Zebrafish Early Macrophages Colonize Cephalic Mesenchyme and Developing Brain, Retina, and Epidermis through a M-CSF Receptor-Dependent Invasive Process

AbstractThe origin of resident (noninflammatory) macrophages in vertebrate tissues is still poorly understood. In the zebrafish embryo, we recently described a specific lineage of early macrophages that differentiate in the yolk sac before the onset of blood circulation. We now show that these early macrophages spread in the whole cephalic mesenchyme, and from there invade epithelial tissues: epidermis, retina, and brain—especially the optic tectum. In the panther mutant, which lacks a functional fms (M-CSF receptor) gene, early macrophages differentiate and behave apparently normally in the yolk sac, but then fail to invade embryonic tissues. Our video recordings then document for the first time the behavior of macrophages in the invaded tissues, revealing the striking propensity of early macrophages in epidermis and brain to wander restlessly among epithelial cells. This unexpected behavior suggests that tissue macrophages may be constantly “patrolling” for immune and possibly also developmental and trophic surveillance. At 60 h post-fertilization, all macrophages in the brain and retina undergo a specific phenotypic transformation, into “early (amoeboid) microglia”: they become more highly endocytic, they down-regulate the L-plastin gene, and abruptly start expressing high levels of apolipoprotein E, a well-known neurotrophic lipid carrier

The Expression Pattern of the Mouse Receptor Tyrosine Kinase GeneMDK1Is Conserved through Evolution and RequiresHoxa-2for Rhombomere-Specific Expression in Mouse Embryos

AbstractSegmentation of the hindbrain has been conserved throughout the vertebrate species and results in the transient formation of rhombomeres, which are lineage-restricted compartments. Studies on the molecular mechanisms underlying the segmentation process have revealed that rhombomeric boundaries coincide with the expression limits of several evolutionary conserved genes such as the zinc-finger transcription factorKrox-20and homeobox genes which are expressed in a specific spatial and temporal order and have been shown to be important regulators of segmental identity. In addition toKrox-20and Hox genes, several members of the Eph subfamily of receptor protein tyrosine kinase (RTK) genes are also expressed in a segment-restricted manner in the hindbrain, suggesting that these receptors may act in concert with Hox genes to establish regional identity. In the cascade of regulatory interactions leading to segmental identity,Krox-20appears to act “upstream” of Hox genes, but the identity of the “downstream” effectors has not yet been identified. We report here the isolation of the zebrafish orthologue of the mouse RTK geneMDK1which belongs to the Eph receptor subfamily and show that the major expression domains of the mouse and the zebrafish genes have been conserved through evolution. Since the coincident spatial and temporal expression ofHoxa-2andMDK1in the mouse hindbrain suggested a possible regulatory link between them, we analyzed the expression of theMDK1inHoxa-2null mutant embryos. A selective lack ofMDK1expression in rhombomere 3 ofHoxa-2mutant hindbrains together with an overall altered expression pattern in the other rhombomeres was observed, thus demonstrating thatMDK1lies downstream ofHoxa-2in the morphogenetic signaling cascade

Abca12-mediated lipid transport and Snap29-dependent trafficking of lamellar granules are crucial for epidermal morphogenesis in a zebrafish model of ichthyosis.

Zebrafish (Danio rerio) can serve as a model system to study heritable skin diseases. The skin is rapidly developed during the first 5-6 days of embryonic growth, accompanied by expression of skin-specific genes. Transmission electron microscopy (TEM) of wild-type zebrafish at day 5 reveals a two-cell-layer epidermis separated from the underlying collagenous stroma by a basement membrane with fully developed hemidesmosomes. Scanning electron microscopy (SEM) reveals an ordered surface contour of keratinocytes with discrete microridges. To gain insight into epidermal morphogenesis, we have employed morpholino-mediated knockdown of the abca12 and snap29 genes, which are crucial for secretion of lipids and intracellular trafficking of lamellar granules, respectively. Morpholinos, when placed on exon-intron junctions, were \u3e90% effective in preventing the corresponding gene expression when injected into one- to four-cell-stage embryos. By day 3, TEM of abca12 morphants showed accumulation of lipid-containing electron-dense lamellar granules, whereas snap29 morphants showed the presence of apparently empty vesicles in the epidermis. Evaluation of epidermal morphogenesis by SEM revealed similar perturbations in both cases in the microridge architecture and the development of spicule-like protrusions on the surface of keratinocytes. These morphological findings are akin to epidermal changes in harlequin ichthyosis and CEDNIK syndrome, autosomal recessive keratinization disorders due to mutations in the ABCA12 and SNAP29 genes, respectively. The results indicate that interference of independent pathways involving lipid transport in the epidermis can result in phenotypically similar perturbations in epidermal morphogenesis, and that these fish mutants can serve as a model to study the pathomechanisms of these keratinization disorders

The abcc6a Gene Expression Is Required for Normal Zebrafish Development

Pseudoxanthoma elasticum (PXE) is caused by mutations in the ABCC6 gene, which encodes a putative efflux transporter, ABCC6. The zebrafish (Danio rerio) has two ABCC6-related sequences. To study the function of abcc6 during zebrafish development, the mRNA expression levels were measured using RT-PCR and in situ hybridization. The abcc6a showed a relatively high level of expression at 5 days post-fertilization (d.p.f.) and the expression was specific to the Kupffer's vesicles. The abcc6b expression was evident at 6hours post-fertilization (h.p.f.) and remained high up to 8 d.p.f., corresponding to embryonic kidney proximal tubules. Morpholinos were designed to both genes to prevent pre-mRNA splicing and block translation. Injection of the abcc6a morpholinos into 1–4 cell zebrafish embryos decreased gene expression by 54–81%, and induced a phenotype, pericardial edema and curled tail associated with death at around 8 d.p.f. Microinjecting zebrafish embryos with full-length mouse Abcc6 mRNA together with the morpholino completely rescued this phenotype. No phenotypic changes were observed when the abcc6b gene morpholino was injected into embryos with knock-down efficiency of 100%. These results suggest that abcc6a is an essential gene for normal zebrafish development and provide insight into the function of ABCC6, the gene mutated in PXE

Genetic Interactions in Zebrafish Midline Development

AbstractMutational analyses have shown that the genesno tail(ntl, Brachyuryhomolog),floating head(flh,aNothomeobox gene), andcyclops(cyc) play direct and essential roles in the development of midline structures in the zebrafish. In bothntlandflhmutants a notochord does not develop, and incycmutants the floor plate is nearly entirely missing. We made double mutants to learn how these genes might interact. Midline development is disrupted to a greater extent incyc;flhdouble mutants than in eithercycorflhsingle mutants; their effects appear additive. Both the notochord and floor plate are completely lacking, and other phenotypic disturbances suggest that midline signaling functions are severely reduced. On the other hand, trunk midline defects inflh;ntldouble mutants are not additive, but are most often similar to those inntlsingle mutants. This finding reveals that loss ofntlfunction can suppress phenotypic defects due to mutation atflh,and we interpret it to mean that the wild-type allele ofntl(ntl+) functions upstream toflhin a regulatory hierarchy. Loss of function ofntlalso strongly suppresses the floor plate deficiency incycmutants, for we found trunk floor plate to be present incyc;ntldouble mutants. From these findings we propose thatntl+plays an early role in cell fate choice at the dorsal midline, mediated by the Ntl protein acting to antagonize floor plate development as well as to promote notochord development

The cdx Genes and Retinoic Acid Control the Positioning and Segmentation of the Zebrafish Pronephros

Kidney function depends on the nephron, which comprises a blood filter, a tubule that is subdivided into functionally distinct segments, and a collecting duct. How these regions arise during development is poorly understood. The zebrafish pronephros consists of two linear nephrons that develop from the intermediate mesoderm along the length of the trunk. Here we show that, contrary to current dogma, these nephrons possess multiple proximal and distal tubule domains that resemble the organization of the mammalian nephron. We examined whether pronephric segmentation is mediated by retinoic acid (RA) and the caudal (cdx) transcription factors, which are known regulators of segmental identity during development. Inhibition of RA signaling resulted in a loss of the proximal segments and an expansion of the distal segments, while exogenous RA treatment induced proximal segment fates at the expense of distal fates. Loss of cdx function caused abrogation of distal segments, a posterior shift in the position of the pronephros, and alterations in the expression boundaries of raldh2 and cyp26a1, which encode enzymes that synthesize and degrade RA, respectively. These results suggest that the cdx genes act to localize the activity of RA along the axis, thereby determining where the pronephros forms. Consistent with this, the pronephric-positioning defect and the loss of distal tubule fate were rescued in embryos doubly-deficient for cdx and RA. These findings reveal a novel link between the RA and cdx pathways and provide a model for how pronephric nephrons are segmented and positioned along the embryonic axis

Unexpected Novel Relational Links Uncovered by Extensive Developmental Profiling of Nuclear Receptor Expression

Nuclear receptors (NRs) are transcription factors that are implicated in several biological processes such as embryonic development, homeostasis, and metabolic diseases. To study the role of NRs in development, it is critically important to know when and where individual genes are expressed. Although systematic expression studies using reverse transcriptase PCR and/or DNA microarrays have been performed in classical model systems such as Drosophila and mouse, no systematic atlas describing NR involvement during embryonic development on a global scale has been assembled. Adopting a systems biology approach, we conducted a systematic analysis of the dynamic spatiotemporal expression of all NR genes as well as their main transcriptional coregulators during zebrafish development (101 genes) using whole-mount in situ hybridization. This extensive dataset establishes overlapping expression patterns among NRs and coregulators, indicating hierarchical transcriptional networks. This complete developmental profiling provides an unprecedented examination of expression of NRs during embryogenesis, uncovering their potential function during central nervous system and retina formation. Moreover, our study reveals that tissue specificity of hormone action is conferred more by the receptors than by their coregulators. Finally, further evolutionary analyses of this global resource led us to propose that neofunctionalization of duplicated genes occurs at the levels of both protein sequence and RNA expression patterns. Altogether, this expression database of NRs provides novel routes for leading investigation into the biological function of each individual NR as well as for the study of their combinatorial regulatory circuitry within the superfamily

Établissement des axes embryonnaires au cours du développement du poisson zèbre

La définition des axes embryonnaires au cours du développement du poisson zèbre s’effectue au stade blastula. L’embryon consiste alors en une couche de cellules recouvrant le vitellus. Les cellules, initialement toutes identiques, vont progressivement acquérir une identité propre en fonction de leurs coordonnées spatiales (dorsoventrale et antéro-postérieure) dans l’embryon. Ces coordonnées sont définies via deux gradients d’activité de molécules sécrétées appartenant à la superfamille des TGFβ : les bone morphogenetic protein (BMP, pour l’axe D/V) et les facteurs activine et nodal (pour l’axe A/P). Ces gradients d’activité sont finement réglés par des facteurs sécrétés agissant comme des inhibiteurs. L’activité des BMP est contrôlée au niveau de son expression via les FGF (FGF8) ainsi que par noggin et chordin qui se fixent sur les BMP et les empêchent de stimuler leur récepteur spécifique. Pour les facteurs activine et nodal, l’activité est réglée par un autre TGFβ (antivin) qui agit comme un inhibiteur compétitif de la fixation de ces ligands sur leur récepteur. Les recherches actuelles et futures visent à examiner les interactions entre ces voies ainsi qu’avec d’autres voies de signalisation impliquées lors de l’embryogenèse précoce : les Wnt et les rétinoïdes

Three Different noggin Genes Antagonize the Activity of Bone Morphogenetic Proteins in the Zebrafish Embryo

International audienceThe dorsoventral polarity of the vertebrate embryo is established through interactions between ventrally expressed bone morphogenetic proteins and their organizer-borne antagonists Noggin, Chordin, and Follistatin. While the opposing interactions between Short Gastrulation/Chordin and Decapentaplegic/BMP4 have been evolutionarily conserved in arthropods and vertebrates, there has been up to now no functional evidence of an implication of Noggin in the early patterning of organisms other than Xenopus. We have studied the contribution of Noggin to the embryonic development of the zebrafish. While single-copy noggin genes have been characterized in several vertebrate species, we report that the zebrafish genome harbors three noggin homologues. Overexpression experiments show that Noggin1, Noggin2, and Noggin3 can antagonize ventralizing BMPs. While all three factors have similar biological activities, their embryonic expression is different. The combined expression of the three genes recapitulates the different aspects of the expression of the single-copy noggin genes of other organisms. This suggests that the three zebrafish noggin genes and the single noggin genes of other vertebrates have evolved from a common ancestor and that subsequent differential loss of tissue-specific elements in the promoters of the different zebrafish genes accounts for their more restricted spatiotemporal expression. Finally we show that noggin1 is expressed in the fish organizer and able to dorsalize the embryo, suggesting its implication in the dorsoventral patterning of the zebrafish