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The formation of vascular tubes is driven by extensive changes in endothelial cell (EC) shape. Here, we have identified a role of the actin-binding protein, Marcksl1, in modulating the mechanical properties of EC cortex to regulate cell shape and vessel structure during angiogenesis. Increasing and depleting Marcksl1 expression level in vivo results in an increase and decrease, respectively, in EC size and the diameter of microvessels. Furthermore, endothelial overexpression of Marcksl1 induces ectopic blebbing on both apical and basal membranes, during and after lumen formation, that is suppressed by reduced blood flow. High resolution imaging reveals that Marcksl1 promotes the formation of linear actin bundles and decreases actin density at the EC cortex. Our findings demonstrate that a balanced network of linear and branched actin at the EC cortex is essential in conferring cortical integrity to resist the deforming forces of blood flow to regulate vessel structure., During lumen formation in blood vessels, endothelial cells become exposed to hemodynamic forces that induce membrane blebbing and changes in cell shape. Here, the authors show endothelial cells develop an actin-based protective mechanism in the cell cortex that prevents excessive blebbing to control cell shape and vessel diameter.

Autism spectrum disorders (ASDs) are characterized by abnormal behavioral traits arising from neural circuit dysfunction. While a number of genes have been implicated in ASDs, in most cases, a clear understanding of how mutations in these genes lead to circuit dysfunction and behavioral abnormality is absent. The autism susceptibility candidate 2 (AUTS2) gene is one such gene, associated with ASDs, intellectual disability and a range of other neurodevelopmental conditions. However, the role of AUTS2 in neural development and circuit function is not at all known. Here, we undertook functional analysis of Auts2a, the main homolog of AUTS2 in zebrafish, in the context of the escape behavior. Escape behavior in wild-type zebrafish is critical for survival and is therefore, reliable, rapid, and has well-defined kinematic properties. auts2a mutant zebrafish are viable, have normal gross morphology and can generate escape behavior with normal kinematics. However, the behavior is unreliable and delayed, with high trial-to-trial variability in the latency. Using calcium imaging we probed the activity of Mauthner neurons during otic vesicle (OV) stimulation and observed lower probability of activation and reduced calcium transients in the mutants. With direct activation of Mauthner by antidromic stimulation, the threshold for activation in mutants was higher than that in wild-type, even when inhibition was blocked. Taken together, these results point to reduced excitability of Mauthner neurons in auts2a mutant larvae leading to unreliable escape responses. Our results show a novel role for Auts2a in regulating neural excitability and reliability of behavior.

Autism spectrum disorders (ASDs) are characterized by abnormal behavioral traits arising from neural circuit dysfunction. While a number of genes have been implicated in ASDs, in most cases, a clear understanding of how mutations in these genes lead to circuit dysfunction and behavioral abnormality is absent. Theautism susceptibility candidate 2(AUTS2) gene is one such gene, associated with ASDs, intellectual disability and a range of other neurodevelopmental conditions. Yet, the function of AUTS2 in neural development and circuit function is not at all known. Here, we undertook functional analysis of Auts2a, the main homolog of AUTS2 in zebrafish, in the context of the escape behavior. Escape behavior in wild type zebrafish is critical for survival and is therefore, reliable, rapid, and has well-defined kinematic properties.Auts2a−/−zebrafish are viable, have normal gross morphology and can generate escape behavior with normal kinematics. However, the behavior is unreliable and delayed, with high trial-to-trial variability in the latency. We demonstrate that this is due to the reduced excitability of Mauthner neurons resulting in unreliable firing with stimuli that normally elicit the escape response. Combined with previous studies that show Auts2-regulation of the transcription of ion channel proteins, our results suggest that Auts2 sets the excitability of neurons by activating a set transcriptional program.Significance statementAUTS2 is one among recently identified autism susceptibility candidate genes, whose function in neuronal circuits is unclear. Using zebrafish as a model organism, we probe the function of Auts2a (homolog of mammalian AUTS2) at the cellular, network and behavioral levels. The escape behavior of Auts2a mutant zebrafish is highly variable with normal short latency escapes, long latency escapes and total failures across trials in the same fish. This occurs because neuronal excitability is inappropriately set in the Mauthner neurons of mutants leading to the large trial-to-trial variability in responses. The behavioral variability is fully explained by variability in firing action potentials in the Mauthner neuron, providing an integrative understanding of how behavioral variability arises from mutations at the genetic level.

Autism spectrum disorders (ASDs) are characterized by abnormal behavioral traits arising from neural circuit dysfunction. While a number of genes have been implicated in ASDs, in most cases, a clear understanding of how mutations in these genes lead to circuit dysfunction and behavioral abnormality is absent. The autism susceptibility candidate 2 (AUTS2) gene is one such gene, associated with ASDs, intellectual disability and a range of other neurodevelopmental conditions. Yet, the role of AUTS2 in neural development and circuit function is not at all known. Here, we undertook functional analysis of Auts2a, the main homolog of AUTS2 in zebrafish, in the context of the escape behavior. Escape behavior in wild type zebrafish is critical for survival and is therefore, reliable, rapid, and has well-defined kinematic properties. Auts2a mutant zebrafish are viable, have normal gross morphology and can generate escape behavior with normal kinematics. However, the behavior is unreliable and delayed, with high trial-to-trial variability in the latency. Using calcium imaging we probed the activity of Mauthner neurons during otic vesicle stimulation and observed lower probability of activation and reduced calcium transients in the mutants. With direct activation of Mauthner by antidromic stimulation, the threshold for activation in mutants was higher than that in wild type, even when inhibition was blocked. Taken together, these results point to reduced excitability of Mauthner neurons in auts2a mutant larvae leading to unreliable escape responses. Our results show a novel role for Auts2a in regulating neural excitability and reliability of behavior.Significance statementAUTS2 is one among recently identified autism susceptibility candidate genes, whose function in neuronal circuits is unclear. Using zebrafish as a model organism, we probe the function of Auts2a (homolog of mammalian AUTS2) at the cellular, network and behavioral levels. The escape behavior of Auts2a mutant zebrafish is highly variable with normal short latency escapes, long latency escapes and total failures across trials in the same fish. This occurs because neuronal excitability is inappropriately set in the Mauthner neurons of mutants leading to large trial-to-trial variability in responses. The behavioral variability is fully explained by variability in firing action potentials in the Mauthner neuron, providing an integrative understanding of how behavioral variability arises from mutations at the genetic level.

The emergence of jawed vertebrates (gnathostomes) from jawless vertebrates was accompanied by major morphological and physiological innovations, such as hinged jaws, paired fins and immunoglobulin-based adaptive immunity. Gnathostomes subsequently diverged into two groups, the cartilaginous fishes and the bony vertebrates. Here we report the whole-genome analysis of a cartilaginous fish, the elephant shark (Callorhinchus milii). We find that the C. milii genome is the slowest evolving of all known vertebrates, including the 'living fossil' coelacanth, and features extensive synteny conservation with tetrapod genomes, making it a good model for comparative analyses of gnathostome genomes. Our functional studies suggest that the lack of genes encoding secreted calcium-binding phosphoproteins in cartilaginous fishes explains the absence of bone in their endoskeleton. Furthermore, the adaptive immune system of cartilaginous fishes is unusual: it lacks the canonical CD4 co-receptor and most transcription factors, cytokines and cytokine receptors related to the CD4 lineage, despite the presence of polymorphic major histocompatibility complex class II molecules. It thus presents a new model for understanding the origin of adaptive immunity.

The advent of genomics in the study of developmental mechanisms has brought a trove of information on gene datasets and regulation during development, where the Zic family of zinc-finger proteins plays an important role. Genomic analysis of the modes of action of Zic3 in pluripotent cells demonstrated its requirement for maintenance of stem cells pluripotency upon binding to the proximal regulatory regions (promoters) of genes associated with cell pluripotency (Nanog, Sox2, Oct4, etc.) as well as cell cycle, proliferation, oncogenesis and early embryogenesis. In contrast, during gastrulation and neurulation Zic3 acts by binding the distal regulatory regions (enhancers, etc) associated with control of gene transcription in the Nodal and Wnt signaling pathways, including genes that act to break body symmetry. This illustrates a general role of Zic3 as a transcriptional regulator that acts not only alone, but in many instances in conjunction with other transcription factors. The latter is done by binding to adjacent sites in the context of multi-transcription factor complexes associated with regulatory elements.

The cardiac conduction system (CCS) propagates and coordinates the electrical excitation that originates from the pacemaker cells, throughout the heart, resulting in rhythmic heartbeat. Its defects result in life-threatening arrhythmias and sudden cardiac death. Understanding of the factors involved in the formation and function of the CCS remains incomplete. By transposon assisted transgenesis, we have developed enhancer trap (ET) lines of zebrafish that express fluorescent protein in the pacemaker cells at the sino-atrial node (SAN) and the atrio-ventricular region (AVR), termed CCS transgenics. This expression pattern begins at the stage when the heart undergoes looping morphogenesis at 36 h post fertilization (hpf) and is maintained into adulthood. Using the CCS transgenics, we investigated the effects of perturbation of cardiac function, as simulated by either the absence of endothelium or hemodynamic stimulation, on the cardiac conduction cells, which resulted in abnormal compaction of the SAN. To uncover the identity of the gene represented by the EGFP expression in the CCS transgenics, we mapped the transposon integration sites on the zebrafish genome to positions in close proximity to the gene encoding fibroblast growth homologous factor 2a (fhf2a). Fhf2a is represented by three transcripts, one of which is expressed in the developing heart. These transgenics are useful tools for studies of development of the CCS and cardiac disease.

The brain ventricular system is essential for neurogenesis and brain homeostasis. Its neuroepithelial lining effects these functions, but the underlying molecular pathways remain to be understood. We found that the potassium channels expressed in neuroepithelial cells determine the formation of the ventricular system. The phenotype of a novel zebrafish mutant characterized by denudation of neuroepithelial lining of the ventricular system and hydrocephalus is mechanistically linked to Kcng4b, a homologue of the 'silent' voltage-gated potassium channel α-subunit K

The brain ventricular system is essential for neurogenesis and brain homeostasis. Its neuroepithelial lining effects these functions, but the underlying molecular pathways remain to be understood. We found that the potassium channels expressed in neuroepithelial cells determine the formation of the ventricular system. The phenotype of a novel zebrafish mutant characterized by denudation of neuroepithelial lining of the ventricular system and hydrocephalus is mechanistically linked to Kcng4b, a homologue of the ‘silent’ voltage-gated potassium channel α-subunit Kv6.4. We demonstrated that Kcng4b modulates proliferation of cells lining the ventricular system and maintains their integrity. The gain of Kcng4b function reduces the size of brain ventricles. Electrophysiological studies suggest that Kcng4b mediates its effects via an antagonistic interaction with Kcnb1, the homologue of the electrically active delayed rectifier potassium channel subunit Kv2.1. Mutation of kcnb1 reduces the size of the ventricular system and its gain of function causes hydrocephalus, which is opposite to the function of Kcng4b. This demonstrates the dynamic interplay between potassium channel subunits in the neuroepithelium as a novel and crucial regulator of ventricular development in the vertebrate brain.

Optically translucent embryos of model vertebrates expressing transgenic fluorescent proteins provide a possibility to unravel developmental events, particularly when combined with live imaging. An introduction of transposon-mediated transgenesis resulted in generation of a number of transgenics expressing cytosolic green fluorescent protein in a tissue-specific manner. The recent generation of photodynamic and differentially tagged fluorescent proteins opened a possibility not only to mix-and-match living markers of different color, but also to employ them as powerful experimental tools for studies of cell physiology. Using this approach, transgenic lines expressing membrane-tagged KillerRed (memKR), a genetically encoded photosensitizer, with little or no inducible phototoxicity under confocal imaging were generated. Phototoxicity is only induced by intense green or white light generated by the mercury lamp in a widefield mode. Here, we discuss new ideas born from experimentation using the zebrafish Tol2 transposon-mediated enhancer trap transgenic lines expressing memKR. Because of accumulation on the cell membrane, memKR reveals fine details of cellular morphology. In combination with cytosolic green fluorescent protein, the multicolor in vivo imaging of memKR transgenics reveals complex developmental processes and provides a possibility to manipulate them by regulated generation of reactive oxygen species.

Venkatesh; Byrappa et al.-- This work is licensed under a Creative Commons Attribution- NonCommercial-Share Alike 3.0 Unported licence., The emergence of jawed vertebrates (gnathostomes) from jawless vertebrates was accompanied by major morphological and physiological innovations, such as hinged jaws, paired fins and immunoglobulin-based adaptive immunity. Gnathostomes subsequently diverged into two groups, the cartilaginous fishes and the bony vertebrates. Here we report the whole-genome analysis of a cartilaginous fish, the elephant shark (Callorhinchus milii). We find that the C. milii genome is the slowest evolving of all known vertebrates, including the 'living fossil' coelacanth, and features extensive synteny conservation with tetrapod genomes, making it a good model for comparative analyses of gnathostome genomes. Our functional studies suggest that the lack of genes encoding secreted calcium-binding phosphoproteins in cartilaginous fishes explains the absence of bone in their endoskeleton. Furthermore, the adaptive immune system of cartilaginous fishes is unusual: it lacks the canonical CD4 co-receptor and most transcription factors, cytokines and cytokine receptors related to the CD4 lineage, despite the presence of polymorphic major histocompatibility complex class II molecules. It thus presents a new model for understanding the origin of adaptive immunity. © 2014 Macmillan Publishers Limited.

Our first Zebrafish Enhancer TRAP lines database (ZETRAP) generated a few years ago was a web-based system informing the scientific community about the developmental, genetic, and genomic aspects of transgenic zebrafish lines expressing the cytosolic version of EGFP. These transgenic lines were obtained in a primary screen using Tol2 transposon-mediated transgenesis. Following that, several hundreds transgenics were generated by a systematic "rejump" of the transposon from the two distinct genomic sites. This collection was expanded further by generation of transgenics expressing the membrane-tethered version of a novel red protein KillerRed. These KR transgenics are useful not only to complement the cytosolic GFP in compound GFP/KR transgenics for improved bioimaging. They also could be used to affect cells physiology by tissue-specific optogenetic generation of reactive oxygen species. We have compiled the genomic data and expression patterns of these novel ET transgenic lines in an updated online database--the Zebrafish Enhancer TRAP lines database version 2.0 (ZETRAP 2.0). This improved and expanded version contains the sequence of regions flanking the insertion sites, links to genes in zebrafish genome, and confocal images of embryos/larvae of these transgenics.

Using the transposon-mediated enhancer trap (ET), we generated 18 cardiac enhancer trap (CET) transgenic zebrafish lines. They exhibit EGFP expression in defined cell types—the endocardium, myocardium, and epicardium—or in anatomical regions of the heart—the atrium, ventricle, valves, or bulbus arteriosus. Most of these expression domains are maintained into adulthood. The genomic locations of the transposon insertions were determined by thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). The expression pattern of EGFP in some CETs is unique and recapitulates expression of genes flanking the transposon insertion site. The CETs enabled us to capture the dynamics of the embryonic heart beating in vivo using fast scanning confocal microscopy coupled with image reconstruction, producing three-dimensional movies in time (4D) illustrating region-specific features of heart contraction. This collection of CET lines represents a toolbox of markers for in vivo studies of heart development, physiology, and drug screening. Developmental Dynamics 239:914–926, 2010. © 2010 Wiley-Liss, Inc.

Background Recently we have performed a detailed analysis of early development of zebrafish swimbladder, a homologous organ of tetrapod lung; however, the events of swimbladder development are still poorly characterized. Many studies have implicated the role of vascular system in development of many organs in vertebrates. As the swimbladder is lined with an intricate network of blood capillaries, it is of interest to investigate the role of the vascular system during early development of swimbladder. Results To investigate the role of endothelial cells (ECs) and blood circulation during development of the swimbladder, phenotypes of swimbladder were analysed at three different stages (~2, 3 and 5 dpf [day postfertilization]) in cloche (clo) mutant and Tnnt2 morphants, in the background of transgenic lines Et(krt4:EGFP) sq33-2 and Et(krt4:EGFP) sqet3 which express EGFP in the swimbladder epithelium and outer mesothelium respectively. Analyses of the three tissue layers of the swimbladder were performed using molecular markers hb9, fgf10a, acta2, and anxa5 to distinguish epithelium, mesenchyme, and outer mesothelium. We showed that the budding stage was independent of ECs and blood flow, while early epithelial growth, mesenchymal organization and its differentiation into smooth muscle, as well as outer mesothelial organization, were dependent on ECs. Blood circulation contributed to later stage of epithelial growth, smooth muscle differentiation, and organization of the outer mesothelium. Inflation of the swimbladder was also affected as a result of absence of ECs and blood flow. Conclusion Our data demonstrated that the vascular system, though not essential in swimbladder budding, plays an important role in the development of the swimbladder starting from the early growth stage, including mesenchyme organization and smooth muscle differentiation, and outer mesothelial organization, which in turn may be essential for the function of the swimbladder as reflected in its eventual inflation.

The swimbladder is a hydrostatic organ in fish postulated as a homolog of the tetrapod lung. While lung development has been well studied, the molecular mechanism of swimbladder development is essentially uncharacterized. In the present study, swimbladder development in zebrafish was analyzed by using several molecular markers: hb9 (epithelium), fgf10a and acta2 (mesenchyme), and anxa5 (mesothelium), as well as in vivo through enhancer trap transgenic lines Et(krt4:EGFP)(sq33-2) and Et(krt4:EGFP)(sqet3) that showed strong EGFP expression in the swimbladder epithelium and outer mesothelium respectively. We defined three phases of swimbladder development: epithelial budding between 36 and 48 hpf, growth with the formation of two additional mesodermal layers up to 4.5 dpf, and inflation of posterior and anterior chambers at 4.5 and 21 dpf respectively. Similar to those in early lung development, conserved expression of Hedgehog (Hh) genes, shha and ihha, in the epithelia, and Hh receptor genes, ptc1 and ptc2, as well as fgf10a in mesenchyme was observed. By analyzing several mutants affecting Hh signaling and Ihha morphants, we demonstrated an essential role of Hh signaling in swimbladder development. Furthermore, time-specific Hh inhibition by cyclopamine revealed different requirements of Hh signaling in the formation and organization of all three tissue layers of swimbladder.

BackgroundThe choroid plexus (ChP), a component of the blood-brain barrier (BBB), produces the cerebrospinal fluid (CSF) and as a result plays a role in (i) protecting and nurturing the brain as well as (ii) in coordinating neuronal migration during neurodevelopment. Until now ChP development was not analyzed in living vertebrates due to technical problems.Methodology/principal findingsWe have analyzed the formation of the fourth ventricle ChP of zebrafish in the GFP-tagged enhancer trap transgenic line SqET33-E20 (Gateways) by a combination of in vivo imaging, histology and mutant analysis. This process includes the formation of the tela choroidea (TC), the recruitment of cells from rhombic lips and, finally, the coalescence of TC resulting in formation of ChP. In Notch-deficient mib mutants the first phase of this process is affected with premature GFP expression, deficient cell recruitment into TC and abnormal patterning of ChP. In Hedgehog-deficient smu mutants the second phase of the ChP morphogenesis lacks cell recruitment and TC cells undergo apoptosis.Conclusions/significanceThis study is the first to demonstrate the formation of ChP in vivo revealing a role of Notch and Hedgehog signalling pathways during different developmental phases of this process.

Nature 505, 174–179 (2014); doi:10.1038/nature12826 The ranges of accession numbers JW861113–JW881738 and KA353634–KA353668 (20,661 in total) cited in this Article include 3,630 sequences that were unrelated and hence removed from the NCBI GenBank database before the analysis of the data and submission of the manuscript to Nature.

Zic3 regulates early embryonic patterning in vertebrates. Loss of Zic3 function is known to disrupt gastrulation, left-right patterning, and neurogenesis. However, molecular events downstream of this transcription factor are poorly characterized. Here we use the zebrafish as a model to study the developmental role of Zic3 in vivo, by applying a combination of two powerful genomics approaches – ChIP-seq and microarray. Besides confirming direct regulation of previously implicated Zic3 targets of the Nodal and canonical Wnt pathways, analysis of gastrula stage embryos uncovered a number of novel candidate target genes, among which were members of the non-canonical Wnt pathway and the neural pre-pattern genes. A similar analysis in zic3-expressing cells obtained by FACS at segmentation stage revealed a dramatic shift in Zic3 binding site locations and identified an entirely distinct set of target genes associated with later developmental functions such as neural development. We demonstrate cis-regulation of several of these target genes by Zic3 using in vivo enhancer assay. Analysis of Zic3 binding sites revealed a distribution biased towards distal intergenic regions, indicative of a long distance regulatory mechanism; some of these binding sites are highly conserved during evolution and act as functional enhancers. This demonstrated that Zic3 regulation of developmental genes is achieved predominantly through long distance regulatory mechanism and revealed that developmental transitions could be accompanied by dramatic changes in regulatory landscape., Author Summary The Zic3 transcription factor regulates early embryonic patterning, and the loss of its function leads to defects in left-right body asymmetry. Previous studies have only identified a small number of Zic3 targets, which renders the molecular mechanism underlying its activity insufficiently understood. Utilizing two genomics technologies, next generation sequencing and microarray, we profile the genome-wide binding sites of Zic3 and identified its target genes in the developing zebrafish embryo. Our results show that Zic3 regulates its target genes predominantly through regulatory elements located far from promoters. Among the targets of Zic3 are the Nodal and Wnt pathways known to regulate gastrulation and left-right body asymmetry, as well as neural pre-pattern genes regulating proliferation of neural progenitors. Using enhancer activity assay, we further show that genomic regions bound by Zic3 function as enhancers. Our study provides a genome-wide view of the regulatory landscape of Zic3 and its changes during vertebrate development.

Tissue organization in epithelial organs is achieved during development by the combined processes of cell differentiation and morphogenetic cell movements. In the kidney, the nephron is the functional organ unit. Each nephron is an epithelial tubule that is subdivided into discrete segments with specific transport functions. Little is known about how nephron segments are defined or how segments acquire their distinctive morphology and cell shape. Using live, in vivo cell imaging of the forming zebrafish pronephric nephron, we found that the migration of fully differentiated epithelial cells accounts for both the final position of nephron segment boundaries and the characteristic convolution of the proximal tubule. Pronephric cells maintain adherens junctions and polarized apical brush border membranes while they migrate collectively. Individual tubule cells exhibit basal membrane protrusions in the direction of movement and appear to establish transient, phosphorylated Focal Adhesion Kinase–positive adhesions to the basement membrane. Cell migration continued in the presence of camptothecin, indicating that cell division does not drive migration. Lengthening of the nephron was, however, accompanied by an increase in tubule cell number, specifically in the most distal, ret1-positive nephron segment. The initiation of cell migration coincided with the onset of fluid flow in the pronephros. Complete blockade of pronephric fluid flow prevented cell migration and proximal nephron convolution. Selective blockade of proximal, filtration-driven fluid flow shifted the position of tubule convolution distally and revealed a role for cilia-driven fluid flow in persistent migration of distal nephron cells. We conclude that nephron morphogenesis is driven by fluid flow–dependent, collective epithelial cell migration within the confines of the tubule basement membrane. Our results establish intimate links between nephron function, fluid flow, and morphogenesis., Author Summary The kidney's job is to maintain blood ion and metabolite concentrations in a narrow range that supports the function of all other organs. Blood is filtered and essential solutes are recovered in a structure called the nephron. Human kidneys have one million nephrons, while simpler kidneys like the zebrafish larval kidney have only two. Nephrons are segmented epithelial tubules; each segment takes on a particular shape (such as convoluted, straight, or U-shaped) and plays a specific role in recovering filtered solutes. How the nephron is proportioned into segments and how some tubule segments become convoluted is not known. This work takes advantage of the simple zebrafish kidney to image living cells during nephron formation. Unexpectedly, we found that nephron cells are actively migrating “upstream” toward the filtering end of the nephron. The cells remain connected to each other and migrate as an intact tube. This is similar to a process called “collective cell migration.” We find that collective cell migration establishes the final position of nephron segment boundaries and drives convolution of the tubule. We also find that cell migration is dependent on fluid flow in the tubules, supporting the idea that organ function is important in defining its final form., Epithelial cell shape, tubule convolution, and segment boundary position along the kidney nephron unexpectedly involve the migration of fully differentiated epithelial cells against the flow of lumenal fluid.

Background Tol2, a member of the hAT family of transposons, has become a useful tool for genetic manipulation of model animals, but information about its interactions with vertebrate genomes is still limited. Furthermore, published reports on Tol2 have mainly been based on random integration of the transposon system after co-injection of a plasmid DNA harboring the transposon and a transposase mRNA. It is important to understand how Tol2 would behave upon activation after integration into the genome. Results We performed a large-scale enhancer trap (ET) screen and generated 338 insertions of the Tol2 transposon-based ET cassette into the zebrafish genome. These insertions were generated by remobilizing the transposon from two different donor sites in two transgenic lines. We found that 39% of Tol2 insertions occurred in transcription units, mostly into introns. Analysis of the transposon target sites revealed no strict specificity at the DNA sequence level. However, Tol2 was prone to target AT-rich regions with weak palindromic consensus sequences centered at the insertion site. Conclusion Our systematic analysis of sequential remobilizations of the Tol2 transposon from two independent sites within a vertebrate genome has revealed properties such as a tendency to integrate into transcription units and into AT-rich palindrome-like sequences. This information will influence the development of various applications involving DNA transposons and Tol2 in particular.

Materials and methods We generated a range of random transgenics by means of enhancer trapping using the Tol2 transposon that carries the EGFP gene controlled by a partial keratin8 promoter. It inserts randomly and reveals activity of enhancers in tissue-specific manner in stable transgenic lines. (Parinov et al., 2004; Choo et al.2006). One of these lines, ET33 expressed GFP in the roof plate, dorsal interneurons and meninx. The injection of the transposase mRNA into ET33 embryos resulted in transposition of Tol2 in germ cells and many novel transgenic lines including ET33E20 ("Gateways") were generated. Gateways embryos demonstrate the GFP expression pattern mainly restricted to the brain and spinal cord, similar to that of several genes located close to the insertion site that we cloned in an attempt to define which genes expression is recapitulated by GFP expression in Gateways. Interestingly, the early GFP expression is localized to sites, where several cranial blood vessels develop later on. GFP also appears in the roof plate and in groups of cells at the midline in contact with brain ventricles, including the choroid plexus. Using the enhancer trap lines we studied the morphogenesis of the choroid plexus of IV ventricle using a combination of histology, immunohistochemistry and in vivo analysis during normal development.