14279732
Restricted expression of reggie genes and proteins during early zebrafish development
Reggies are plasma membrane‐associated proteins and characteristic markers of lipid‐raft microdomains. They are highly conserved from flies to humans and have been implicated in axon regeneration and cell process and contact formation, possibly providing functional platforms for cell‐signaling in neurons and other cell types. We analyzed reggie mRNA and protein expression patterns during early zebrafish development. All three zebrafish genes, re‐1a, ‐2a, and ‐2b, span a considerably diverse set of expression patterns, and their proteins are induced maternally, showing ubiquitous expression at early stages. Although re‐2a mRNA can be observed in differentiating neurons in the brain, spinal cord, and neurogenic placodes, re‐2b is transcribed mainly in head mesoderm, in neural crest derivates, and along somite boundaries. re‐1a mRNA is present at high levels in expression domains that overlap with the combined expression pattern of both re‐2 genes except at the somites, where it complements the pattern of re‐2b. Immunostaining on embryos reveals reggie protein localization at the cell membrane, at cell–cell contacts, and along all early axon tracts. The early phase of reggie expression suggests a basic and ubiquitous function during the first stages of embryogenesis and into the gastrula period. Upon segmentation, a second phase of expression shows distinctly localized expression patterns, indicating tissue‐specific roles and an involvement of re‐1a/re‐2a in neural development. J. Comp. Neurol. 482:257–272, 2005. © 2004 Wiley‐Liss, Inc.
1522502
Reggie-1 and reggie-2, two cell surface proteins expressed by retinal ganglion cells during axon regeneration.
Fish--in contrast to mammals--regenerate retinal ganglion cell axons when the optic nerve is severed. Optic nerve injury leads to reexpression of proteins, which typically are first expressed in newly differentiated retinal ganglion cells and axons. Here we identified two new proteins of fish retinal ganglion cells, reggie-1 and reggie-2, with monoclonal antibody M802 and molecular cloning techniques. In normal fish, M802 stained the few retinal axons derived from newborn ganglion cells which in fish are added lifelong to the retinal margin. After optic nerve injury, however, M802 labeled all retinal ganglion cells and retinal axons throughout their path into tectum. Consistent with M802 staining, reggie-1 and reggie-2 mRNAs were present in lesioned retinal ganglion cells, as demonstrated by in situ hybridization, but were not detectable in their normal mature counterparts. In western blots with membrane proteins of the adult goldfish brain, M802 recognizes a 48x10(3) Mr protein band. At the amino acid level, 48x10(3) Mr reggie-1 and reggie-2 are 44% identical, lack transmembrane and membrane anchor domains, but appear membrane associated by ionic interactions. Reggie-1 and reggie-2 are homologous to 35x10(3) Mr ESA (human epidermal surface antigen) but are here identified as neuronal surface proteins, present on newly differentiated ganglion cells at the retinal margin and which are reexpressed in mature ganglion cells upon injury and during axonal regeneration.
14014423
Identification of reggie-1 and reggie-2 as plasmamembrane-associated proteins which cocluster with activated GPI-anchored cell adhesion molecules in non-caveolar micropatches in neurons.
Neurons are believed to possess plasmalemmal microdomains and proteins analogous to the caveolae and caveolin of nonneuronal cells. Caveolae are plasmalemmal invaginations where activated glycosyl-phosphatidylinositol (GPI)-anchored proteins preferentially assemble and where transmembrane signaling may occur. Molecular cloning of rat reggie-1 and -2 (80% identical to goldfish reggie proteins) shows that reggie-2 is practically identical to mouse flotillin-1. Flotillin-1 and epidermal surface antigen (ESA) (flotillin-2) are suggested to represent possible membrane proteins in caveolae. Rat reggie-1 is 99% homologous to ESA in overlapping sequences but has a 49-amino-acid N-terminus not present in ESA. Antibodies (ABs) which recognize reggie-1 or -2 reveal that both proteins cluster at the plasmamembrane and occur in micropatches in neurons [dorsal root ganglia (DRGs), retinal ganglion, and PC-12 cells] and in nonneuronal cells. In neurons, reggie micropatches occur along the axon and in lamellipodia and filopodia of growth cones, but they do not occur in caveolae. By quantitative electronmicroscopic analysis we demonstrate the absence of caveolae in (anti-caveolin negative) neurons and show anti-reggie-1 immunogold-labeled clusters at the plasmamembrane of DRGs. When ABs against the GPI-anchored cell adhesion molecules (CAMs) F3 and Thy-1 are applied to live DRGs, the GPI-linked CAMs sequester into micropatches. Double immunofluorescence shows a colocalization of the CAMs with micropatches of anti-reggie antibodies. Thus, reggie-1 and reggie-2 identify sites where activated GPI-linked CAMs preferentially accumulate and which may represent noncaveolar micropatches (domains).
41092355
Flotillin and Epidermal Surface Antigen Define a New Family of Caveolae-associated Integral Membrane Proteins*
Caveolae are plasmalemmal microdomains that are involved in vesicular trafficking and signal transduction. We have sought to identify novel integral membrane proteins of caveolae. Here we describe the identification and molecular cloning of flotillin. By several independent methods, flotillin behaves as a resident integral membrane protein component of caveolae. Furthermore, we have identified epidermal surface antigen both as a flotillin homologue and as a resident caveolar protein. Significantly, flotillin is a marker for the Triton-insoluble, buoyant membrane fraction in brain, where to date mRNA species for known caveolin gene family members have not been detected.
1892029
Glycosylphosphatidyl inositol-anchored proteins and fyn kinase assemble in noncaveolar plasma membrane microdomains defined by reggie-1 and -2.
Using confocal laser scanning and double immunogold electron microscopy, we demonstrate that reggie-1 and -2 are colocalized in < or =0.1-microm plasma membrane microdomains of neurons and astrocytes. In astrocytes, reggie-1 and -2 do not occur in caveolae but clearly outside these structures. Microscopy and coimmunoprecipitation show that reggie-1 and -2 are associated with fyn kinase and with the glycosylphosphatidyl inositol-anchored proteins Thy-1 and F3 that, when activated by antibody cross-linking, selectively copatch with reggie. Jurkat cells, after cross-linking of Thy-1 or GM1 (with the use of cholera toxin), exhibit substantial colocalization of reggie-1 and -2 with Thy-1, GM1, the T-cell receptor complex and fyn. This, and the accumulation of reggie proteins in detergent-resistant membrane fractions containing F3, Thy-1, and fyn imparts to reggie-1 and -2 properties of raft-associated proteins. It also suggests that reggie-1 and -2 participate in the formation of signal transduction centers. In addition, we find reggie-1 and -2 in endolysosomes. In Jurkat cells, reggie-1 and -2 together with fyn and Thy-1 increase in endolysosomes concurrent with a decrease at the plasma membrane. Thus, reggie-1 and -2 define raft-related microdomain signaling centers in neurons and T cells, and the protein complex involved in signaling becomes subject to degradation.
13737047
Identification, sequence and developmental expression of invertebrate flotillins from Drosophila melanogaster.
Caveolae are vesicular organelles that represent a sub-compartment of the plasma membrane. Caveolins (Cav-1, -2 and -3) and flotillins (FLO-1 and FLO-2 [also known as epidermal surface antigens (ESAs)] are two families of mammalian caveolae-associated integral membrane proteins. Although a caveolin gene family has recently been described in the invertebrate Caenorhabditis elegans, it remains unknown as to whether flotillin homologues exist in invertebrates. Here, we report the identification, cDNA sequence and embryonic expression pattern of the first invertebrate flotillin, i.e. flotillin from Drosophila melanogaster (FLODm). FLODm is most closely related to mammalian flotillin-1. Remarkably, the invertebrate FLODm protein behaves like mammalian flotillins and is targeted to the caveolae-enriched membrane fraction after transient expression in mammalian cells. Localization of the FLODm message in D. melanogaster embryos reveals that expression of FLODm is confined primarily to the developing nervous system. This is consistent with our previous observation that mammalian flotillin-1 mRNA and protein is expressed abundantly in brain tissue. Interestingly, the FLODm gene is localized to chromosomal region 52 B1-B2. In addition, we find that at least two flotillin-related genes are expressed in D. melanogaster. Our current results provide a starting point and systematic basis for dissecting the role of flotillin in caveolae and neuronal development using Drosophila as a genetic system.
8824524
Stomatin, flotillin-1, and flotillin-2 are major integral proteins of erythrocyte lipid rafts.
Lipid rafts are sphingolipid- and cholesterol-rich membrane microdomains that are insoluble in nonionic detergents, have a low buoyant density, and preferentially contain lipid-modified proteins, like glycosyl phosphatidylinositol (GPI)-anchored proteins. The lipid rafts were isolated from human erythrocytes and major protein components were identified. Apart from the GPI-anchored proteins, the most abundant integral proteins were found to be the distantly related membrane proteins stomatin (band 7.2b), flotillin-1, and flotillin-2. Flotillins, already described as lipid raft components in neurons and caveolae-associated proteins in A498 kidney cells, have not been recognized as red cell components yet. In addition, it was shown that the major cytoskeletal proteins, spectrin, actin, band 4.1, and band 4.2, are partly associated with the lipid rafts. Stomatin and the flotillins are present as independently organized high-order oligomers, suggesting that these complexes act as separate scaffolding components at the cytoplasmic face of erythrocyte lipid rafts.
17678861
The lipid raft microdomain-associated protein reggie-1/flotillin-2 is expressed in human B cells and localized at the plasma membrane and centrosome in PBMCs.
Reggie-1/flotillin-2 is a plasma membrane-associated cytoplasmic protein, which defines non-caveolar raft microdomains. Reggie-1/flotillin-2 is enriched in detergent insoluble (TX100) membrane fractions (DIG), co-localizes with activated GPI-linked proteins and the fyn-kinase in neurons and T cells, and thus apparently participates in the assembly of protein complexes essential for signal transduction. In T cells activated by crosslinking the GPI-linked protein Thy-1 or by crosslinking the ganglioside GM1, reggie-1/flotillin-2 co-localizes with the T cell receptor. To determine whether reggie-1/flotillin-2 is also expressed in B cells, primary B cells from human blood and cell lines representing the developmental stages of pro, pre, mature and plasma B cells were analyzed by Western blotting, RT-PCR and immunofluorescence. Here, we show that reggie-1/flotillin-2 is expressed throughout B cell development, as well as in primary B cells, purified by cell sorting. On non-activated mature B cell Raji cell line we found reggie-1/flotillin-2 are exclusively in the detergent (TX100) insoluble membrane fractions that are staining positive for the raft marker GM1. Immunofluorescence microscopy showed that reggie-1/flotillin-2 is localized at the plasma membrane and marks intracellular spots in PBMCs. Confocal co-localization studies showed that reggie-1/flotillin-2 is associated with the plasma membrane, and the centrosomes (microtubule organizing centers) in these PBMCs. Comparison of reggie-1/flotillin-2 cDNA sequences with the genomic sequence database allowed us to determine the exon/intron structures in mouse and human. The gene organizations are highly conserved suggesting an important function of reggie-1/flotillin-2. Since reggie/flotillin proteins co-cluster with the T cell receptor and fyn kinases upon T cell stimulation, our findings of reggie-1/flotillin-2 in B cells suggest a similar role in B cell function.
17349429
Asymmetric localization of flotillins/reggies in preassembled platforms confers inherent polarity to hematopoietic cells
Hematopoietic cells have long been defined as round, nonpolar cells that show uniform distribution of cell surface-associated molecules. However, recent analyses of the immunological synapse and the importance of lipid microdomains in signaling have shed new light on the aspect of lymphocyte polarization during the activation processes, but none of the molecules implicated so far in either the activation process or the microdomain residency are known to have a preferential localization in nonactivated cells. Chemical crosslinking and fluorescence resonance energy transfer methods have allowed the visualization of certain glycosylphosphatidylinositol-anchored proteins in lipid rafts but so far no microdomain resident protein has been shown to exist as visible stable platforms in the membrane. We report here that two lipid microdomain resident proteins, flotillins/reggies, form preassembled platforms in hematopoietic cells. These platforms recruit signaling molecules upon activation through lipid rafts. The preassembled platforms significantly differ from the canonical cholesterol-dependent “lipid rafts,” as they are resistant to cholesterol-disrupting agents. Most evidence for the functional relevance of microdomains in living cells remains indirect. Using laser scanning confocal microscopy, we show that these proteins exist as stable, microscopically patent domains localizing asymmetrically to one pole of the cell. We present evidence that the asymmetric concentration of these microdomain resident proteins is built up during cytokinesis.
6359957
Evolution of Duplicated reggie Genes in Zebrafish and Goldfish
Invertebrates, tetrapod vertebrates, and fish might be expected to differ in their number of gene copies, possibly due the occurrence of genome duplication events during animal evolution. Reggie (flotillin) genes code for membrane-associated proteins involved in growth signaling in developing and regenerating axons. Until now, there appeared to be only two reggie genes in fruitflies, mammals, and fish. The aim of this research was to search for additional copies of reggie genes in fishes, since a genome duplication might have increased the gene copy number in this group. We report the presence of up to four distinct reggie genes (two reggie-1 and two reggie-2 genes) in the genomes of zebrafish and goldfish. Phylogenetic analyses show that the zebrafish and goldfish sequence pairs are orthologous, and that the additional copies could have arisen through a genome duplication in a common ancestor of bony fish. The presence of novel reggie mRNAs in fish embryos indicates that the newly discovered gene copies are transcribed and possibly expressed in the developing and regenerating nervous system. The intron/exon boundaries of the new fish genes characterized here correspond with those of human genes, both in location and phase. An evolutionary scenario for the evolution of reggie intron-exon structure, where loss of introns appears to be a distinctive trait in invertebrate reggie genes, is presented.
29135902
Flotillin-1/Reggie-2 Traffics to Surface Raft Domains via a Novel Golgi-independent Pathway
Flotillins are lipid raft-associated proteins, which have been implicated in neuronal regeneration and insulin signaling. We now show that newly synthesized flotillin-1 reaches the plasma membrane via a Sar1-independent and brefeldin A-resistant targeting pathway. Consistent with post-translational membrane association of flotillin, protease sensitivity experiments suggest that flotillin-1 is not a transmembrane protein but is associated with the cytoplasmic face of the plasma membrane. The N terminus of flotillin contains a prohibitin-like domain (PHB), which shows homology to a number of proteins associated with raft domains including stomatin, podocin, and prohibitin. We show that the PHB domain of flotillin can efficiently target a heterologous protein, green fluorescent protein, to the plasma membrane. Another PHB-containing protein, stomatin, traffics to the plasma membrane via the conventional secretory pathway. Plasma membrane association of both full-length flotillin and the green fluorescent protein-tagged PHB domain of flotillin is dependent on palmitoylation and requires a conserved cysteine residue, Cys-34, in the PHB domain. The results identify a novel targeting mechanism for plasma membrane association of flotillin-1 involving a Golgi-independent trafficking pathway, the PHB domain, and palmitoylation.
11267242
Membrane and raft association of reggie-1/flotillin-2: role of myristoylation, palmitoylation and oligomerization and induction of filopodia by overexpression.
The reggie protein family consists of two proteins, reggie-1 and -2, also called flotillins, which are highly ubiquitous and evolutionarily conserved. Both reggies have been shown to be associated with membrane rafts and are involved in various cellular processes such as T-cell activation, phagocytosis and insulin signalling. However, the exact molecular function of these proteins remains to be determined. In addition, the mechanism of membrane association of reggie-1, which does not contain any transmembrane domain, is not known. In this study, we have produced a fusion protein of reggie-1 with enhanced green fluorescent protein and generated targeted substitutions for the inactivation of putative palmitoylation and myristoylation sites. We were able to show that reggie-1 is myristoylated and multiply palmitoylated and that lipid modifications are necessary for membrane association of reggie-1. Overexpression of reggie-1 resulted in the induction of numerous filopodia-like protrusions in various cell lines, suggesting a role for reggie-1 as a signalling protein in actin-dependent processes.
14547234
Identification of teleost Thy-1 and association with the microdomain/lipid raft reggie proteins in regenerating CNS axons
During regeneration, retinal ganglion cell axons in fish upregulate a cell surface protein that is recognized by the monoclonal antibody (mAB) M802. M802 antigen appeared to be linked to the intracellular, membrane-associated lipid raft/microdomain proteins reggie-1 and reggie-2 that were previously shown to be reexpressed in axon-regenerating neurons [Development 124 (1997), 577]. Here, we report the isolation of the M802 antigen and its identification as the teleost homolog of mammalian Thy-1. Fish Thy-1 is detected in the same detergent-insoluble lipid raft fractions from a fibroblast cell line and from axon regenerating retinae as reggie-1 and 2. Importantly, mAB M802 coimmunoprecipitates reggie-1 and 2 from this lipid raft fraction, implying that fish Thy-1 and reggies interact. This correlates with their colocalization in growing cell processes after M802 antigen/Thy-1 activation with mAB M802. These findings suggest a role of clustered M802 antigen/Thy-1 in reggie raft microdomains for cell growth and axon regeneration.
4334519
CAP defines a second signalling pathway required for insulin-stimulated glucose transport
Insulin stimulates the transport of glucose into fat and muscle cells. Although the precise molecular mechanisms involved in this process remain uncertain, insulin initiates its actions by binding to its tyrosine kinase receptor, leading to the phosphorylation of intracellular substrates. One such substrate is the Cbl protooncogene product. Cbl is recruited to the insulin receptor by interaction with the adapter protein CAP, through one of three adjacent SH3 domains in the carboxy terminus of CAP. Upon phosphorylation of Cbl, the CAP–Cbl complex dissociates from the insulin receptor and moves to a caveolin-enriched, triton-insoluble membrane fraction. Here, to identify a molecular mechanism underlying this subcellular redistribution, we screened a yeast two-hybrid library using the amino-terminal region of CAP and identified the caveolar protein flotillin. Flotillin forms a ternary complex with CAP and Cbl, directing the localization of the CAP–Cbl complex to a lipid raft subdomain of the plasma membrane. Expression of the N-terminal domain of CAP in 3T3-L1 adipocytes blocks the stimulation of glucose transport by insulin, without affecting signalling events that depend on phosphatidylinositol-3-OH kinase. Thus, localization of the Cbl–CAP complex to lipid rafts generates a pathway that is crucial in the regulation of glucose uptake.
36986575
The sorbin homology domain: A motif for the targeting of proteins to lipid rafts
On phosphorylation of Cbl, the c-Cbl-associated protein (CAP)/Cbl complex dissociates from the insulin receptor and translocates to a lipid raft membrane fraction to form a ternary complex with flotillin. Deletion analyses of the CAP gene identified a 115-aa region responsible for flotillin binding. This region is homologous to the peptide sorbin and is referred to as the sorbin homology (SoHo) domain. This domain is present in two other proteins, vinexin and ArgBP2. Vinexin also interacted with flotillin, and deletion of its SoHo domain similarly blocked flotillin binding. The overexpression of a CAP mutant in which the SoHo domain had been deleted (CAPΔSoHo) prevented the translocation of Cbl to lipid rafts and subsequently blocked the recruitment of CrkII and C3G. Moreover, overexpression of CAPΔSoHo prevented the stimulation of glucose transport and GLUT4 translocation by insulin. These results suggest a mechanism for localization of signaling proteins to the lipid raft that mediates the compartmentalization of crucial signal transduction pathways.
17945414
Vinexin, CAP/ponsin, ArgBP2: a novel adaptor protein family regulating cytoskeletal organization and signal transduction.
Adaptor proteins, composed of two or more protein-protein interacting modules without enzymatic activity, regulate various cellular functions. Vinexin, CAP/ponsin, and ArgBP2 constitute a novel adaptor protein family. They have a novel conserved region homologous to the active peptide sorbin, as well as three SH3 (src homology 3) domains. A number of proteins binding to this adaptor family have been identified. There is accumulating evidence that this protein family regulates cell adhesion, cytoskeletal organization, and growth factor signaling. This review will summarize the structure and the function of proteins in this family.
9378716
Lipid Rafts Mediate Chemotropic Guidance of Nerve Growth Cones
Axon guidance requires signal transduction of extracellular cues through the plasma membrane for directional motility. Here we present evidence that cholesterol- and sphingolipid-enriched membrane microdomains (lipid rafts) mediate specific guidance responses of nerve growth cones. Disruption of lipid rafts by various approaches targeting cholesterol or gangliosides selectively abolished growth cone attraction and repulsion in BDNF and netrin-1 gradients, respectively, without affecting glutamate-induced attraction. Interestingly, local raft disruption on one side of the growth cone in bath BDNF or netrin-1 produced opposite turning responses to that induced by the gradients. Raft manipulation also blocked Semaphorin 3A-induced growth cone repulsion, inhibition, and collapse. Finally, guidance responses appeared to involve raft-dependent activation of p42/p44 MAPK and ligand-induced receptor recruitment to lipid rafts. Together with the observation of asymmetric receptor-raft associations at the growth cone in guidance gradients, our findings indicate that localized signaling through membrane rafts plays a role in mediating guidance actions of extracellular cues on developing axons.
10914632
Lipid rafts and signal transduction
Signal transduction is initiated by complex protein–protein interactions between ligands, receptors and kinases, to name only a few. It is now becoming clear that lipid micro-environments on the cell surface — known as lipid rafts — also take part in this process. Lipid rafts containing a given set of proteins can change their size and composition in response to intra- or extracellular stimuli. This favours specific protein–protein interactions, resulting in the activation of signalling cascades.
39612796
Visualization of Cranial Motor Neurons in Live Transgenic Zebrafish Expressing Green Fluorescent Protein Under the Control of the Islet-1 Promoter/Enhancer
We generated germ line-transmitting transgenic zebrafish that express green fluorescent protein (GFP) in the cranial motor neurons. This was accomplished by fusing GFP sequences to Islet-1promoter/enhancer sequences that were sufficient for neural-specific expression. The expression of GFP by the motor neurons in the transgenic fish enabled visualization of the cell bodies, main axons, and the peripheral branches within the muscles. GFP-labeled motor neurons could be followed at high resolution for at least up to day four, when most larval neural circuits become functional, and larvae begin to swim and capture prey. Using this line, we analyzed axonal outgrowth by the cranial motor neurons. Furthermore, by selective application of DiI to specific GFP-positive nerve branches, we showed that the two clusters of trigeminal motor neurons in rhombomeres 2 and 3 innervate different peripheral targets. This finding suggests that the trigeminal motor neurons in the two clusters adopt distinct fates. In future experiments, this transgenic line of zebrafish will allow for a genetic analysis of cranial motor neuron development.
31261076
The zebrafish midblastula transition.
The zebrafish midblastula transition (MBT) begins at cycle 10. It is characterized by cell cycle lengthening, loss of cell synchrony, activation of transcription and appearance of cell motility. Superceding a 15 minute oscillator that controls the first nine cycles, the nucleocytoplasmic ratio appears to govern the MBT. This timing mechanism operates cell autonomously: clones of labeled cells initiate cell cycle lengthening independently of neighbors but dependent on immediate lineal ancestors. Unequal divisions, when they occur, produce asymmetric cell cycle lengthening based on the volume of each daughter. During the several cycles after the MBT begins, cycle length is correlated with the reciprocal of the blastomere volume, suggesting a continuation of cell cycle regulation by the nucleocytoplasmic ratio during an interval that we term the 'MBT period'.
1398611
The development of a simple scaffold of axon tracts in the brain of the embryonic zebrafish, Brachydanio rerio.
We have examined neuronal differentiation and the formation of axon tracts in the embryonic forebrain and midbrain of the zebrafish, between 1 and 2 days postfertilisation. Axons were visualised with three techniques; immunocytochemistry (using HNK-1 and antiacetylated tubulin antibodies) and horseradish peroxidase (HRP) labelling in whole-mounted brains, and transmission electron microscopy. Differentiation was monitored by histochemical staining for acetylcholinesterase (AChE). These independent methods demonstrated that a simple grid of tracts and commissures forms the initial axon scaffold of the brain. At 1 day, the olfactory nerve, four commissures, their associated tracts and three other non-commissural tracts are present. By 2 days, these tracts and commissures have all greatly enlarged and, in addition, the optic nerve and tract, and three new commissures and their associated tracts have been added. Small applications of HRP at various sites revealed the origins and projections of some of these earliest axons. Retrogradely labelled cell bodies originated from regions that were also positive for AChE activity. At 1 day, HRP-labelled axons were traced: (1) from the olfactory placode through the olfactory nerve to the dorsal telencephalon; (2) from the telencephalon into the tract of the anterior commissure and also to the postoptic region of the diencephalon; (3) from the hindbrain through the ventral midbrain and diencephalon to the postoptic commissure; (4) from the dorsal diencephalon (in or near the epiphysis) to the tract of the postoptic commissure; (5) from ventral and rostral midbrain through the posterior commissure. Three new projections were demonstrated at 2 days: (1) from the retina through the tract of the postoptic commissure to the tectum; (2) from the telencephalon to the contralateral diencephalon; and (3) from the telencephalon to the ventral flexure. These results show that at 1 day, the zebrafish brain is impressively simple, with a few small, well-separated tracts but by 2 days the brain is already considerably more complex. Most of the additional axons added onto pre-existent tracts rather than pioneered new ones supporting the notion that other axons play a crucial role in the guidance of early central nervous system (CNS) axons.
7503746
Axonogenesis and morphogenesis in the embryonic zebrafish brain
We have examined early neuronal differentiation and axonogenesis in the fore- and midbrain of zebrafish embryos to address general issues of early vertebrate brain development. AChE expression and HNK-1 antibody immunoreactivity were used as markers for differentiated neurons and axons, respectively. The pattern of neuronal differentiation followed a stereotyped sequence. AChE-positive cells first appeared between 14 and 16 hr in three small, isolated, bilaterally symmetrical clusters on the surface of the brain. The three clusters--the dorsorostral, ventrorostral, and ventrocaudal clusters--proved to be the progenitors of the telencephalon, ventral diencephalon, and mesencephalic tegmentum, respectively. With further development, more cells were added to these three clusters, and new clusters appeared in the anlage of the epiphysis (18 hr) and in the pituitary and dorsal mesencephalon (by 24 hr). Subsequently, as more neurons differentiated, the gaps of unlabeled cells were reduced; by 48 hr, the cluster boundaries were indistinguishable. Axonogenesis also followed a stereotyped sequence. The first HNK-1-labeled processes arose from the first three clusters of AChE-positive cells and connected the clusters. The earliest axonal growth cones appeared at 16 hr, directed caudally from two to three neurons of the ventrocaudal cluster and pioneering the ventral longitudinal tract. By 18 hr, the tract of the postoptic commissure was initiated by growth cones directed caudally from the ventrorostral cluster toward the ventrocaudal cluster. By 20 hr, axons from the dorsorostral cluster projected ventrally to form the supraoptic tract. The other dorsoventral tracts (the dorsoventral diencephalic tract and the tract of the posterior commissure) became evident between 20 and 24 hr. These observations provide a continuous record of the topological distortions involved in the conversion of the tubular embryonic brain into the contorted adult form. The telencephalon, ventral diencephalon, and hypothalamus originate from the same rostrocaudal level of the neural tube. The pattern of differentiation demonstrated that the early development of the rostral neural tube occurs simultaneously in several independent centers, similar to the overtly segmental development of the hindbrain.
30568117
Regulatory gene expression boundaries demarcate sites of neuronal differentiation in the embryonic zebrafish forebrain
During development of the zebrafish forebrain, a simple scaffold of axon pathways is pioneered by a small number of neurons. We show that boundaries of expression domains of members of the eph, forkhead, pax, and wnt gene families correlate with the positions at which these neurons differentiate and extend axons. Analysis of genetically or experimentally altered forebrains indicates that if a boundary is maintained, there is appropriate neural differentiation with respect to the boundary. Conversely, in the absence of a boundary, there is concomitant disruption of neural patterning. We also show that a strip of cells within the dorsal diencephalon shares features with ventral midline cells. This strip of cells fails to develop in mutant fish in which specification of the ventral CNS is disrupted, suggesting that its development may be regulated by the same inductive pathways that pattern the ventral midline.
20769193
Motoneuron fate specification revealed by patterned LIM homeobox gene expression in embryonic zebrafish.
In zebrafish, individual primary motoneurons can be uniquely identified by their characteristic cell body positions and axonal projection patterns. The fate of individual primary motoneurons remains plastic until just prior to axogenesis when they become committed to particular identities. We find that distinct primary motoneurons express particular combinations of LIM homeobox genes. Expression precedes axogenesis as well as commitment, suggesting that LIM homeobox genes may contribute to the specification of motoneuronal fates. By transplanting them to new spinal cord positions, we demonstrate that primary motoneurons can initiate a new program of LIM homeobox gene expression, as well as the morphological features appropriate for the new position. We conclude that the patterned distribution of different primary motoneuronal types within the zebrafish spinal cord follows the patterned expression of LIM homeobox genes, and that this reflects a highly resolved system of positional information controlling gene transcription.
8253715
Retinotopic organization of the developing retinotectal projection in the zebrafish embryo
Developing retinal axons in the zebrafish embryo were stained with HRP or with the fluorescent dyes dil and diO to study the formation of the retinotectal projection. Retinal axons leave the eye at 34–36 hr postfertilization (PF), invade the tectum at 46–48 hr PF, and innervate the tectal neuropil at 70–72 hr PF. Dorsal and ventral axons occupy separate aspects of the optic nerve and tract and pass into their retinotopically appropriate ventral and dorsal hemitectum, respectively. Nasal and temporal axons are segregated in the nerve, mixed in the tract, and are coextensive over the rostral half of tectum until 56 hr PF. They then segregate again, due to the progression of nasal axons into the open caudal tectum. Thus, at 70–72 hr PF, dorsal and ventral as well as temporal and nasal axons occupy their retinotopically appropriate tectal quadrants. After ablation of the temporal retina prior to the time of axonal outgrowth, the nasal axons bypass the vacant rostral tectum to terminate in the caudal tectal half. Temporal axons in the absence of nasal axons remain restricted to their appropriate rostral tectal half, suggesting that nasal and temporal axons possess a preference for their retinotopically appropriate tectal domains. Measurements of individual terminal arbors and the tectal areas in embryos and in adult zebrafish showed that individual arbors are large with respect to the embryonic tectum but are about 14–15 times smaller than in the adult. However, the proportion of tectum covered by embryonic arbors is about 7 times larger than in the adult, suggesting that a higher precision of the adult projection is achieved as a result of a greater enlargement of the tectum than of the arbors.
1380064
Spatiotemporal pattern of retinal ganglion cell differentiation revealed by the expression of neurolin in embryonic zebrafish.
The expression of neurolin, the fish homologue of the cell adhesion molecule DM-GRASP/BEN/SC-1, is dynamically regulated. Here we demonstrate that the expression of neurolin correlates with early events of retinal ganglion cell (RGC) differentiation in zebrafish embryos. Neurolin mRNA first appears [28 h postfertilization, (PF)] in nasoventral cells, representing the first RGCs, then in dorsal, central (34 to 40 h PF) and temporal RGCs. After differentiation of RGCs in the central portion of the retina, RGCs exhibiting neurolin mRNA form rings. These rings move toward the retinal periphery and encompass older (central) RGCs. Thereafter, such as at 3.5 days PF, neurolin mRNA expressing RGCs are confined to the annular growth zone at the retinal peripheral margin. Two hours after onset of mRNA expression, RGCs acquire antineurolin immunoreactivity on the surface of their somata and on their axons as they extend to the tectum. The mRNA signal in RGCs decreases significantly within 20 h after its appearance, which correlates with the arrival of axons in the tectum. This is followed by weakening of neurolin immunoreactivity on RGCs and axons. This pattern of RGC differentiation in zebrafish revealed by the expression of neurolin is unique among vertebrates. The spatiotemporal expression pattern of neurolin suggests a functional significance of this cell adhesion molecule in RGC recognition and RGC axon growth.
248517
Midline Signals Regulate Retinal Neurogenesis in Zebrafish
In zebrafish, neuronal differentiation progresses across the retina in a pattern that is reminiscent of the neurogenic wave that sweeps across the developing eye in Drosophila. We show that expression of a zebrafish homolog of Drosophila atonal, ath5, sweeps across the eye predicting the wave of neuronal differentiation. By analyzing the regulation of ath5 expression, we have elucidated the mechanisms that regulate initiation and spread of neurogenesis in the retina. ath5 expression is lost in Nodal pathway mutant embryos lacking axial tissues that include the prechordal plate. A likely role for axial tissue is to induce optic stalk cells that subsequently regulate ath5 expression. Our results suggest that a series of inductive events, initiated from the prechordal plate and progressing from the optic stalks, regulates the spread of neuronal differentiation across the zebrafish retina.
42989324
Segmental homologies among reticulospinal neurons in the hindbrain of the zebrafish larva
We have examined the morphology of identified reticulospinal neurons in larval zebrafish by retrogradely labeling them with horseradish peroxidase. We describe the morphology of 27 different types of reticulospinal neurons found in the hindbrain 5 days after fertilization. Nineteen of these types are present as single identified neurons on each side of the brain; the other are present as pairs or small groups of cells.
24161255
Segmental pattern of development of the hindbrain and spinal cord of the zebrafish embryo.
In the ventral hindbrain and spinal cord of zebrafish embryos, the first neurones that can be identified appear as single cells or small clusters of cells, distributed periodically at intervals equal to the length of a somite. In the hindbrain, a series of neuromeres of corresponding length is present, and the earliest neurones are located in the centres of each neuromere. Young neurones within both the hindbrain and spinal cord were identified in live embryos using Nomarski optics, and histochemically by labelling for acetylcholinesterase activity and expression of an antigen recognized by the monoclonal antibody zn-1. Among them are individually identified hindbrain reticulospinal neurones and spinal motoneurones. These observations suggest that early development in these regions of the CNS reflects a common segmental pattern. Subsequently, as more neurones differentiate, the initially similar patterning of the cells in these two regions diverges. A continuous longitudinal column of developing neurones appears in the spinal cord, whereas an alternating series of large and small clusters of neurones is present in the hindbrain.
44973469
Organization of hindbrain segments in the zebrafish embryo
To learn how neural segments are structured in a simple vertebrate, we have characterized the embryonic zebrafish hindbrain with a library of monoclonal antibodies. Two regions repeat in an alternating pattern along a series of seven segments. One, the neuromere centers, contains the first basal plate neurons to develop and the first neuropil. The other region, surrounding the segment boundaries, contains the first neurons to develop in the alar plate. The projection patterns of these neurons differ: those in the segment centers have descending axons, while those in the border regions form ventral commissures. A row of glial fiber bundles forms a curtain-like structure between each center and border region. Specific features of the individual hindbrain segments in the series arise within this general framework. We suggest that a cryptic simplicity underlies the eventual complex structure that develops from this region of the CNS.
1152278
Development of branchiomotor neurons in zebrafish.
The mechanisms underlying neuronal specification and axonogenesis in the vertebrate hindbrain are poorly understood. To address these questions, we have employed anatomical methods and mutational analysis to characterize the branchiomotor neurons in the zebrafish embryo. The zebrafish branchiomotor system is similar to those in the chick and mouse, except for the location of the nVII and nIX branchiomotor neurons. Developmental analyses of genes expressed by branchiomotor neurons suggest that the different location of the nVII neurons in the zebrafish may result from cell migration. To gain insight into the mechanisms underlying the organization and axonogenesis of these neurons, we examined the development of the branchiomotor pathways in neuronal mutants. The valentino b337 mutation blocks the formation of rhombomeres 5 and 6, and severely affects the development of the nVII and nIX motor nuclei. The cyclops b16 mutation deletes ventral midline cells in the neural tube, and leads to a severe disruption of most branchiomotor nuclei and axon pathways. These results demonstrate that rhombomere-specific cues and ventral midline cells play important roles in the development of the branchiomotor pathways.
19786122
Anatomy of the posterior lateral line system in young larvae of the zebrafish
We studied the anatomy of neuromasts, afferent sensory neurons, and efferent neurons of the midbody branch of the posterior lateral line in larvae of the zebrafish (Brachydanio rerio), 5 days after fertilization. This simple sensory system consists often or 11 neuromasts, 15–20 sensory neurons, and about nine efferent neurons. The neuromasts are typical free neuromasts and both afferent and efferent synapses are present on hair cells within them. The sensory neurons project into a single longitudinal column of neuropil in the hindbrain. The sensory terminals appear by light microscopy to contact the dorsolateral dendrite of the ipsilateral Mauthner cell. Three types of efferent neurons are present; two types in the hindbrain and one type in the diencephalon. We provide several lines of evidence that demonstrate that these central neurons are efferent to the lateral line.
1252947
Organization of the lateral line system in embryonic zebrafish
We describe the organization of lateral line nerves and ganglia in the embryonic zebrafish, Danio rerio. Two lateral line nerves are found anterior to the otic vesicle: the anterodorsal nerve innervates neuromasts of the supraorbital, infraorbital, and otic lines, whereas the anteroventral nerve innervates the mandibular and opercular lines. An additional two lateral line nerves are found posterior to the otic vesicle: the middle lateral line nerve innervates the middle line, whereas the posterior nerve innervates the occipital dorsal and posterior trunk lines. Preotic nerves converge on a single entry zone into the central nervous system at the facial motor root (mVII), as do axons of the octaval nerve. Postotic nerves converge to a posterior entry zone at the glossopharyngeal root. Both lateral line ganglia and neuromasts develop on a stereotypical schedule. To examine the segmental relationships among cranial ganglia, neural crest, and hindbrain, lateral line organization was analyzed in valentino mutants, which have disruptions in the development of rhombomeres 5–7 and in the third arch neural crest, and are missing glossopharyngeal motor neurons. The proposed corresponding lateral line nerve for this head segment, the middle lateral line, appears to develop normally. However, the middle and posterior nerves do not form a posterior entry zone in the absence of a glossopharyngeal root in val mutants, but instead course anteriorly to join the preotic nerves. J. Comp. Neurol. 421:189–198, 2000. © 2000 Wiley‐Liss, Inc.
18574330
Identification of spinal neurons in the embryonic and larval zebrafish
Previous studies indicated that the developing fish spinal cord was a simple system containing a small number of distinguishable neuronal cell types (Eisen et al., Nature 320:269–271, '86; Kuwada, Science, 233:740–746, '86). To verify this we have characterized the cellular anatomy of the spinal cord of developing zebrafish in order to determine the number, identities, and organization of the spinal neurons. Spinal neurons were labeled by intracellular dye injections, application of an axonal tracer dye to all or subsets of the axonal tracts, and application of antibodies which recognize embryonic neurons. We found that nine classes of neurons could be identified based on soma size and position, pattern of dendrites, axonal trajectory, and time of axonogenesis. These are two classes of axial motor neurons, which have been previously characterized (Myers, J. Comp. Neurol. 236:555–561, '85), one class of sensory neurons, and six classes of interneurons. One of the interneuron classes could be subclassified as primary and secondary based on criteria similar to those used to classify the axial motor neurons into primary and secondary classes.
20617931
Temporal separation in the specification of primary and secondary motoneurons in zebrafish.
In zebrafish there are two populations of motoneurons, primary and secondary, that are temporally separate in their development. To determine if midline cells play a role in the specification of these neurons, we analyzed both secondary and primary motoneurons in mutants lacking floor plate, notochord, or both floor plate and notochord. Our data show that the specification of secondary motoneurons, those most similar to motoneurons in birds and mammals, depends on the presence of either a differentiated floor plate or notochord. In the absence of both of these structures, secondary motoneurons fail to form. In contrast, primary motoneurons, early developing motoneurons found in fish and amphibians, can develop in the absence of both floor plate and notochord. A spatial correspondence is found between secondary motoneurons and sonic hedgehog-expressing floor plate and notochord. In contrast, primary motoneuronal specification depends on the presence of sonic hedgehog in gastrula axial mesoderm, the tissue that will give rise to the notochord. These results suggest that both primary and secondary motoneurons are specified by signals from midline tissues, but at very different stages of embryonic development.
250082
Hedgehog signaling is required for primary motoneuron induction in zebrafish.
Sonic hedgehog (Shh) is crucial for motoneuron development in chick and mouse. However, zebrafish embryos homozygous for a deletion of the shh locus have normal numbers of motoneurons, raising the possibility that zebrafish motoneurons may be specified differently. Unlike other vertebrates, zebrafish express three hh genes in the embryonic midline: shh, echidna hedgehog (ehh) and tiggywinkle hedgehog (twhh). Therefore, it is possible that Twhh and Ehh are sufficient for motoneuron formation in the absence of Shh. To test this hypothesis we have eliminated, or severely reduced, all three Hh signals using mutations that directly or indirectly reduce Hh signaling and antisense morpholinos. Our analysis shows that Hh signals are required for zebrafish motoneuron induction. However, each of the three zebrafish Hhs is individually dispensable for motoneuron development because the other two can compensate for its loss. Our results also suggest that Twhh and Shh are more important for motoneuron development than Ehh.
8656186
Rapid in vivo labeling of identified zebrafish neurons
We report a simple and rapid method to label individual neurons in live zebrafish embryos and to examine their gene expression profiles. Injection of plasmid DNA encoding an α‐tubulin promotor driving GFP expression results in mosaic embryos containing a limited number of GFP‐positive neurons. Labeled neurons express GFP in their soma and axon, providing the opportunity to analyze pathfinding behaviors of identified neurons in vivo. Moreover, the presence of only a small subset of GFP tagged neurons permits the rapid anatomical identification of these neurons based on soma position and axonal trajectory. Analysis of injected embryos reveals that most, if not all, spinal cord cell types and many other neuronal cell types elsewhere in the nervous system can be GFP tagged. Finally, by combining GFP labeling of individual neurons with fluorescent in situ hybridization, we demonstrate the potential of this method to elucidate gene expression patterns at single cell resolution. genesis 2:196–202, 2002. © 2002 Wiley‐Liss, Inc.
22263580
Segment and cell type lineage restrictions during pharyngeal arch development in the zebrafish embryo.
In zebrafish, the segmental series of pharyngeal arches is formed predominantly by two migratory cell types, neural crest and paraxial mesoderm, which arise in the early embryo. Neural crest cells migrate ventrally out of the neuroepithelium and into the arches to form cartilage, neurons, glia and pigment cells. Surrounding mesoderm generates muscles and endothelia. We labeled individual pharyngeal precursor cells with fluorescent dyes and found that their clonal progeny were confined to single segments and generated single cell types. When a neural crest or mesodermal cell was marked before migration into the pharynx, its progeny dispersed but generally remained confined to a single arch primordium. Such segmental restrictions arose first in the most rostral arches, mandibular and hyoid, and progressed caudally. The phenotypes of progeny generated by single cells were examined in the mandibular arch. Clones derived from premigratory neural crest cells generally did not contribute to more than one cell type. Further, the progenitors of some cell types were spatially separated in the premigratory crest. In particular, neurogenic crest cells were situated further laterally than cells that generate cartilage and connective tissues, while pigment and glial cell progenitors were more evenly distributed. Based on these results we suggest that arch precursors may be specified as to their eventual fates before the major morphogenetic movements that form the arch primordia. Further, cell movements are restricted during segmentation establishing a group of arch precursors as a unit of developmental patterning, as in the fashion of vertebrate rhombomeres or segmental lineage compartments in Drosophila.
31281655
Tenascin-C mRNA is expressed in cranial neural crest cells, in some placodal derivatives, and in discrete domains of the embryonic zebrafish brain.
A partial zebrafish tenascin-C cDNA clone was isolated from an embryonic zebrafish cDNA library on the basis of homology to mouse tenascin-C. The expression pattern in the head of embryonic zebrafish was analyzed by in situ hybridization. Tenascin-C mRNA was detected in neural crest cells during the period of their migration and differentiation. Expression also occurred in differentiating placodal tissues and in mesodermal cells. In the developing brain, tenascin-C mRNA was expressed in specific domains. In the hindbrain the pattern of the domains was dynamic. At 18 to 22 h postfertilization, expression was widespread in rhombomeres 3, 5, and 6, confined to periventricular cells in rhombomere 2, and not detectable in rhombomere 4. At 32 h postfertilization, tenascin-C was expressed at the rhombomere boundaries. In contrast to the hindbrain, the pattern in the forebrain and midbrain did not show any major changes between 22 and 32 h postfertilization. Domains expressing tenascin-C alternated with regions devoid of it. The most anterior domain of expression was observed at the telencephalic-diencephalic border, surrounding the optic recess. A second domain, at the border between the diencephalon and the midbrain, and a third domain, in the caudal midbrain tegmentum, appeared restricted to the basal plate. Additionally, expression of tenascin-C mRNA was detected in the hypothalamus and in the developing epiphysis. These expression patterns suggest that tenascin-C may play a role in neural crest cell migration and during the differentiation of neural crest, placodal, and mesodermal derivatives. In the developing brain, tenascin-C may be involved in the consolidation of different regional identities.
2591788
Musculoskeletal patterning in the pharyngeal segments of the zebrafish embryo.
The head skeleton and muscles of the zebrafish develop in a stereotyped pattern in the embryo, including seven pharyngeal arches and a basicranium underlying the brain and sense organs. To investigate how individual cartilages and muscles are specified and organized within each head segment, we have examined their early differentiation using Alcian labeling of cartilage and expression of several molecular markers of muscle cells. Zebrafish larvae begin feeding by four days after fertilization, but cartilage and muscle precursors develop in the pharyngeal arches up to 2 days earlier. These chondroblasts and myoblasts lie close together within each segment and differentiate in synchrony, perhaps reflecting the interdependent nature of their patterning. Initially, cells within a segment condense and gradually become subdivided into individual dorsal and ventral structures of the differentiated arch. Cartilages or muscles in one segment show similar patterns of condensation and differentiation as their homologues in another, but vary in size and shape in the most anterior (mandibular and hyoid) and posterior (tooth-bearing) arches, possibly as a consequence of changes in the timing of their development. Our results reveal a segmental scaffold of early cartilage and muscle precursors and suggest that interactions between them coordinate their patterning in the embryo. These data provide a descriptive basis for genetic analyses of craniofacial patterning.
45508281
Regulation of flotillin‐1 in the establishment of NIH‐3T3 cell–cell interactions 
1
Flotillin‐1 is a lipid raft‐associated protein involved in neuronal regeneration, insulin signaling in adipocytes, and phagosome maturation in macrophages. We detected abundant flotillin‐1 mRNA expression in confluent cultures of mouse NIH‐3T3 fibroblasts. These cells were used as a model to assess the regulation of flotillin‐1 mRNA and protein expression by cell–cell interactions. The disruption of cell–cell interactions triggered the downregulation of the flotillin‐1 gene. However, the establishment of new cell interactions, and finally confluence, increased both the flotillin‐1 transcript and its protein. Despite flotillin‐1 having been described as an integral protein of the cell membrane, it is also very conspicuous in the hydrophilic fraction (the non‐membrane fraction) of confluent cell extracts. The promoter region of the flotillin‐1 gene was isolated and analyzed by transfection experiments, and the regulatory domains containing motifs for binding transcription factors involved in cell differentiation were identified.
21972578
Activity and distribution of paxillin, focal adhesion kinase, and cadherin indicate cooperative roles during zebrafish morphogenesis.
We investigated the focal adhesion proteins paxillin and Fak, and the cell-cell adhesion protein cadherin in developing zebrafish (Danio rerio) embryos. Cadherins are expressed in presomitic mesoderm where they delineate cells. The initiation of somite formation coincides with an increase in the phosphorylation of Fak, and the accumulation of Fak, phosphorylated Fak, paxillin, and fibronectin at nascent somite boundaries. In the notochord, cadherins are expressed on cells during intercalation, and phosphorylated Fak accumulates in circumferential rings where the notochord cells contact laminin in the perichordal sheath. Subsequently, changes in the orientations of collagen fibers in the sheath suggest that Fak-mediated adhesion allows longitudinal expansion of the notochord, but not lateral expansion, resulting in notochord elongation. Novel observations showed that focal adhesion kinase and paxillin concentrate at sites of cell-cell adhesion in the epithelial enveloping layer and may associate with actin cytoskeleton at epithelial junctions containing cadherins. Fak is phosphorylated at these epithelial junctions but is not phosphorylated on Tyr397, implicating a noncanonical mechanism of regulation. These data suggest that Fak and paxillin may function in the integration of cadherin-based and integrin-based cell adhesion during the morphogenesis of the early zebrafish embryo.
11678131
Rules of order in the retinotectal fascicles of goldfish
Individual fascicles of retinal axons were labeled in the goldfish tectum with horseradish peroxidase (HRP). The contralateral retina was later processed for HRP histochemistry to mark the cells that had axons in the fascicles. Labeled cells were found in a partial half anulus in ventral hemiretina, centered on the optic disk. The distance of the partial anulus from the disk depended on which tectal fascicle had been labeled; the more rostrocentral the fascicle, the smaller was the annular radius. The angular subtense of the partial anulus with respect to the disk depended on where (along its tectal course) the fascicle had been labeled; the more rostral the label site, the longer was the angular subtense. These results were interpreted in the context of retinotectal growth, and it was inferred that the axons followed two rules: (1) grow in along the edge of the tectum and (2) exit and terminate in order, axons from temporal retina first, nasal retina last. These rules would produce a retinotopic projection in peripheral tectum, but they require that some of the terminals already in place must shift as the tectum grows.