ABSTRACT
In zebrafish, sensory epithelia and neuroblasts of the inner ear form simultaneously in abutting medial and lateral domains, respectively, in the floor of the otic vesicle. Previous studies support regulatory roles for Fgf and Wnt, but how signaling is coordinated is poorly understood. We investigated this problem using pharmacological and transgenic methods to alter Fgf or Wnt signaling from early placodal stages to evaluate later changes in growth and patterning. Blocking Fgf at any stage reduces proliferation of otic tissue and terminates both sensory and neural specification. Wnt promotes proliferation in the otic vesicle but is not required for sensory or neural development. However, sustained overactivation of Wnt laterally expands sensory epithelia and blocks neurogenesis. pax2a, sp5a and sp5l are coregulated by Fgf and Wnt and show overlapping expression in the otic placode and vesicle. Gain- and loss-of-function studies show that these genes are together required for Wnt's suppression of neurogenesis, as well as some aspects of sensory development. Thus, pax2a, sp5a and sp5l are critical for mediating Fgf and Wnt signaling to promote spatially localized sensory and neural development.
Subject(s)
Ear, Inner , Zebrafish , Animals , Zebrafish/genetics , Gene Expression Regulation, Developmental , Fibroblast Growth Factors/metabolism , Ear, Inner/metabolism , Wnt Signaling Pathway , Zebrafish Proteins/genetics , PAX2 Transcription Factor/genetics , PAX2 Transcription Factor/metabolismABSTRACT
The process that partitions the nascent vertebrate central nervous system into forebrain, midbrain, hindbrain, and spinal cord after neural induction is of fundamental interest in developmental biology, and is known to be dependent on Wnt/ß-catenin signaling at multiple steps. Neural induction specifies neural ectoderm with forebrain character that is subsequently posteriorized by graded Wnt signaling: embryological and mutant analyses have shown that progressively higher levels of Wnt signaling induce progressively more posterior fates. However, the mechanistic link between Wnt signaling and the molecular subdivision of the neural ectoderm into distinct domains in the anteroposterior (AP) axis is still not clear. To better understand how Wnt mediates neural AP patterning, we performed a temporal dissection of neural patterning in response to manipulations of Wnt signaling in zebrafish. We show that Wnt-mediated neural patterning in zebrafish can be divided into three phases: (I) a primary AP patterning phase, which occurs during gastrulation, (II) a mes/r1 (mesencephalon-rhombomere 1) specification and refinement phase, which occurs immediately after gastrulation, and (III) a midbrain-hindbrain boundary (MHB) morphogenesis phase, which occurs during segmentation stages. A major outcome of these Wnt signaling phases is the specification of the major compartment divisions of the developing brain: first the MHB, then the diencephalic-mesencephalic boundary (DMB). The specification of these lineage divisions depends upon the dynamic changes of gene transcription in response to Wnt signaling, which we show primarily involves transcriptional repression or indirect activation. We show that otx2b is directly repressed by Wnt signaling during primary AP patterning, but becomes resistant to Wnt-mediated repression during late gastrulation. Also during late gastrulation, Wnt signaling becomes both necessary and sufficient for expression of wnt8b, en2a, and her5 in mes/r1. We suggest that the change in otx2b response to Wnt regulation enables a transition to the mes/r1 phase of Wnt-mediated patterning, as it ensures that Wnts expressed in the midbrain and MHB do not suppress midbrain identity, and consequently reinforce formation of the DMB. These findings integrate important temporal elements into our spatial understanding of Wnt-mediated neural patterning and may serve as an important basis for a better understanding of neural patterning defects that have implications in human health.
Subject(s)
Body Patterning/physiology , Neural Plate/physiology , Wnt Signaling Pathway/physiology , Animals , Diencephalon/metabolism , Ectoderm/metabolism , Embryo, Nonmammalian/metabolism , Fibroblast Growth Factors/metabolism , Gastrula/metabolism , Gastrulation/physiology , Gene Expression Regulation, Developmental/genetics , Homeodomain Proteins/metabolism , Mesencephalon/metabolism , Nervous System/metabolism , Neural Plate/metabolism , Rhombencephalon/metabolism , Zebrafish/metabolism , Zebrafish Proteins/metabolismABSTRACT
One of the earliest patterning events in the vertebrate neural plate is the specification of mes/r1, the territory comprising the prospective mesencephalon and the first hindbrain rhombomere. Within mes/r1, an interface of gene expression defines the midbrain-hindbrain boundary (MHB), a lineage restriction that separates the mesencephalon and rhombencephalon. wnt1 is critical to mes/r1 development and functions within the MHB as a component of the MHB gene regulatory network (GRN). Despite its importance to these critical and early steps of vertebrate neurogenesis, little is known about the factors responsible for wnt1 transcriptional regulation. In the zebrafish, wnt1 and its neighboring paralog, wnt10b, are expressed in largely overlapping patterns, suggesting co-regulation. To understand wnt1 and wnt10b transcriptional control, we used a comparative genomics approach to identify relevant enhancers. We show that the wnt1-wnt10b locus contains multiple cis-regulatory elements that likely interact to generate the wnt1 and wnt10b expression patterns. Two of 11 conserved enhancers tested show activity restricted to the midbrain and MHB, an activity that is conserved in the distantly related spotted gar orthologous elements. Three non-conserved elements also play a likely role in wnt1 regulation. The identified enhancers display dynamic modes of chromatin accessibility, suggesting controlled deployment during embryogenesis. Our results suggest that the control of wnt1 and wnt10b expression is under complex regulation involving the interaction of multiple enhancers.
Subject(s)
Brain/embryology , Regulatory Elements, Transcriptional , Wnt Proteins/genetics , Wnt1 Protein/genetics , Zebrafish Proteins/genetics , Zebrafish/embryology , Animals , Chromatin , Embryo, Nonmammalian/metabolism , Fish Proteins/genetics , Fish Proteins/metabolism , Fishes/embryology , Fishes/genetics , Gene Expression Regulation, Developmental , Genomics , Mice , Promoter Regions, Genetic , Wnt Proteins/metabolism , Wnt1 Protein/metabolism , Zebrafish/metabolism , Zebrafish Proteins/metabolismABSTRACT
The establishment of anteroposterior identity in the vertebrate neural plate has been a subject of investigation for decades, but molecular explanations of posteriorization were only revealed beginning in the late 1980s. A model has emerged from several key studies that identifies Wnt signaling as a key posteriorizing agent, which evidence suggests specifies anteroposterior fates in a concentration-dependent manner. In this review, we consider the historical context of posteriorization studies and evaluate models for Wnt-dependent posteriorization. With new information about the mode of delivery of many signaling ligands, we propose alternative scenarios to reconcile the Wnt gradient model with the complex process of gastrulation and potential non-secretory mechanisms of Wnt delivery.
Subject(s)
Models, Biological , Neural Plate/embryology , Wnt Proteins/metabolism , Wnt Signaling Pathway/physiology , Animals , Humans , Neural Plate/cytology , Wnt Proteins/geneticsABSTRACT
wnt8a Is essential for normal patterning during vertebrate embryonic development, and either gain or loss-of-function gene dysregulation results in severe axis malformations. The zebrafish wnt8a locus is structured such that transcripts may possess two regulatory 3' untranslated regions (UTRs), raising the possibility of post-transcriptional regulation as an important mode of wnt8a signaling control. To determine whether both UTRs contribute to post-transcriptional wnt8a gene regulation, each UTR (UTR1 and UTR2) was tested in transient and transgenic reporter assays. Both UTRs suppress EGFP reporter expression in cis, with UTR2 exhibiting a more pronounced effect. UTR2 contains a 6 base sequence necessary for UTR2 regulatory function that is complementary to the seed of the microRNA, miR-430. A target protector morpholino that overlaps the seed complement stabilizes both reporter mRNAs and wnt8a mRNAs, and produces phenotypic abnormalities consistent with wnt8a gain-of-function. In rescue assays, specific functions can be attributed to each of the two wnt8a proteins encoded by the locus. An interplay of wnt8a.1 and wnt8a.2 regulates neural and mesodermal patterning and morphogenesis as well as patterning between brain subdivisions. Thus, post-transcriptional control of wnt8a is essential to fine tune the balance of the signaling outputs of the complex wnt8a locus.
Subject(s)
Cytoskeletal Proteins/genetics , Gene Expression Regulation, Developmental , RNA Processing, Post-Transcriptional , Wnt Proteins/genetics , Zebrafish Proteins/genetics , Zebrafish/embryology , Animals , Base Sequence , Body Patterning , Green Fluorescent Proteins/metabolism , MicroRNAs/metabolism , Molecular Sequence Data , Neurons/metabolism , Phenotype , RNA, Messenger/metabolism , Signal Transduction , Transgenes , Zebrafish/geneticsABSTRACT
BACKGROUND: Vertebrate axis development depends upon wnt8a transcription in a dynamic pool of mesoderm progenitors at the posterior pole of the gastrulating embryo. The transcriptional mechanisms controlling wnt8a expression are not understood, but previous studies identified two phases of wnt8a expression in zebrafish: Nodal-dependent activation during early gastrulation (phase I) and No tail (Ntl)-dependent regulation from mid gastrula stages (phase II). RESULTS: We identified two upstream cis-regulatory regions, proximal and distal, each of which possesses a promoter. The proximal regulatory region contains a margin-specific enhancer that is required for both the Nodal and Ntl responses. Phase I expression requires Nodal activation of the margin enhancer in combination with the transcription factor Zbtb4 and the distal regulatory region. Phase II expression requires Ntl regulation of the margin enhancer in the context of the proximal regulatory region. An additional mechanism is required to ensure the transition from phase I to phase II regulation. Analysis of stickleback wnt8a suggests this mechanism of regulation may be conserved. CONCLUSIONS: The seemingly simple wnt8a expression pattern reflects complex interactions of multiple regulatory inputs.
Subject(s)
Body Patterning/physiology , Cytoskeletal Proteins/metabolism , Gastrula/embryology , Gene Expression Regulation, Developmental/physiology , Regulatory Elements, Transcriptional/physiology , Wnt Proteins/metabolism , Zebrafish Proteins/metabolism , Zebrafish/embryology , Animals , Base Sequence , Binding Sites/genetics , Cytoskeletal Proteins/genetics , DNA Primers/genetics , Fetal Proteins , Gastrula/metabolism , Gene Expression Regulation, Developmental/genetics , Mesoderm/embryology , Mesoderm/metabolism , Molecular Sequence Data , Nodal Protein/metabolism , Promoter Regions, Genetic/genetics , Regulatory Elements, Transcriptional/genetics , Smegmamorpha/embryology , Smegmamorpha/metabolism , Species Specificity , T-Box Domain Proteins/metabolism , Wnt Proteins/genetics , Zebrafish/metabolism , Zebrafish Proteins/geneticsABSTRACT
BACKGROUND: ZNF143 is a sequence-specific DNA-binding protein that stimulates transcription of both small RNA genes by RNA polymerase II or III, or protein-coding genes by RNA polymerase II, using separable activating domains. We describe phenotypic effects following knockdown of this protein in developing Danio rerio (zebrafish) embryos by injection of morpholino antisense oligonucleotides that target znf143 mRNA. RESULTS: The loss of function phenotype is pleiotropic and includes a broad array of abnormalities including defects in heart, blood, ear and midbrain hindbrain boundary. Defects are rescued by coinjection of synthetic mRNA encoding full-length ZNF143 protein, but not by protein lacking the amino-terminal activation domains. Accordingly, expression of several marker genes is affected following knockdown, including GATA-binding protein 1 (gata1), cardiac myosin light chain 2 (cmlc2) and paired box gene 2a (pax2a). The zebrafish pax2a gene proximal promoter contains two binding sites for ZNF143, and reporter gene transcription driven by this promoter in transfected cells is activated by this protein. CONCLUSIONS: Normal development of zebrafish embryos requires ZNF143. Furthermore, the pax2a gene is probably one example of many protein-coding gene targets of ZNF143 during zebrafish development.
Subject(s)
Trans-Activators/metabolism , Zebrafish Proteins/metabolism , Zebrafish/metabolism , Animals , Base Sequence , Cardiac Myosins/genetics , Cardiac Myosins/metabolism , Embryo, Nonmammalian/metabolism , Embryonic Development , GATA1 Transcription Factor/genetics , GATA1 Transcription Factor/metabolism , Morpholinos , Myosin Light Chains/genetics , Myosin Light Chains/metabolism , PAX2 Transcription Factor/genetics , PAX2 Transcription Factor/metabolism , Promoter Regions, Genetic , Trans-Activators/antagonists & inhibitors , Trans-Activators/genetics , Zebrafish/embryology , Zebrafish Proteins/antagonists & inhibitors , Zebrafish Proteins/geneticsABSTRACT
Vertebrate wnt8a links anteroposterior and dorsoventral axis patterning, but the regulation of wnt8a expression and its relationship to mesoderm induction and maintenance pathways is unclear. To address this, we have generated zebrafish transgenic for a modified genomic PAC clone that expresses EGFP from the wnt8a locus. The EGFP reporter transgene is expressed in a pattern nearly identical to wnt8a, including maternal deposition, expression in the ventrolateral mesoderm and in the yolk syncytial layer. Loss of function studies show that wnt8a expression is under biphasic control by Nodal and No Tail/Brachyury, whereby early phase expression is Nodal-dependent but late phase expression is Ntl/Bra dependent. EGFP fluorescence persists in cells that transcribe the reporter, thus comprising a tracer for ventrolaterally derived mesodermal lineages. We use this property to show that wnt8a expression marks Nodal-independent tail mesoderm formation and that Ntl/Bra predominantly regulates wnt8a in paraxial mesoderm progenitors.
Subject(s)
Chromosomes, Artificial, P1 Bacteriophage/genetics , Cytoskeletal Proteins/genetics , Mesoderm/embryology , Wnt Proteins/genetics , Zebrafish Proteins/genetics , Zebrafish/embryology , Animals , Animals, Genetically Modified , Body Patterning/genetics , Body Patterning/physiology , Cytoskeletal Proteins/metabolism , Embryo, Nonmammalian , Gene Expression Regulation, Developmental/physiology , Gene Regulatory Networks/physiology , Genes, Reporter/physiology , Mesoderm/metabolism , Models, Biological , Stem Cells/metabolism , Stem Cells/physiology , Tail/embryology , Tail/metabolism , Vertebrates/embryology , Vertebrates/genetics , Vertebrates/metabolism , Wnt Proteins/metabolism , Zebrafish/genetics , Zebrafish Proteins/metabolismABSTRACT
Live imaging of zebrafish embryos that maintains normal development can be difficult to achieve due to a combination of sample mounting, immobilization, and phototoxicity issues that, once overcome, often still results in image quality sufficiently poor that computer-aided analysis or even manual analysis is not possible. Here, we describe our mounting strategy for imaging the zebrafish midbrain-hindbrain boundary (MHB) with light sheet fluorescence microscopy (LSFM) and pilot experiments to create a study-specific set of parameters for semiautomatically tracking cellular movements in the embryonic midbrain primordium during zebrafish segmentation.
Subject(s)
Zebrafish Proteins , Zebrafish , Animals , Mesencephalon , Microscopy, Fluorescence , RhombencephalonABSTRACT
Vertebrate dorsoventral patterning requires both Wnt8 and BMP signaling. Because of their multiple interactions, discerning roles attributable specifically to Wnt8 independent of BMP has been a challenge. For example, Wnt8 represses the dorsal organizer that negatively regulates ventral BMP signals, thus Wnt8 loss-of-function phenotypes may reflect the combined effects of reduced Wnt8 and BMP signaling. We have taken a loss-of-function approach in the zebrafish to generate embryos lacking expression of both Wnt8 and the BMP antagonist Chordin. wnt8;chordin loss-of-function embryos show rescued BMP signaling, thereby allowing us to identify Wnt8-specific requirements. Our analysis shows that Wnt8 is uniquely required to repress prechordal plate specification but not notochord, and that Wnt8 signaling is not essential for specification of tailbud progenitors but is required for normal expansion of posterior mesoderm cell populations. Thus, Wnt8 and BMP signaling have independent roles during vertebrate ventrolateral mesoderm development that can be identified through loss-of-function analysis.
Subject(s)
Body Patterning/physiology , Bone Morphogenetic Proteins/metabolism , Embryo, Nonmammalian/metabolism , Mesoderm/metabolism , Wnt Proteins/metabolism , Zebrafish Proteins/metabolism , Animals , Body Patterning/genetics , Bone Morphogenetic Proteins/genetics , Gene Expression Regulation, Developmental/genetics , Gene Expression Regulation, Developmental/physiology , Glycoproteins/genetics , Glycoproteins/metabolism , In Situ Hybridization , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Wnt Proteins/genetics , Zebrafish , Zebrafish Proteins/geneticsABSTRACT
We introduce a computational approach to build three-dimensional (3D) surface mesh models of the early-stage zebrafish brain primordia from time-series microscopy images. The complexity of the early-stage brain primordia and lack of recognizable landmarks pose a distinct challenge for feature segmentation and 3D modeling. Additional difficulty arises because of noise and variations in pixel intensity. We overcome these by using a hierarchical approach in which simple geometric elements, such as "beads" and "bonds," are assigned to represent local features and their connectivity is used to smoothen the surface while retaining high-curvature regions. We apply our method to build models of two zebrafish embryo phenotypes at discrete time points between 19 and 28 h post-fertilization and collect measurements to quantify development. Our approach is fast and applicable to building models of other biological systems, as demonstrated by models from magnetic resonance images of the human fetal brain. The source code, input scripts, sample image files, and generated outputs are publicly available on GitHub.
ABSTRACT
From the combined perspective of biologists, microscope instrumentation developers, imaging core facility scientists, and high performance computing experts, we discuss the challenges faced when selecting imaging and analysis tools in the field of light-sheet microscopy. Our goal is to provide a contextual framework of basic computing concepts that cell and developmental biologists can refer to when mapping the peculiarities of different light-sheet data to specific existing computing environments and image analysis pipelines. We provide our perspective on efficient processes for tool selection and review current hardware and software commonly used in light-sheet image analysis, as well as discuss what ideal tools for the future may look like.
ABSTRACT
Promoters for vertebrate small nuclear RNA (snRNA) genes contain a relatively simple array of transcriptional control elements, divided into proximal and distal regions. Most of these genes are transcribed by RNA polymerase II (e.g., U1, U2), whereas the U6 gene is transcribed by RNA polymerase III. Previously identified vertebrate U6 snRNA gene promoters consist of a proximal sequence element (PSE) and TATA element in the proximal region, plus a distal region with octamer (OCT) and SphI postoctamer homology (SPH) elements. We have found that zebrafish U6 snRNA promoters contain the SPH element in a novel proximal position immediately upstream of the TATA element. The zebrafish SPH element is recognized by SPH-binding factor/selenocysteine tRNA gene transcription activating factor/zinc finger protein 143 (SBF/Staf/ZNF143) in vitro. Furthermore, a zebrafish U6 promoter with a defective SPH element is inefficiently transcribed when injected into embryos.
Subject(s)
Promoter Regions, Genetic , RNA, Small Nuclear/genetics , Zebrafish/genetics , 5' Flanking Region , Animals , Base Sequence , Consensus Sequence , Humans , Molecular Sequence Data , RNA, Small Nuclear/biosynthesis , Sequence Alignment , Trans-Activators/metabolism , Transcription, GeneticABSTRACT
Chromodomain-Helicase-DNA binding protein 8 (CHD8) is a member of a large family of eukaryotic ATP-dependent chromatin remodeling complexes. Loss of function alleles of human chd8 are correlated with autism spectrum disorder. The CHD subfamily members contain a tandem pair of chromodomains that are adjacent to a centrally located Snf2-like helicase domain. An alternatively spliced variant mRNA of CHD8 was identified years ago in mammals that encode a truncated form of the protein, called Duplin, that lacks the helicase domain and everything else in the carboxyl direction. We are using zebrafish to explore the functions of CHD8, especially the truncated form that we refer to as CHD8short (CHD8S). The mRNA for CHD8S is expressed differentially during embryonic development. Using a PCR assay we detected expression of putative zebrafish chd8s mRNA that is barely detectable during early embryogenesis (shield stage at 6h), but increases markedly soon thereafter at 80-90% epiboly (9h) and bud stages (10h), with a return to low levels in 16-somite (17h) and 24hpf embryos. Except for high expression during the shield stage, steady-state levels of chd8l (long) mRNA are relatively constant during the same period of development. We subcloned both chd8l and chd8s cDNAs into expression vector plasmids for use in transient transfection experiments in zebrafish ZF4 cells. In some experiments the luciferase reporter gene was driven by a synthetic promoter that is responsive to activation by ZNF143 activator protein, a known interacting protein with CHD8 in mammalian cells. Whereas CHD8L was a modest coactivator, CHD8S was a potent coactivator, a surprising result since CHD8S is lacking a critical domain to function as a chromatin remodeler enzyme. CHD8S coactivator function is dependent on a region of the protein within the first 50 amino-terminal amino acids. In transient transfection experiments using a Lef1/ß-catenin reporter gene, CHD8S was a modest repressor, but deletion of 50 or more amino-terminal amino acids converted it to a coactivator. When synthetic chd8s mRNA was injected into zebrafish embryos in order to overexpress CHD8S, we observed significant brain disruption phenotypes.
Subject(s)
Chromatin/genetics , DNA Helicases/genetics , DNA-Binding Proteins/genetics , Protein Domains/genetics , Transcription Factors/genetics , Transcription, Genetic/genetics , Zebrafish Proteins/genetics , Alternative Splicing/genetics , Animals , Cell Line , Gene Expression Regulation, Developmental/genetics , Genes, Reporter/genetics , HEK293 Cells , Humans , Lymphoid Enhancer-Binding Factor 1/genetics , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , Transfection/methods , Zebrafish , beta Catenin/geneticsABSTRACT
A constriction in the neural tube at the junction of the midbrain and hindbrain is a conserved feature of vertebrate embryos. The constriction is a defining feature of the midbrain-hindbrain boundary (MHB), a signaling center that patterns the adjacent midbrain and rostral hindbrain and forms at the junction of two gene expression domains in the early neural plate: an anterior otx2/wnt1 positive domain and a posterior gbx/fgf8 positive domain. otx2 and gbx genes encode mutually repressive transcription factors that create a lineage restriction boundary at their expression interface. Wnt and Fgf genes form a mutually dependent feedback system that maintains their expression domains on the otx2 or gbx side of the boundary, respectively. Constriction morphogenesis occurs after these conserved gene expression domains are established and while their mutual interactions maintain their expression pattern; consequently, mutant studies in zebrafish have led to the suggestion that constriction morphogenesis should be considered a unique phase of MHB development. We analyzed MHB morphogenesis in fgf8 loss of function zebrafish embryos using a reporter driven by the conserved wnt1 enhancer to visualize anterior boundary cells. We found that fgf8 loss of function results in a re-activation of wnt1 reporter expression posterior to the boundary simultaneous with an inactivation of the wnt1 reporter in the anterior boundary cells, and that these events correlate with relaxation of the boundary constriction. In consideration of other results that correlate the boundary constriction with Wnt and Fgf expression, we propose that the maintenance of an active Wnt-Fgf feedback loop is a key factor in driving the morphogenesis of the MHB constriction.
ABSTRACT
Wnt signaling is known to be required for the normal development of the vertebrate midbrain and hindbrain, but genetic loss of function analyses in the mouse and zebrafish yield differing results regarding the relative importance of specific Wnt loci. In the zebrafish, Wnt1 and Wnt10b functionally overlap in their control of gene expression in the ventral midbrain-hindbrain boundary (MHB), but they are not required for the formation of the MHB constriction. Whether other wnt loci are involved in zebrafish MHB development is unclear, although the expression of at least two wnts, wnt3a and wnt8b, is maintained in wnt1/wnt10b mutants. In order to address the role of wnt3a in zebrafish, we have isolated a full length cDNA and examined its expression and function via knockdown by morpholino antisense oligonucleotide (MO)-mediated knockdown. The expression pattern of wnt3a appears to be evolutionarily conserved between zebrafish and mouse, and MO knockdown shows that Wnt3a, while not uniquely required for MHB development, is required in the absence of Wnt1 and Wnt10b for the formation of the MHB constriction. In zebrafish embryos lacking Wnt3a, Wnt1 and Wnt10b, the expression of engrailed orthologs, pax2a and fgf8 is not maintained after mid-somitogenesis. In contrast to acerebellar and no isthmus mutants, in which midbrain and hindbrain cells acquire new fates but cell number is not significantly affected until late in embryogenesis, zebrafish embryos lacking Wnt3a, Wnt1 and Wnt10b undergo extensive apoptosis in the midbrain and cerebellum anlagen beginning in mid-somitogenesis, which results in the absence of a significant portion of the midbrain and cerebellum. Thus, the requirement for Wnt signaling in forming the MHB constriction is evolutionarily conserved in vertebrates and it is possible in zebrafish to dissect the relative impact of multiple Wnt loci in midbrain and hindbrain development.
Subject(s)
Brain/embryology , Brain/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Zebrafish/embryology , Zebrafish/metabolism , Amino Acid Sequence , Animals , Animals, Genetically Modified , Apoptosis , Brain/cytology , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/metabolism , Gene Expression Regulation, Developmental , Humans , In Situ Hybridization , Intercellular Signaling Peptides and Proteins/chemistry , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/isolation & purification , Molecular Sequence Data , Phylogeny , Proteins/chemistry , Proteins/genetics , Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Alignment , Signal Transduction , Somites/metabolism , Wnt Proteins , Wnt1 Protein , Wnt3 Protein , Wnt3A Protein , Zebrafish/geneticsABSTRACT
During embryogenesis, presumptive brain compartments are patterned by dynamic networks of gene expression. The spatiotemporal dynamics of these networks, however, have not been characterized with sufficient resolution for us to understand the regulatory logic resulting in morphogenetic cellular behaviors that give the brain its shape. We have developed a new, integrated approach using ultrashort pulse microscopy [a high-resolution, two-photon fluorescence (2PF)-optical coherence microscopy (OCM) platform using 10-fs pulses] and image registration to study brain patterning and morphogenesis in zebrafish embryos. As a demonstration, we used time-lapse 2PF to capture midbrain-hindbrain boundary morphogenesis and a wnt1 lineage map from embryos during brain segmentation. We then performed in situ hybridization to deposit NBT/BCIP, where wnt1 remained actively expressed, and reimaged the embryos with combined 2PF-OCM. When we merged these datasets using morphological landmark registration, we found that the mechanism of boundary formation differs along the dorsoventral axis. Dorsally, boundary sharpening is dominated by changes in gene expression, while ventrally, sharpening may be accomplished by lineage sorting. We conclude that the integrated visualization of lineage reporter and gene expression domains simultaneously with brain morphology will be useful for understanding how changes in gene expression give rise to proper brain compartmentalization and structure.
Subject(s)
Brain/embryology , Gene Expression Profiling/methods , Imaging, Three-Dimensional/methods , Microscopy, Fluorescence, Multiphoton/methods , Tomography, Optical Coherence/methods , Animals , Brain/metabolism , Brain Chemistry/physiology , Embryo, Nonmammalian , In Situ Hybridization , Wnt Signaling Pathway , ZebrafishABSTRACT
During vertebrate mesoderm formation, fates are established according to position in the dorsoventral (D/V) axis of the embryo. Initially, maternal signaling divides nascent mesoderm into axial (dorsal) and non-axial (ventral) domains. Although the subsequent subdivision of non-axial mesoderm into multiple D/V fate domains is known to involve zygotic Wnt8 and BMP signaling as well as the Vent/Vox/Ved family of transcriptional repressors, how levels of signaling activity are translated into differential regulation of fates is not well understood. To address this question, we have analyzed zebrafish embryos lacking Wnt8 and BMP2b. Zebrafish wnt8; swr (bmp2b) double mutants display a progressive loss of non-axial mesoderm and a concomitant expansion of axial mesoderm during gastrulation. Mesoderm induction and specification of the axial domain occur normally in wnt8; swr mutants, but dorsal mesoderm genes eventually come to be expressed throughout the mesoderm, suggesting that the establishment of non-axial mesoderm identity requires continual repression of dorsal mesoderm factors, including repressors of ventral genes. Loss-of-function for Vent, Vox, and Ved phenocopies the wnt8; swr mutant phenotype, consistent with Wnt8 and BMP2b maintaining non-axial mesoderm identity during gastrulation through the regulation of these three transcriptional repressors. We postulate that timely differentiation of the mesoderm requires the maintenance of non-axial mesoderm identity by Wnt8 and BMP2b at the onset of gastrulation followed by subdivision of the non-axial mesoderm into different functional domains during gastrulation.
Subject(s)
Bone Morphogenetic Proteins/metabolism , Cytoskeletal Proteins/metabolism , Gastrula/metabolism , Mesoderm/metabolism , Wnt Proteins/metabolism , Zebrafish Proteins/metabolism , Zebrafish/metabolism , Animals , Body Patterning , Bone Morphogenetic Protein 2 , Bone Morphogenetic Proteins/genetics , Cytoskeletal Proteins/genetics , Embryonic Induction , Gastrula/cytology , Gene Expression Regulation, Developmental , Mesoderm/cytology , Mutation , Phenotype , Wnt Proteins/genetics , Zebrafish/embryology , Zebrafish/genetics , Zebrafish Proteins/geneticsABSTRACT
Dorsoventral (DV) patterning of vertebrate embryos requires the concerted action of the Bone Morphogenetic Protein (BMP) and Wnt signaling pathways. In contrast to our understanding of the role of BMP in establishing ventral fates, our understanding of the role of Wnts in ventralizing embryos is less complete. Wnt8 is required for ventral patterning in both Xenopus and zebrafish; however, its mechanism of action remains unclear. We have used the zebrafish to address the requirement for Wnt8 in restricting the size of the dorsal organizer. Epistasis experiments suggest that Wnt8 achieves this restriction by regulating the early expression of the transcriptional repressors Vent and Vox. Our data show that vent and vox are direct transcriptional targets of Wnt8/beta-catenin. Additionally, we show that Wnt8 and Bmp2b co-regulate vent and vox in a dynamic fashion. Thus, whereas both Wnt8 and zygotic BMP are ventralizing agents that regulate common target genes, their temporally different modes of action are necessary to pattern the embryo harmoniously along its DV axis.
Subject(s)
Homeodomain Proteins/metabolism , Organizers, Embryonic/metabolism , Proteins/metabolism , Repressor Proteins/metabolism , Zebrafish Proteins/metabolism , Animals , Body Patterning/genetics , Body Patterning/physiology , Bone Morphogenetic Protein 2 , Bone Morphogenetic Proteins/genetics , Bone Morphogenetic Proteins/metabolism , Cytoskeletal Proteins/metabolism , Gene Expression Regulation, Developmental/physiology , Homeodomain Proteins/genetics , Proteins/genetics , RNA, Messenger/metabolism , Repressor Proteins/genetics , Trans-Activators/metabolism , Wnt Proteins , Zebrafish/embryology , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/genetics , beta CateninABSTRACT
The zebrafish wnt8 locus differs from its tetrapod counterparts in that it produces two functionally overlapping but distinct Wnt8 proteins. Studies of zebrafish wnt8 have suggested that the two major Wnt8 proteins produced are functionally similar yet may behave differently depending on the assay context. To determine whether the bicistronic wnt8 and its accompanying unique protein activities found in zebrafish are more widespread (and perhaps universal) among teleosts, we have extended our studies to the pufferfish Takifugu rubripes. We have found that Takifugu wnt8 is also bicistronic, indicating that the wnt8 duplication occurred before the divergence of these teleosts approximately 150 million years ago. Furthermore, overexpression assays in zebrafish embryos show that functional differences between the zebrafish Wnt8.1 and Wnt8.2 proteins are conserved in their Takifugu orthologs. Thus, despite the fact that Wnt8.1 and Wnt8.2 proteins are as similar to each other as each is to Xenopus Xwnt-8, Wnt8 family members can behave quite differently in the context of zebrafish embryos. This finding suggests that zebrafish (and possibly teleost in general) Wnt8 receptors are able to discriminate between highly related ligands.