Anti-inflammatory effects of aloe vera on soy meal-induced intestinal inflammation in zebrafish.

Soybean meal is one of the most promising alternatives to replace fishmeal in the aquaculture industry. However, its ingestion triggers an intestinal inflammatory process that compromises fish health and nutrition. Therefore, finding strategies that reduce the deleterious effects of a soy protein-based diet are relevant. In this work we analyzed the effects of an aloe vera (Aloe barbadensis miller, AV) extract on intestinal inflammation and innate immunity of zebrafish by adding it to the water and by supplementing it in a soybean meal-based diet. To search for potential immunomodulatory effects of AV, we tested its effectiveness in two inflammation assays and compared fish fed with either fishmeal or soybean meal-based feed supplemented with AV. Our results show a strong anti-inflammatory effect of AV. Furthermore, while soy-based meal strongly induces the expression of inflammation markers, supplementation with AV reverted this effect. Finally, we show that fish fed with a soy meal diet are highly susceptible to bacterial infection, but that this condition is significantly reduced when the soy meal is supplemented with AV. Our results suggest that AV is a good candidate to be incorporated as an additive in farmed fish diets to facilitate the replacement of fishmeal by soybean meal, maintaining intestinal health.

CNBP mediates neural crest cell expansion by controlling cell proliferation and cell survival during rostral head development.

Striking conservation in various organisms suggests that cellular nucleic acid binding protein (CNBP) plays a fundamental biological role across different species. Recently, it was reported that CNBP is required for forebrain formation during chick and mouse embryogenesis. In this study, we have used the zebrafish model system to expand and contextualize the basic understanding of the molecular mechanisms of CNBP activity during vertebrate head development. We show that zebrafish cnbp is expressed in the anterior CNS in a similar fashion as has been observed in early chick and mouse embryos. Using antisense morpholino oligonucleotide knockdown assays, we show that CNBP depletion causes forebrain truncation while trunk development appears normal. A substantial reduction in cell proliferation and an increase in cell death were observed in the anterior regions of cnbp morphant embryos, mainly within the cnbp expression territory. In situ hybridization assays show that CNBP depletion does not affect CNS patterning while it does cause depletion of neural crest derivatives. Our data suggest an essential role for CNBP in mediating neural crest expansion by controlling proliferation and cell survival rather than via a cell fate switch during rostral head development. This possible role of CNBP may not only explain the craniofacial anomalies observed in zebrafish but also those reported for mice and chicken and, moreover, demonstrates that CNBP plays an essential and conserved role during vertebrate head development.

The DXD motif is required for GM2 synthase activity but is not critical for nucleotide binding.

We tested the importance of the aspartate-any residue-aspartate (DXD) motif for the enzymatic activity and nucleotide binding capacity of the Golgi glycosyltransferase GM2 synthase. We prepared point mutations of the motif, which is found in the sequence 352-VLWVDDDFV, and analyzed cells that stably expressed the mutated proteins. Whereas the folding of the mutated proteins was not seriously disrupted as judged by assembly into homodimers, Golgi localization, and secretion of a soluble form of the enzyme, exchange of the highly conserved aspartic acid residues at position 356 or 358 with alanine or asparagine reduced enzyme activity to background levels. In contrast, the D356E and D357N mutations retained weak activity, while the activity of V352A and W354A mutants was 167% and 24% that of wild-type enzyme, respectively. Despite the major effect of the DXD motif on enzymatic activity, nucleotide binding was not altered in the triple mutant D356N/D357N/D358N as revealed by binding to UDP-beads and labeling with the photoaffinity reagent, P(3)-(4-azidoanilido)uridine 5'-triphosphate (AAUTP). In summary, rather than being critical for nucleotide binding, this motif may function during catalysis in GM2 synthase, as has been proposed elsewhere for the SpsA glycosyltransferase based on its crystal structure.

Mice expressing only monosialoganglioside GM3 exhibit lethal audiogenic seizures.

Gangliosides are a family of glycosphingolipids that contain sialic acid. Although they are abundant on neuronal cell membranes, their precise functions and importance in the central nervous system (CNS) remain largely undefined. We have disrupted the gene encoding GD3 synthase (GD3S), a sialyltransferase expressed in the CNS that is responsible for the synthesis of b-series gangliosides. GD3S-/- mice, even with an absence of b-series gangliosides, appear to undergo normal development and have a normal life span. To further restrict the expression of gangliosides, the GD3S mutant mice were crossbred with mice carrying a disrupted GalNAcT gene encoding beta1,4-N-acetylgalactosaminyltransferase. These double mutant mice expressed GM3 as their major ganglioside. In contrast to the single mutant mice, the double mutants displayed a sudden death phenotype and were extremely susceptible to induction of lethal seizures by sound stimulus. These results demonstrate unequivocally that gangliosides play an essential role in the proper functioning of the CNS.

Disulfide bonds of GM2 synthase homodimers. Antiparallel orientation of the catalytic domains.

GM2 synthase is a homodimer in which the subunits are joined by lumenal domain disulfide bond(s). To define the disulfide bond pattern of this enzyme, we analyzed a soluble form by chemical fragmentation, enzymatic digestion, and mass spectrometry and a full-length form by site-directed mutagenesis. All Cys residues of the lumenal domain of GM2 synthase are disulfide bonded with Cys(429) and Cys(476) forming a disulfide-bonded pair while Cys(80) and Cys(82) are disulfide bonded in combination with Cys(412) and Cys(529). Partial reduction to produce monomers converted Cys(80) and Cys(82) to free thiols while the Cys(429) to Cys(476) disulfide remained intact. CNBr cleavage at amino acid 330 produced a monomer-sized band under nonreducing conditions which was converted upon reduction to a 40-kDa fragment and a 24-kDa myc-positive fragment. Double mutation of Cys(80) and Cys(82) to Ser produced monomers but not dimers. In summary these results demonstrate that Cys(429) and Cys(476) form an intrasubunit disulfide while the intersubunit disulfides formed by both Cys(80) and Cys(82) with Cys(412) and Cys(529) are responsible for formation of the homodimer. This disulfide bond arrangement results in an antiparallel orientation of the catalytic domains of the GM2 synthase homodimer.

Evidence supporting a late Golgi location for lactosylceramide to ganglioside GM3 conversion.

Ganglioside GM2 synthase and other enzymes required for complex ganglioside synthesis were localized recently to the trans Golgi network (TGN). However, there are conflicting reports as to the location of GM3 synthase; originally this enzyme was detected in the early Golgi of rat liver but a recent report localized it to the late Golgi. We have used chimeric forms of ganglioside GM2 synthase to determine if the location of lactosylceramide (LacCer) to GM3 conversion in Chinese hamster ovary (CHO) cells was the early or late Golgi. Our approach tested whether GM3 could be utilized as a substrate by GM2 synthase chimeras which were targeted to compartments earlier than the trans Golgi, i.e., GM3 produced in the cis Golgi should be utilized by GM2 synthase located anywhere in the Golgi whereas GM3 produced in the trans Golgi should only be used by GM2 synthase located in the trans Golgi or TGN. Comparison of cell lines stably expressing these chimeras revealed that the in vivo functional activity of GM2 synthase decreased progressively as the enzyme was targeted to earlier compartments; specifically, the percentage of GM3 converted to GM2 was 83-86% for wild type enzyme, 70% for the medial Golgi targeted enzyme, 13% for the ER and cis Golgi targeted enzyme, and only 1.7% for the ER targeted enzyme. Thus, these data are consistent with a late Golgi location for LacCer to GM3 conversion in these cells.

Chitin oligosaccharides as candidate patterning agents in zebrafish embryogenesis.

In this work we investigate the possible function of N-acetyl-chitooligosaccharides (NACOs) produced during zebrafish (Danio rerio) development. First, we show that NACOs are synthesized in vivo during early embryogenesis in the zebrafish. Second, we demonstrate that injection of a pure bacterial chitinase into one-cell stage embryos elicits developmental defects in which the posterior trunk and tail of developing embryo are severely affected. In addition, an endogenous chitinase activity detected both intra- and extracellularly is described, suggesting that cells may secrete it into the extracellular space. Moreover, this compartmentalization appears to be functionally relevant as inhibition of the extracellular, but not the intracellular, endogenous chitinase activity causes morphological defects similar to those seen in embryos injected with chitinase 63. Finally, analysis of the expression of the zebrafish ZDG42 gene, which has been suggested to be involved in synthesis of NACOs, is described. Transcripts are detected from late blastula stage, during gastrulation, and move as an anterior-posterior wave of expression in adaxial mesoderm during somitogenesis.

Ganglioside expression during differentiation of chick retinal cells in vitro.

The neural retina has been widely used to study the developmental patterns of ganglioside metabolism. Recent findings about in vitro differentiating chick embryo retina cells showed that: a) GD3 and GD1a ganglioside patterns undergo the most dramatic changes; b) when the cells emit neurites, GD3 ganglioside and a group of complex gangliotetraosylgangliosides (GTOG) are transiently coexpressed; c) synchronized developmental phenomena are dissociated by anti-GM1 antibodies; d) GD3 remains as a major ganglioside in differentiated neurons, though it is almost not immunoexpressed; e) GTOG affect antibody binding to GD3; f) the content of gangliosides involved in neural differentiation modifies their immunostain localization on cell membrane; g) after exogenous GTOG uptake, immature neurons mimic GD3 immunofluorescent localization of mature cells; h) a subset of purified retinal ganglion cells express GTOG characteristic of mature neurons.

Reevaluating the effect of Brefeldin A (BFA) on ganglioside synthesis: the location of GM2 synthase cannot be deduced from the inhibition of GM2 synthesis by BFA.

Brefeldin A reversibly disassembles the Golgi complex, causing mixing of the Golgi cisternae with the ER while the trans Golgi network persists as part of a separate endosomal membrane system. Because of this compartmental separation, Brefeldin A treatment has been used to map the sub-Golgi locations of several Golgi enzymes including GM2 synthase. We previously proposed that GM2 synthase might be located in a distal portion of the Golgi complex which in the presence of Brefeldin A would be separated from the substrate ganglioside GM3 present in the mixed ER-Golgi membrane system. In the present study we show using GM2 synthase chimeras that GM2 synthesis was blocked by Brefeldin A when GM2 synthase was distributed throughout all Golgi subcompartments or even when it was restricted to the medial Golgi. Because these findings opposed our speculation regarding a distal location of this enzyme, we sought an alternative explanation for the inhibition of ganglioside synthesis by Brefeldin A. However, Brefeldin A did not degrade GM2 synthase, prevent its homodimerization, or inhibit its in vitro activity. Brefeldin A did result in the conversion of a portion of membrane bound GM2 synthase into a soluble form which has minimal capability to produce GM2 in whole cells. However, this conversion was not sufficient to explain the nearly total loss of GM2 production in intact cells in the presence of Brefeldin A. Nevertheless, the results of this study indicate that Brefeldin A-induced inhibition of ganglioside synthesis cannot be used to deduce the location of GM2 synthase.

not really finished is crucial for development of the zebrafish outer retina and encodes a transcription factor highly homologous to human Nuclear Respiratory Factor-1 and avian Initiation Binding Repressor.

Not really finished (nrf), a larval-lethal mutation in zebrafish generated by retroviral insertion, causes specific retinal defects. Analysis of mutant retinae reveals an extensive loss of photoreceptors and their precursors around the onset of visual function. These neurons undergo apoptosis during differentiation, affecting all classes of photoreceptors, suggesting an essential function of nrf for the development of all types of photoreceptors. In the mutant, some photoreceptors escape cell death, are functional and, as judged by opsin expression, belong to at least three classes of cones and one class of rods. The protein encoded by nrf is a close homologue of human Nuclear Respiratory Factor 1 and avian Initiation Binding Repressor, transcriptional regulators binding the upstream consensus sequence RCGCRYGCGY. At 24 hours of development, prior to neuronal differentiation, nrf is expressed ubiquitously throughout the developing retina and central nervous system. At 48 hours of development, expression of nrf is detected in the ganglion cell layer, in the neurons of the inner nuclear layer, and in the optic nerve and optic tracts, and, at 72 hours of development, is no longer detectable by in situ hybridization. Mutants contain no detectable nrf mRNA and die within 2 weeks postfertilization as larvae with reduced brain size. On the basis of its similarity with NRF-1 and IBR, nrf is likely involved in transcriptional regulation of multiple target genes, including those that encode mitochondrial proteins, growth factor receptors and other transcription factors. This demonstrates the power of insertional mutagenesis as a means for characterizing novel genes necessary for vertebrate retinal development.

Two soluble glycosyltransferases glycosylate less efficiently in vivo than their membrane bound counterparts.

Many Golgi glycosyltransferases are type II membrane proteins which are cleaved to produce soluble forms that are released from cells. Cho and Cummings recently reported that a soluble form of alpha1, 3-galactosyltransferase was comparable to its membrane bound counterpart in its ability to galactosylate newly synthesized glycoproteins (Cho,S.K. and Cummings,R.D. (1997) J. Biol. Chem., 272, 13622-13628). To test the generality of their findings, we compared the activities of the full length and soluble forms of two such glycosyltransferases, ss1,4 N-Acetylgalactosaminyltransferase (GM2/GD2/ GA2 synthase; GalNAcT) and beta galactoside alpha2,6 sialyltransferase (alpha2,6-ST; ST6Gal I), for production of their glycoconjugate products in vivo . Unlike the full length form of GalNAcT which produced ganglioside GM2 in transfected cells, soluble GalNAcT did not produce detectable GM2 in vivo even though it possessed in vitro GalNAcT activity comparable to that of full length GalNAcT. When compared with cells expressing full length alpha2,6-ST, cells expressing a soluble form of alpha2,6-ST contained 3-fold higher alpha2,6-ST mRNA levels and secreted 7-fold greater alpha2,6-ST activity as measured in vitro , but in striking contrast contained 2- to 4-fold less of the alpha2,6-linked sialic acid moiety in cellular glycoproteins in vivo . In summary these results suggest that unlike alpha1,3-galactosyltransferase the soluble forms of these two glycosyltransferases are less efficient at glycosylation of membrane proteins and lipids in vivo than their membrane bound counterparts.

Expression of Rhizobium chitin oligosaccharide fucosyltransferase in zebrafish embryos disrupts normal development.

In this report we present data about the effect of the Rhizobium NodZ enzyme on zebrafish development. We injected zebrafish embryos with a plasmid expressing NodZ protein, and we confirmed that the enzyme is active and has chitin oligosaccharide fucosyltransferase (NodZ) activity in vitro. In addition, the embryos injected with the NodZ-expressing plasmid, but not with a control plasmid, showed malformations or bends in the tail, and in some cases shunted tail structures and fused somites. These results clearly indicate that the likely substrates for this enzyme, chitin oligosaccharides and free N-glycans, have essential functions during early vertebrate embryogenesis.

Relationships among msx gene structure and function in zebrafish and other vertebrates.

The zebrafish genome contains at least five msx homeobox genes, msxA, msxB, msxC, msxD, and the newly isolated msxE. Although these genes share structural features common to all Msx genes, phylogenetic analyses of protein sequences indicate that the msx genes from zebrafish are not orthologous to the Msx1 and Msx2 genes of mammals, birds, and amphibians. The zebrafish msxB and msxC are more closely related to each other and to the mouse Msx3. Similarly, although the combinatorial expression of the zebrafish msx genes in the embryonic dorsal neuroectoderm, visceral arches, fins, and sensory organs suggests functional similarities with the Msx genes of other vertebrates, differences in the expression patterns preclude precise assignment of orthological relationships. Distinct duplication events may have given rise to the msx genes of modern fish and other vertebrate lineages whereas many aspects of msx gene functions during embryonic development have been preserved.

Appearance of beta 1,4 N-acetylgalactosaminyltransferase (glycosphingolipids GA2/GM2/GD2 synthase) in embryonic chicken vitreous humor during development.

PURPOSE: beta 1,4 N-Acetylgalactosaminyltransferase (GalNAcT) is a type II integral membrane protein of the Golgi apparatus that catalyzes the synthesis of the glycosphingolipids GM2, GD2, and GA2. The activity of GalNAcT in chick retinal cells increases 6-fold between embryonic days 7 and 14. Because GalNAcT, like many Golgi glycosyltransferases, is proteolytically cleaved from Golgi membranes to release a soluble form into the culture medium of cells transfected with the cloned human enzyme, we tested whether GalNAcT might be released from embryonic retinal cells into the vitreous humor. METHODS: Samples of vitreous humor and plasma and extracts of retinal cells were assayed for GalNAcT activity. RESULTS: The activity of a soluble form of GalNAcT in embryonic chick vitreous humor was nearly undetectable until embryonic day 10, then increased more than six fold until day 16, and remained at that level until birth. The activity was identified as authentic GalNAcT based on a requirement for Mn++, GSL substrate specificity, and product characterization. GalNAcT activity in embryonic plasma was roughly 10% that of the corresponding vitreous humor, suggesting that the plasma was not the source of the activity in the vitreous. CONCLUSIONS: GalNAcT in embryonic chicken vitreous humor is likely due either to a specific release from neural retinal cells or due to non-specific lysis of these cells during apoptosis associated with the development of the retina. Regardless of the source, GalNAcT in the vitreous humor has the potential to function as a lectin by binding to gangliosides GD3 and GM3 on the surface of retinal cells and, thereby, to influence neuronal development.

Insertional mutagenesis in zebrafish identifies two novel genes, pescadillo and dead eye, essential for embryonic development.

Recently our laboratory described an efficient method for generating retroviral provirus insertions in the zebrafish germ line, and we showed that provirus insertions induce embryonic mutations at a frequency of roughly one mutant per 70 insertions. To date we have isolated four insertional mutants and, using the proviruses as a molecular tag, have cloned the genes disrupted in three of them. The proviruses in all three mutants lie within or just 5' of the first coding exon, point in the opposite transcriptional orientation from the gene, and disrupt transcription. Here we present a molecular characterization of two genes identified by this method and describe the associated mutant phenotypes. The pescadillo (pes) gene is predicted to encode a protein of 582 amino acids with no recognizable functional motifs, which is highly conserved from yeast to humans. pes mRNA is expressed widely and dynamically during the first 3 days of embryogenesis. Prominent sites of expression are the eyes and optic tectum on day 1, the fin buds, liver primordium, and gut on day 2, and the branchial arches on day 3. Beginning at day 3 of embryogenesis, pes mutant embryos exhibit small eyes, a reduced brain and visceral skeleton, shortened fins, and a lack of expansion of the liver and gut, and then die on the sixth day of development. The dead eye (dye) gene encodes a protein of 820 amino acids that is homologous to genes of unknown function in human, mouse, and Xenopus, and that has weak homology with the yeast NIC96 (nucleoporin-interacting component) gene. dye mutants can be recognized on day 2 of embryogenesis by the presence of necrotic cells in the tectum and eyes. dye mutants die on day 5 of development. These results demonstrate the power of insertional mutagenesis in zebrafish for rapidly finding and characterizing novel genes essential for embryonic development. Using our current methodology, we estimate that our laboratory could screen approximately 25,000 insertions in 2-3 years, identifying perhaps 250-350 embryonic lethal genes. Assuming that all genes are accessible to proviral insertion, the wider application of this approach could lead to the rapid identification of the majority of genes that are required for embryonic development of this vertebrate.

Mutations affecting the formation of the notochord in the zebrafish, Danio rerio.

In a large scale screen for mutants with defects in the embryonic development of the zebrafish we identified mutations in four genes,floating head (flh), momo (mom), no tail (ntl), and doc, that are required for early notochord formation. Mutations in flh and ntl have been described previously, while mom and doc are newly identified genes. Mutant mom embryos lack a notochord in the trunk, and trunk somites from the right and left side of the embryo fuse underneath the neural tube. In this respect mom appears similar to flh. In contrast, notochord precursor cells are present in both ntl and doc embryos. In order to gain a greater understanding of the phenotypes, we have analysed the expression of several axial mesoderm markers in mutant embryos of all four genes. In flh and mom, Ntl expression is normal in the germ ring and tailbud, while the expression of Ntl and other notochord markers in the axial mesodermal region is disrupted. Ntl expression is normal in doc embryos until early somitic stages, when there is a reduction in expression which is first seen in anterior regions of the embryo. This suggests a function for doc in the maintenance of ntl expression. Other notochord markers such as twist, sonic hedgehog and axial are not expressed in the axial mesoderm of ntl embryos, their expression parallels the expression of ntl in the axial mesoderm of mutant doc, flh and mom embryos, indicating that ntl is required for the expression of these markers. The role of doc in the expression of the notochord markers appears indirect via ntl. Floor plate formation is disrupted in most regions in flh and mom mutant embryos but is present in mutant ntl and doc embryos. In mutant embryos with strong ntl alleles the band of cells expressing floor plate markers is broadened. A similar broadening is also observed in the axial mesoderm underlying the floor plate of ntl embryos, suggesting a direct involvement of the notochord precursor cells in floor plate induction. Mutations in all of these four genes result in embryos lacking a horizontal myoseptum and muscle pioneer cells, both of which are thought to be induced by the notochord. These somite defects can be traced back to an impairment of the specification of the adaxial cells during early stages of development. Transplantation of wild-type cells into mutant doc embryos reveals that wild-type notochord cells are sufficient to induce horizontal myoseptum formation in the flanking mutant tissue. Thus doc, like flh and ntl, acts cell autonomously in the notochord. In addition to the four mutants with defects in early notochord formation, we have isolated 84 mutants, defining at least 15 genes, with defects in later stages of notochord development. These are listed in an appendix to this study.

Mutations affecting somite formation and patterning in the zebrafish, Danio rerio.

Somitogenesis is the basis of segmentation of the mesoderm in the trunk and tail of vertebrate embryos. Two groups of mutants with defects in this patterning process have been isolated in our screen for zygotic mutations affecting the embryonic development of the zebrafish (Danio rerio). In mutants of the first group, boundaries between individual somites are invisible early on, although the paraxial mesoderm is present. Later, irregular boundaries between somites are present. Mutations in fused somites (fss) and beamter (bea) affect all somites, whereas mutations in deadly seven (des), after eight (aei) and white tail (wit) only affect the more posterior somites. Mutants of all genes but wit are homozygous viable and fertile. Skeletal stainings and the expression pattern of myoD and snail1 suggest that anteroposterior patterning within individual somites is abnormal. In the second group of mutants, formation of the horizontal myoseptum, which separates the dorsal and ventral part of the myotome, is reduced. Six genes have been defined in this group (you-type genes). you-too mutants show the most severe phenotype; in these the adaxial cells, muscle pioneers and the primary motoneurons are affected, in addition to the horizontal myoseptum. The horizontal myoseptum is also missing in mutants that lack a notochord. The similarity of the somite phenotype in mutants lacking the notochord and in the you-type mutants suggests that the genes mutated in these two groups are involved in a signaling pathway from the notochord, important for patterning of the somites.

Developmental regulation of zebrafish MyoD in wild-type, no tail and spadetail embryos.

We describe the isolation of the zebrafish MyoD gene and its expression in wild-type embryos and in two mutants with altered somite development, no tail (ntl) and spadetail (spt). In the wild-type embryo, MyoD expression first occurs in an early phase, extending from mid-gastrula to just prior to somite formation, in which cells directly adjacent to the axial mesoderm express the gene. In subsequent phases, during the anterior-to-posterior wave of somite formation and maturation, expression occurs within particular regions of each somite. In spt embryos, which lack normal paraxial mesoderm due to incorrect cell migration, early MyoD expression is not observed and transcripts are instead first detected in small groups of trunk cells that will develop into aberrant myotomal-like structures. In ntl embryos, which lack notochords and tails, the early phase of MyoD expression is also absent. However, the later phase of expression within the developing somites appears to occur at the normal time in the ntl mutants, indicating that the presomitogenesis and somitogenesis phases of MyoD expression can be uncoupled. In addition, we demonstrate that the entire paraxial mesoderm of wild-type embryos has the potential to express MyoD when Sonic hedgehog is expressed ubiquitously in the embryo, and that this potential is lost in some of the cells of the paraxial mesoderm lineage in no tail and spadetail embryos. We also show that MyoD expression precedes myogenin expression and follows or is coincident with expression of snaill in some regions that express this gene.

Complex gangliosides affect GD3 accessibility to antibody in developing neuronal cells.

Ganglioside expression of embryonic chick retina cells developed in vitro was analyzed by indirect immunofluorescence. Immature neurons were GD3 positive cells and the labeling was chiefly distributed all over their cell membrane. Mature neurons became GD3 negative and expressed complex gangliosides of the a- and b-pathways; nevertheless, the content of GD3 accounted for approximately 40% of the total gangliosides in these cells. Neuraminidase hydrolysis pointed out that GD3 was located in membrane of differentiated cells. The frequency of cells with the GD3 immunostain localized in restricted area of membrane of undifferentiated neurons increased significantly after adding a mixture of bovine brain gangliosides (largely complex gangliosides). Antibody binding to immobilized GD3 showed a dose-dependent inhibition by adding a mixture of bovine brain gangliosides, GM1, GD1a or asialo-GM1. Glycosphingolipids with shorter oligosaccharide chains, as cerebrosides or sulfatides, did not affect this binding. These results suggest that, concomitant with the accretion of content of complex gangliosides, a rearrangement in the membrane would occur, which progressively masks GD3 to its antibody. This rearrangement might affect putative ganglioside functions involved in neuronal differentiation.

The expression pattern of two zebrafish achaete-scute homolog (ash) genes is altered in the embryonic brain of the cyclops mutant.

Two achaete-scute homolog sequences, Zash-1a and Zash-1b, were isolated from a zebrafish embryonic cDNA library. The Zash-1a cDNA encodes a protein very similar to rat Mash-1 and Xenopus Xash-1, with over 94% identity in the C-terminal three-fourths of all three polypeptides. The Zash-1b cDNA encodes a more distantly related protein, with 80% identity of amino acids to Mash-1 in this part of the sequence. At 24 hr, the Zash-1a transcripts are found in the hindbrain in two bilaterally symmetrical lines of cells which mark the boundary between the alar and basal plates and in rhombomere 1 in ventral cells near the floorplate. The gene is also expressed in particular regions of the telencephalon and diencephalon, in the epiphysis, the ventral tegmentum, the neural retina, and in specific cells in the spinal cord. Zash-1b transcripts are found in the hindbrain in segmentally arranged fan-like groups of cells which are located close to the anterior and posterior boundaries of each of rhombomeres 2-6 and in ventral cells close to the floor plate of most rhombomeres. The gene is also expressed at sites distinct from cells expressing Zash-1a in the tegmentum, diencephalon, telencephalon, and spinal cord. In the mutant cyclops, Zash-1a transcripts are absent from the ventral region of the tegmentum and in the ventral cells of rhombomere 1, while more dorsal expression regions are unaffected. The effects of the mutation on Zash-1b expression, however, are more complex. In the hindbrain, the ventral expression zone of this gene is absent, the more dorsal segmented expression is disorganized, and ectopic expression in the alar plate is observed. A dramatic ectopic expression is also observed in the anterior tegmentum. The cyclops gene, therefore, has both positive and negative effects on the CNS of the wild-type embryo: it is required for activation of both Zash-1a and -1b in particular ventral cells, but it also restricts the expression of Zash-1b in other ventral cells and in some dorsal regions. Zash-1a and -1b gene probes will be extremely useful in the analysis of additional mutations affecting development of the central nervous system in zebrafish embryos.