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The Nodal family of signaling molecules includes critical intercellular regulators of early vertebrate development. In a recent issue of Nature, maternal transcripts encoding the zebrafish nodal squint were shown to be localized to the future organizer region by the four-cell stage, providing the earliest evidence of embryonic axis asymmetry in the zebrafish embryo.
Motor growth cones navigate long and complex trajectories to connect with their muscle targets. Experimental studies have shown that this guidance process critically depends on extrinsic cues. In the zebrafish embryo, a subset of mesodermal cells, the adaxial cells, delineates the prospective path of pioneering motor growth cones. Genetic ablation of adaxial cells causes profound pathfinding defects, suggesting the existence of adaxial cell derived guidance factors. Intriguingly, adaxial cells are themselves migratory, and as growth cones approach they migrate away from the prospective axonal path to the lateral surface of the myotome, where they develop into slow-twitching muscle fibers. Genetic screens in embryos stained with an antibody cocktail identified mutants with specific defects in differentiation and migration of adaxial cells/slow muscle fibers, as well as mutants with specific defects in axonal pathfinding, including exit from the spinal cord and pathway selection. Together, the genes underlying these mutant phenotypes define pathways essential for nerve and muscle development and interactions between these two cell types.
Several basic helix-loop-helix (bHLH) transcription factors are known to be involved in vertebrate neurogenesis. To investigate their roles in zebrafish neurogenesis, we isolated cDNAs for homologues of neurogenin and Math(-1)/atonal. The transcription of neurogenin was first detectable in zebrafish nervous system at late gastrulation stage. The expression of zebrafish neurogenin precedes and overlaps that of HuC, one of the earliest neuronal precursor markers. Injection of neurogenin mRNA into early stage zebrafish embryos induced ectopic expression of HuC. These results suggest that neurogenin may participate in the generation of HuC-expressing cells, implying its role in neuronal determination in zebrafish.
Successful cryopreservation of most multicompartmental biological systems has not been achieved. One prerequisite for success is quantitative information on cryoprotectant permeation into and amongst the compartments. This report describes direct measurements of cryoprotectant permeation into a multicompartmental system using chemical shift selective magnetic resonance (MR) microscopy and MR spectroscopy. We used the developing zebrafish embryo as a model for studying these complex systems because these embryos are composed of two membrane-limited compartments: (i) a large yolk (surrounded by the yolk syncytial layer) and (ii) differentiating blastoderm cells (each surrounded by a plasma membrane). MR images of the spatial distribution of three cryoprotectants (dimethyl sulfoxide, propylene glycol, and methanol) demonstrated that methanol permeated the entire embryo within 15 min. In contrast, the other cryoprotectants exhibited little or no permeation over 2.5 h. MR spectroscopy and microinjections of cryoprotectants into the yolk inferred that the yolk syncytial layer plays a critical role in limiting the permeation of some cryoprotectants throughout the embryo. This study demonstrates the power of MR technology combined with micromanipulation for elucidating key physiological factors in cryobiology.
Functional analyses of gene function by knockdown and expression approaches strongly enhance the genetic study of development. In vivo application of the introduction of inhibitors of gene expression, mRNA, and expression constructs in the target region make it possible to perform region- and stage-specific regulation of gene function in a simple manner. As a basic tool for the conditional regulation of gene expression in target tissue, we present methods for the efficient introduction of antisense morpholino oligonucleotide (MO), mRNA, and expression plasmid constructs into early and later stage zebrafish embryo and larva. Lipofection of a neuron-specific expression construct plasmid encoding green fluorescent protein (GFP) into optic vesicle resulted in clear GFP expression in the retinotectal pathway in hatched larva. Co-lipofection of MO and GFP mRNA to the presumptive head region resulted in brain-specific knockdown of the gene in mid-stage embryos.
The effect of external medium composition on chorion and plasma membrane permeability of zebrafish (Danio rerio) embryos was investigated in this study. Initially, survival of embryos spawned into varying strengths (10-40%) of Hank's solution (HBSS) was assessed. Development and hatching rates for embryos spawned into 30% and 40% HBSS were significantly lower than those obtained with embryos spawned into system water. The effect of embryo survival in 30% HBSS with different calcium levels was then investigated. Embryo survival in calcium free 30% HBSS or 30% HBSS with 10x the standard calcium concentration was similar to survival in standard 30% HBSS. Membrane water permeability was determined by measuring the floatation time of embryos in test solutions made up with heavy water (D2O) instead of deionized water. Intact embryos at early developmental stages were less permeable than later stages irrespective of the external medium that they were spawned into. In system water, the floatation time of embryos at one-cell and two-cell stages were 1323+/-83 and 1189+/-55 s, respectively, compared to 432+/-6 and 353+/-10 s at the high and 50% epiboly stages. Change of external medium composition had no effect on membrane permeability of intact embryos at early developmental stages. However, at later stages embryos spawned into 30% HBSS were less permeable than embryos spawned into system water, irrespective of calcium concentration. The flotation time of embryos at the high stage increased from 432+/-6s in system water to 468+/-10s in 30% HBSS. The study on dechorionated embryos showed that change of external medium composition had no effect on plasma membrane permeability.
BACKGROUND: Heparan sulfate (HS) is an ubiquitous component of the extracellular matrix that binds and modulates the activity of growth factors, cytokines and proteases. Animals with defective HS biosynthesis display major developmental abnormalities however the processes that are affected remain to be defined. D-glucuronyl-C5-epimerase (Glce) is a key HS chain modifying enzyme that catalyses the conversion of glucuronic acid into iduronic acid, a biosynthetic step that enhances HS biological activity. In this study the role of Glce during early zebrafish development has been investigated. RESULTS: Two Glce-like proteins (Glce-A and -B) are expressed in zebrafish at all times. They are the products of two distinct genes that, based on chromosomal mapping, are both orthologues of the same single human gene. Transcripts for both proteins were detected in fertilized zebrafish embryos prior to the onset of zygotic transcription indicating their maternal origin. At later developmental stages the epimerases are expressed widely throughout gastrulation and then become restricted to the hindbrain at 24 h post-fertilization. By monitoring the expression of well characterized marker genes during gastrulation, we have found that misexpression of Glce causes a dose-dependent expansion of the ventral structures, whereas protein knockdown using targeted antisense morpholino oligonucleotides promotes axis dorsalization. The ventralizing activity of Bmp2b is enhanced by Glce overexpression whereas Glce knockdown impairs Bmp2b activity. CONCLUSION: Glce activity is an important determinant of of dorso-ventral axis formation and patterning in zebrafish. In particular Glce acts during gastrulation by affecting Bmp-mediated cell specification. The results obtained further corroborate the concept that HS encodes information that affect morphogenesis during early vertebrate development.
Sequence analysis of zebrafish fibronectin (FN) cDNAs indicates that at least two forms of the protein exist in fish. One form (FN1) is very similar to FNs identified in other vertebrates possessing 12 type I, 2 type II, and 17 type III repeats including two alternative splice sites (EIIIA and EIIIB) and a variable region (V). Zebrafish FN1 contains the RGD cell adhesion site in type III(10) and a second cell-binding site (LDV) in the V region. In addition to this conserved form of FN, a novel truncated form of zebrafish FN (FN2) was identified. The predicted structure of FN2 is identical to FN1 at the N-terminal region possessing 9 type I, 2 type II, and the first 3 type III repeats. Following III(3), FN2 contains a unique 20-amino-acid C-terminal tail that is different from the C-terminus of FN1, lacking the two cysteines that are usually involved in the formation of interchain disulfide bonds. Genomic sequence analysis has revealed that FN2 is generated by an alternative RNA splicing pattern that has not been described for FN in other organisms. Reverse transcription-polymerase chain reaction analysis and RNase protection assays reveal that FN2 mRNA is present in the zebrafish embryo throughout development as well as in cultures of an established liver cell line. Experiments conducted with recombinant FN2 synthesized in insect cells demonstrate that the protein promotes the attachment and spreading of fish embryo cells in culture.
Gicerin, a novel cell adhesion molecule which belongs to the immunoglobulin superfamily, is expressed temporally and spatially in the developing chick brain and retina. The previous in vitro experiments using transfected cells showed that gicerin can function as a cell adhesion molecule which has both homophilic and heterophilic binding activities. For the in vivo analyses of gicerin in neural development, we tried to utilize a zebrafish system, a vertebrate suitable for studying early development. We generated transient transgenic animals by microinjecting DNA constructs into zebrafish embryos. Chicken gicerin, under control of the neurofilament gene promoter, was preferentially expressed in neuronal cells and gicerin-expressing neurons exhibited a fasciculation formation with neighboring gicerin-positive axons, which may be partly due to homophilic cell adhesion activity of gicerin. These experimental results suggest that this fast and efficient transgenic animal system is useful for studying the functional roles of neuron-specific genes during the development.
Vertebrate body plans have a conserved left-right (LR) asymmetry manifested in the position and anatomy of the heart, visceral organs, and brain. Recent studies have suggested that LR asymmetry is established by asymmetric Ca2+ signaling resulting from cilia-driven flow of extracellular fluid across the node. We report here that inositol 1,3,4,5,6-pentakisphosphate 2-kinase (Ipk1), which generates inositol hexakisphosphate, is critical for normal LR axis determination in zebrafish. Zebrafish embryos express ipk1 symmetrically during gastrulation and early segmentation. ipk1 knockdown by antisense morpholino oligonucleotide injection randomized LR-specific gene expression and organ placement, effects that were associated with reduced intracellular Ca2+ flux in cells surrounding the ciliated Kupffer's vesicle, a structure analogous to the mouse node. Our data suggest that the pathway for inositol hexakisphosphate production is a key regulator of asymmetric Ca(2+) flux during LR specification.
As in other vertebrate species, the zebrafish retina is simpler than other regions of the central nervous system. This relative simplicity along with rapid development, and accessibility to genetic analysis make the zebrafish retina an excellent model system for studies of neurogenesis in the vertebrate CNS. Several genetic screens have led to the isolation of an impressive collection of mutants affecting the retina and the retinotectal projections in zebrafish. A variety of techniques and markers are available to study the isolated mutants. These include several antigen- and transcript-detection methods, retrograde and anterograde labeling of neurons, blastomere transplantations, H3 labeling, and others. As past genetic screens have achieved a rather low level of saturation, the current collection of mutants can only grow in the future. Morphological and behavioral criteria have been successfully applied in zebrafish to search for defects in spinal development. In future genetic screens, progressively more sophisticated screening approaches will make it possible to detect very subtle changes in the retinal development. The remarkable evolutionary conservation of the vertebrate eye provides the basis for using the zebrafish as a model system for the detection and analysis of genetic defects potentially related to human eye disorders. Some of the genetic defects of the zebrafish retina indeed resemble human retinopathies. As the genetic analysis of the vertebrate visual system is far from being complete and new techniques are being introduced at a rapid pace, the zebrafish embryo will become increasingly useful as a model for studies of the vertebrate retina.
Establishing a network of blood vessels has been, for more reasons than one, an exciting field of research. In Development, Isogai et al. now describe in unprecedented resolution the dynamic process of angiogenesis in the trunk of the zebrafish embryo.
Chemical genetics is a potentially powerful tool for studying developmental processes in vertebrate systems. We present data showing Xenopus laevis as a model organism in which systematic chemical genetic screens can be carried out. Previous forward chemical genetic screens, including those with developing zebrafish embryos, have demonstrated the nature and value of biological information gained with this approach. We show how amenable Xenopus is to chemical genetics by investigating a series of compounds either with known biochemical effects, or previously identified to give developmental phenotypes, on a range of biological functions, including the development of pigmentation, the heart and the central nervous system in zebrafish. We have found that the compounds give comparable phenotypes when applied to developing Xenopus embryos. We have also studied the penetrance and expressivity of these chemical genetic phenotypes in relation to genetic variation and the developmental window during which the compound is present. Finally, we assess the feasibility and the potential throughput of a screen in this vertebrate species.
Many proteins are modified by conjugation with Sumo, a gene-encoded, ubiquitin-related peptide, which is transferred to its target proteins via an enzymatic cascade. A central component of this cascade is the E2-conjugating enzyme Ubc9, which is highly conserved across species. Loss-of-function studies in yeast, nematode, fruit fly, and mouse blastocystes point to multiple roles of Ubc9 during cell cycle regulation, maintenance of nuclear architecture, chromosome segregation, and viability. Here we show that in zebrafish embryos, reduction of Ubc9 activity by expression of a dominant negative version causes widespread apoptosis, similar to the effect described in Ubc9-deficient mice. However, antisense-based knock down of zygotic ubc9 leads to much more specific defects in late proliferating tissues, such as cranial cartilage and eyes. Affected cartilaginous elements are of relatively normal size and shape, but consist of fewer and larger cells. Stainings with mitotic markers and 5-Bromo-2'-deoxyuridine incorporation studies indicate that fewer chondrocyte precursors are in mitosis, whereas the proportion of cells in S-phase is unaltered. Consistently, FACS analyses reveal an increase in the number of cells with a DNA content of 4n or even 8n. Our data indicate an in vivo requirement of Ubc9 for G2/M transition and/or progression through mitosis during vertebrate organogenesis. Failed mitosis in the absence of Ubc9 is not necessarily coupled with cell death. Rather, cells can continue to replicate their DNA, grow to a larger size, and finish their normal developmental program.
The goal of this study was to determine the pattern of gamma-aminobutyric acid (GABA) expression in the retina and optic nerve of the zebrafish (Brachydanio rerio) during embryonic development. Zebrafish embryos were fixed at intervals between 1 and 4 days postfertilization, and semithin plastic sections were prepared for postembedding immunocytochemistry with antisera against GABA. Sections were also prepared from several adult zebrafish eyes for comparison. GABA immunoreactivity first appeared in the optic nerve at 2 days postfertilization, and by 2.5 days the inner nuclear layer (INL), inner plexiform layer (IPL), retinal ganglion cell layer, and optic nerve were all positive for GABA. The GABA expression in the retinal ganglion cell layer and optic nerve was transient, however, and these structures were largely unlabeled by 4 days postfertilization. The pattern of GABA immunoreactivity at 4 days resembled that seen in the adult zebrafish: A large population of presumptive amacrine cells was labeled at the base of the INL, and the IPL was positive for GABA, as were occasional cells in the ganglion cell layer. Horizontal cells, particularly at the retinal margins, were also GABA positive beginning at about 3 days postfertilization. The transient expression of GABA in retinal ganglion cells and their axons during the period when synaptic contacts are being established both within the retina and between the retina and central targets suggests that GABA may have a role in the development of this system, in addition to serving as a classical neurotransmitter.
Cadherins are cell surface molecules that mediate cell-cell adhesion through homophilic interactions. Cadherin-2 (also called N-cadherin), a member of classic cadherin subfamily, has been shown to play important roles in development of a variety of tissues and organs, including the nervous system. We recently reported that cadherin-2 was strongly expressed by the majority of cranial ganglia and lateral line system of developing zebrafish. To gain insight into cadherin-2 role in the formation of these structures, we have used several markers to analyze zebrafish embryos injected with a specific cadherin-2 antisense morpholino oligonucleotide (cdh2MO). We find that development of several cranial ganglia, including the trigeminal, facial, and vagal ganglia, and the lateral line ganglia and neuromasts of the cdh2MO-injected embryos are severely disrupted. These phenotypes were confirmed by analyzing a cadherin-2 mutant, glass onion. Our results suggest that cadherin-2 function is crucial for the normal formation of the zebrafish lateral line system and a subset of cranial ganglia.
Convergent extension (CE) cell movements during gastrulation mediate extension of the anterior-posterior body axis of vertebrate embryos. Non-canonical Wnt5 and Wnt11 signalling is essential for normal CE movements in vertebrate gastrulation. Here, we show that morpholino (MO)-mediated double knock-down of the Fyn and Yes tyrosine kinases in zebrafish embryos impaired normal CE cell movements, resembling the silberblick and pipetail mutants, caused by mutations in wnt11 and wnt5, respectively. Co-injection of Fyn/Yes- and Wnt11- or Wnt5-MO was synergistic, but wnt11 or wnt5 RNA did not rescue the Fyn/Yes knockdown or vice versa. Remarkably, active RhoA rescued the Fyn/Yes knockdown as well as the Wnt11 knockdown, indicating that Fyn/Yes and Wnt11 signalling converged on RhoA. Our results show that Fyn and Yes act together with non-canonical Wnt signalling via RhoA in CE cell movements during gastrulation.
MicroRNAs (miRNAs) are 20-23 nucleotide (nt) RNA molecules that regulate gene expression post-transcriptionally. A key step toward understanding the function of the hundreds of miRNAs identified in animals is to determine their expression during development. Here we performed a detailed analysis of conditions for in situ detection of miRNAs in the zebrafish embryo using locked nucleic acid (LNA)-modified DNA probes and report expression patterns for 15 miRNAs in the mouse embryo.