are progressively lost in embryos lacking Tcf7l1, and that the number of proliferative progenitors decreases accordingly. Furthermore, we show that the production of both neuronal and glial secondary derivatives of the pMN progenitor pool requires Tcf7l1. Together, these results indicate that Tcf7l1 plays an important role in spinal cord progenitor maintenance, indicating that this core function is conserved throughout multiple epithelial cell populations.

Unique advantages of zebrafish larvae as a model for spinal cord regeneration.

The regenerative capacity of the spinal cord in mammals ends at birth. In contrast, teleost fish and amphibians retain this capacity throughout life, leading to the use of the powerful zebrafish model system to identify novel mechanisms that promote spinal cord regeneration. While adult zebrafish offer an effective comparison with non-regenerating mammals, they lack the complete array of experimental approaches that have made this animal model so successful. In contrast, the optical transparency, simple anatomy and complex behavior of zebrafish larvae, combined with the known conservation of pro-regenerative signals and cell types between larval and adult stages, suggest that they may hold even more promise as a system for investigating spinal cord regeneration. In this review, we highlight characteristics and advantages of the larval model that underlie its potential to provide future therapeutic approaches for treating human spinal cord injury.

Progenitor expansion in apc mutants is mediated by Jak/Stat signaling.

BACKGROUND: Mutations in APC, a negative regulator of the Wnt/ß-catenin pathway, can cause cancer as well as profound developmental defects. In both cases, affected cells adopt a proliferative progenitor state and fail to differentiate. While the upregulation of some target genes of Wnt/ß-catenin signaling has been shown to mediate these phenotypes in individual tissues, it is unclear whether a common mechanism underlies the defects in APC mutants. RESULTS: Here we show that stat3, a known oncogene and a target of ß-catenin in multiple tissues, is upregulated in apc mutant zebrafish embryos. We further demonstrate that Jak/Stat signaling is necessary for the increased level of proliferation and neural progenitor gene expression observed in apc mutants. CONCLUSIONS: Together, our data suggest that the regulation of Jak/Stat signaling may represent a conserved mechanism explaining the expansion of undifferentiated cells downstream of APC mutations.

Regenerated interneurons integrate into locomotor circuitry following spinal cord injury.

Whereas humans and other adult mammals lack the ability to regain locomotor function after spinal cord injury, zebrafish are able to recover swimming behavior even after complete spinal cord transection. We have previously shown that zebrafish larvae regenerate lost spinal cord neurons within 9 days post-injury (dpi), but it is unknown whether these neurons are physiologically active or integrate into functional circuitry. Here we show that genetically defined premotor interneurons are regenerated in injured spinal cord segments as functional recovery begins. Further, we show that these newly-generated interneurons receive excitatory input and fire synchronously with motor output by 9 dpi. Taken together, our data indicate that regenerative neurogenesis in the zebrafish spinal cord produces interneurons with the ability to integrate into existing locomotor circuitry.

Intrauterine Growth Restriction Causes Abnormal Embryonic Dentate Gyrus Neurogenesis in Mouse Offspring That Leads to Adult Learning and Memory Deficits.

Human infants who suffer from intrauterine growth restriction (IUGR), which is a failure to attain their genetically predetermined weight, are at increased risk for postnatal learning and memory deficits. Hippocampal dentate gyrus (DG) granule neurons play an important role in memory formation; however, it is unknown whether IUGR affects embryonic DG neurogenesis, which could provide a potential mechanism underlying abnormal postnatal learning and memory function. Using a mouse model of the most common cause of IUGR, induced by hypertensive disease of pregnancy, we first assessed adult learning and memory function. We quantified the percentages of embryonic hippocampal DG neural stem cells (NSCs) and progenitor cells and developing glutamatergic granule neurons, as well as hippocampal volumes and neuron cell count and morphology 18 and 40 d after delivery. We characterized the differential embryonic hippocampal transcriptomic pathways between appropriately grown and IUGR mouse offspring. We found that IUGR offspring of both sexes had short-term adult learning and memory deficits. Prenatally, we found that IUGR caused accelerated embryonic DG neurogenesis and Sox2+ neural stem cell depletion. IUGR mice were marked by decreased hippocampal volumes and decreased doublecortin+ neuronal progenitors with increased mean dendritic lengths at postnatal day 18. Consistent with its known molecular role in embryonic DG neurogenesis, we also found evidence for decreased Wnt pathway activity during IUGR. In conclusion, we have discovered that postnatal memory deficits are associated with accelerated NSC differentiation and maturation into glutamatergic granule neurons following IUGR, a phenotype that could be explained by decreased embryonic Wnt signaling.

Chromosomal position mediates spinal cord expression of a dbx1a enhancer.

Dbx homeodomain proteins are important for the production of multiple spinal cord cell types. To examine the regulation of Dbx genes in more detail, we have generated transgenic zebrafish in which fluorescent protein expression is driven by predicted dbx1a enhancers. We identified three areas of sequence conservation upstream of the dbx1a coding sequence and generated fluorescent reporter constructs driven by these predicted enhancer elements and the endogenous dbx1a promoter. In multiple stable insertions of a 3.5-kb enhancer fragment, we observed that there was additional reporter expression in the dorsal spinal cord not normally observed by dbx1a in situ hybridization. In addition, these lines exhibited only transient reporter expression, unlike the endogenous gene. Surprisingly, a single insertion line expressed the reporter in the endogenous pattern, indicating that other local regulatory elements modulate gene expression through the 3.5-kb enhancer.

Bsx Is Essential for Differentiation of Multiple Neuromodulatory Cell Populations in the Secondary Prosencephalon.

The hypothalamus is characterized by great neuronal diversity, with many neuropeptides and other neuromodulators being expressed within its multiple anatomical domains. The regulatory networks directing hypothalamic development have been studied in detail, but, for many neuron types, control of differentiation is still not understood. The highly conserved Brain-specific homeobox (Bsx) transcription factor has previously been described in regulating Agrp and Npy expression in the hypothalamic arcuate nucleus (ARC) in mice. While Bsx is expressed in many more subregions of both tuberal and mamillary hypothalamus, the functions therein are not known. Using genetic analyses in zebrafish, we show that most bsx expression domains are dependent on Nkx2.1 and Nkx2.4 homeodomain transcription factors, while a subset depends on Otp. We show that the anatomical pattern of the ventral forebrain appears normal in bsx mutants, but that Bsx is necessary for the expression of many neuropeptide encoding genes, including agrp, penka, vip, trh, npb, and nts, in distinct hypothalamic anatomical domains. We also found Bsx to be critical for normal expression of two Crh family members, crhb and uts1, as well as crhbp, in the hypothalamus and the telencephalic septal region. Furthermore, we demonstrate a crucial role for Bsx in serotonergic, histaminergic and nitrergic neuron development in the hypothalamus. We conclude that Bsx is critical for the terminal differentiation of multiple neuromodulatory cell types in the forebrain.

Hh and Wnt signaling regulate formation of olig2+ neurons in the zebrafish cerebellum.

The cerebellum, which forms from anterior hindbrain, coordinates motor movements and balance. Sensory input from the periphery is relayed and modulated by cerebellar interneurons, which are organized in layers. The mechanisms that specify the different neurons of the cerebellum and direct its layered organization remain poorly understood. Drawing from investigations of spinal cord, we hypothesized that the embryonic cerebellum is patterned on the dorsoventral axis by opposing morphogens. We tested this using zebrafish. Here we show that expression of olig2, which encodes a bHLH transcription factor, marks a distinct subset of neurons with similarities to eurydendroid neurons, the principal efferent neurons of the teleost cerebellum. In combination with other markers, olig2 reveals a dorsoventral organization of cerebellar neurons in embryos. Disruption of Hedgehog signaling, which patterns the ventral neural tube, produced a two-fold increase in the number of olig2(+) neurons. By contrast, olig2(+) neurons did not develop in embryos deficient for Wnt signaling, which patterns dorsal neural tube, nor did they develop in embryos deficient for both Hedgehog and Wnt signaling. Our data indicate that Hedgehog and Wnt work in opposition across the dorsoventral axis of the cerebellum to regulate formation of olig2(+) neurons. Specifically, we propose that Hedgehog limits the range of Wnt signaling, which is necessary for olig2(+) neuron development.

Proliferation and patterning are mediated independently in the dorsal spinal cord downstream of canonical Wnt signaling.

Canonical Wnt signaling can regulate proliferation and patterning in the developing spinal cord, but the relationship between these functions has remained elusive. It has been difficult to separate the distinct activities of Wnts because localized changes in proliferation could conceivably alter patterning, and gain and loss of function experiments have resulted in both types of defects. To resolve this issue we have investigated canonical Wnt signaling in the zebrafish spinal cord using multiple approaches. We demonstrate that Wnt signaling is required initially for proliferation throughout the entire spinal cord, and later for patterning dorsal progenitor domains. Furthermore, we find that spinal cord patterning is normal in embryos after cell division has been pharmacologically blocked. Finally, we determine the transcriptional mediators of Wnt signaling that are responsible for patterning and proliferation. We show that tcf7 gene knockdown results in dorsal patterning defects without decreasing the mitotic index in dorsal domains. In contrast, tcf3 gene knockdown results in a reduced mitotic index without affecting dorsal patterning. Together, our work demonstrates that proliferation and patterning in the developing spinal cord are separable events that are regulated independently by Wnt signaling.