The role of vasculature and blood circulation in zebrafish swimbladder development

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 <it>cloche </it>(<it>clo</it>) mutant and Tnnt2 morphants, in the background of transgenic lines <it>Et(krt4:EGFP)</it>^{<it>sq33-2 </it>}and <it>Et(krt4:EGFP)</it>^{<it>sqet3 </it>}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 <it>hb9</it>, <it>fgf10a</it>, <it>acta2</it>, and <it>anxa5 </it>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.</p> <p>Conclusion</p> <p>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.</p

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

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

Analysis of early neurodevelopment using insertional transgenics

Ph.DDOCTOR OF PHILOSOPH

Transcriptional Complexity and Distinct Expression Patterns of auts2 Paralogs in Danio rerio

Several genes that have been implicated in autism spectrum disorders (ASDs) have multiple transcripts. Therefore, comprehensive transcript annotation is critical for determining the respective gene function. The autism susceptibility candidate 2 (AUTS2) gene is associated with various neurological disorders, including autism and brain malformation. AUTS2 is important for activation of transcription of neural specific genes, neuronal migration, and neurite outgrowth. Here, we present evidence for significant transcriptional complexity in the auts2 gene locus in the zebrafish genome, as well as in genomic loci of auts2 paralogous genes fbrsl1 and fbrs. Several genes that have been implicated in ASDs are large and have multiple transcripts. Neurons are especially enriched with longer transcripts compared to nonneural cell types. The human autism susceptibility candidate 2 (AUTS2) gene is ∼1.2 Mb long and is implicated in a number of neurological disorders including autism, intellectual disability, addiction, and developmental delay. Recent studies show AUTS2 to be important for activation of transcription of neural specific genes, neuronal migration, and neurite outgrowth. However, much remains to be understood regarding the transcriptional complexity and the functional roles of AUTS2 in neurodevelopment. Zebrafish provide an excellent model system for studying both these questions. We undertook genomic identification and characterization of auts2 and its paralogous genes in zebrafish. There are four auts2 family genes in zebrafish: auts2a, auts2b, fbrsl1, and fbrs. The absence of complete annotation of their structures hampers functional studies. We present evidence for transcriptional complexity of these four genes mediated by alternative splicing and alternative promoter usage. Furthermore, the expression of the various paralogs is tightly regulated both spatially and developmentally. Our findings suggest that auts2 paralogs serve distinct functions in the development and functioning of target tissues

Development of Circumventricular Organs in the Mirror of Zebrafish Enhancer-Trap Transgenics

The circumventricular organs (CVOs) are small structures lining the cavities of brain ventricular system. They are associated with the semitransparent regions of the blood-brain barrier (BBB). Hence it is thought that CVOs mediate biochemical signaling and cell exchange between the brain and systemic blood. Their classification is still controversial and development not fully understood largely due to an absence of tissue-specific molecular markers. In a search for molecular determinants of CVOs we studied the green fluorescent protein (GFP) expression pattern in several zebrafish enhancer trap transgenics including Gateways (ET33-E20) that has been instrumental in defining the development of choroid plexus. In Gateways the GFP is expressed in regions of the developing brain outside the choroid plexus, which remain to be characterized. The neuroanatomical and histological analysis suggested that some previously unassigned domains of GFP expression may correspond to at least six other CVOs–the organum vasculosum laminae terminalis (OVLT), subfornical organ (SFO), paraventricular organ (PVO), pineal (epiphysis), area postrema (AP) and median eminence (ME). Two other CVOs, parapineal and subcommissural organ (SCO) were detected in other enhancer-trap transgenics. Hence enhancer-trap transgenic lines could be instrumental for developmental studies of CVOs in zebrafish and understanding of the molecular mechanism of disease such a hydrocephalus in human. Their future analysis may shed light on general and specific molecular mechanisms that regulate development of CVOs

Characterization of SqET33 transgenic line.

<p>(A), Confocal image of 3 dpf larva of SqET33 line, lateral view. Dashed lines depict the position of transverse sections shown in B–D. (B–D), transverse sections, immunofluorescent staining with anti-GFP antibodies. Arrow indicates the elongated central process of the roof plate cell. (E–J), whole-mount immunofluorescent staining with anti-GFP (midline RP cells and lateral dorsal interneurons) and anti-HuC/HuD (lateral dorsal neurons) antibodies, dorsal view of spinal cord. (K–M), immunofluorescent staining with anti-GFP (green) and anti-GFAP (red) antibodies, transverse section of the spinal cord. Abbreviations: cc, central canal; dt, dorsal thalamus; ha, habenula; m, meninx; nc, notochord; p, pallium; po, preoptic area; rp, roof plate.</p