Igor Kondrychyn

Collective cell migration drives morphogenesis of the kidney nephron.

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.

Development of zebrafish swimbladder: The requirement of Hedgehog signaling in specification and organization of the three tissue layers

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.

Genome-wide analysis of Tol2 transposon reintegration in zebrafish

BackgroundTol2, 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.ResultsWe 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.ConclusionOur 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.

In vivo Analysis of Choroid Plexus Morphogenesis in Zebrafish

Background The 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 Findings We 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/Significance This 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.

Zebrafish cardiac enhancer trap lines: New tools for in vivo studies of cardiovascular development and disease

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.

The role of vasculature and blood circulation in zebrafish swimbladder development

BackgroundRecently 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.ResultsTo 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)sq33-2and Et(krt4:EGFP)sqet3which 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.ConclusionOur 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.

Stretching Morphogenesis of the Roof Plate and Formation of the Central Canal

Background Neurulation is driven by apical constriction of actomyosin cytoskeleton resulting in conversion of the primitive lumen into the central canal in a mechanism driven by F-actin constriction, cell overcrowding and buildup of axonal tracts. The roof plate of the neural tube acts as the dorsal morphogenetic center and boundary preventing midline crossing by neural cells and axons. Methodology/Principal Findings The roof plate zebrafish transgenics expressing cytosolic GFP were used to study and describe development of this structure in vivo for a first time ever. The conversion of the primitive lumen into the central canal causes significant morphogenetic changes of neuroepithelial cells in the dorsal neural tube. We demonstrated that the roof plate cells stretch along the D–V axis in parallel with conversion of the primitive lumen into central canal and its ventral displacement. Importantly, the stretching of the roof plate is well-coordinated along the whole spinal cord and the roof plate cells extend 3× in length to cover 2/3 of the neural tube diameter. This process involves the visco-elastic extension of the roof place cytoskeleton and depends on activity of Zic6 and the Rho-associated kinase (Rock). In contrast, stretching of the floor plate is much less extensive. Conclusions/Significance The extension of the roof plate requires its attachment to the apical complex of proteins at the surface of the central canal, which depends on activity of Zic6 and Rock. The D–V extension of the roof plate may change a range and distribution of morphogens it produces. The resistance of the roof plate cytoskeleton attenuates ventral displacement of the central canal in illustration of the novel mechanical role of the roof plate during development of the body axis.

Yolk syncytial layer formation is a failure of cytokinesis mediated by Rock1 function in the early zebrafish embryo

Summary The yolk syncytial layer (YSL) performs multiple critical roles during zebrafish development. However, little is known about the cellular and molecular mechanisms that underlie the formation of this important extraembryonic structure. Here, we demonstrate by timelapse confocal microscopy of a transgenic line expressing membrane-targeted GFP that the YSL forms as a result of the absence of cytokinesis between daughter nuclei at the tenth mitotic division and the regression of pre-existing marginal cell membranes, thus converting the former margin of the blastoderm into a syncytium. We show that disruption of components of the cytoskeleton induces the formation of an expanded YSL, and identify Rock1 as the regulator of cytoskeletal dynamics that lead to YSL formation. Our results suggest that the YSL forms as a result of controlled cytokinesis failure in the marginal blastomeres, and Rock1 function is necessary for this process to occur. Uncovering the cellular and molecular mechanisms underlying zebrafish YSL formation offers significant insight into syncytial development in other tissues as well as in pathological conditions.

Visualizing Compound Transgenic Zebrafish in Development: A Tale of Green Fluorescent Protein and KillerRed

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.

Functional antagonism of voltage-gated K+ channel α-subunits in the developing brain ventricular system

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. Summary: Voltage-gated K+ channels establish a balance between cell proliferation and barrier properties in the neuroepithelium that lines the ventricles during development of the zebrafish brain ventricular system.