Heat shock genes and the heat shock response in zebrafish embryos

Heat shock genes exhibit complex patterns of spatial and temporal regulation during embryonic development in a wide range of organisms. Our laboratory has initiated an analysis of heat shock protein gene expression in the zebrafish, a model system that is now utilized extensively for the examination of early embryonic development of vertebrates. We have cloned members of the zebrafish hsp47, hsp70, and hsp90 gene families and shown them to be closely related to their counterparts in higher vertebrates. Whole mount in situ hybridization and Northern blot analyses have revealed that these genes are regulated in distinct spatial, temporal, and stress-specific manners. Furthermore, the tissue-specific expression patterns of the hsp47 and hsp90 alpha genes correlate closely with the expression of genes encoding known chaperone targets of Hsp47 and Hsp90 in other systems. The data raise a number of interesting questions regarding the function and regulation of these heat shock genes in zebrafish embryos during normal development and following exposure to environmental stress.     

Biomechanics of wrist injuries in sports

The Hox genes are implicated in conferring regional identity to the anteroposterior axis of the developing embryo. We have characterized the organization and expression of hox genes in the teleost zebrafish (Danio rerio), and compared our findings with those made for the tetrapod vertebrates. We have isolated 32 zebrafish hox genes, primarily via 3'RACE-PCR, and analyzed their linkage relationships using somatic cell hybrids. We find that in comparison to the tetrapods, zebrafish has several additional hox genes, both within and beyond the expected 4 hox clusters (A-D). For example, we have isolated a member of hox paralogue group 8 lying on the hoxa cluster, and a member of hox paralogue group 10 lying on the b cluster, no equivalent genes have been reported for mouse or human. Beyond the 4 clusters (A-D) we have isolated a further 3 hox genes (the hoxx and y genes), which according to their sequence homologies lie in paralogue groups 4, 6, and 9. The hoxx4 and hoxx9 genes occur on the same set of hybrid chromosomes, hinting at the possibility of an additional hox cluster for the zebrafish. Similar to their tetrapod counterparts, zebrafish hox genes (including those with no direct tetrapod equivalent) demonstrate colinear expression along the anteroposterior (AP) axis of the embryo. However, in comparison to the tetrapods, anterior hox expression limits are compacted over a short AP region; some members of adjacent paralogue groups have equivalent limits. It has been proposed that during vertebrate evolution, the anterior limits of Hox gene expression have become dispersed along the AP axis allowing the genes to take on novel patterning roles and thus leading to increased axial complexity. In the teleost zebrafish, axial organization is relatively simple in comparison to that of the tetrapod vertebrates; this may be reflected by the less dispersed expression domains of the zebrafish hox genes.     

The zebrafish Pax3 and Pax7 homologues are highly conserved, encode multiple isoforms and show dynamic segment-like expression in the developing brain

This study describes the isolation and characterization of zebrafish homologues of the mammalian Pax3 and Pax7 genes. The proteins encoded by both zebrafish genes are highly conserved (>83%) relative to the known mammalian sequences. Also the neural expression patterns during embryogenesis are very similar to the murine homologues. However, observed differences in neural crest and mesodermal expression relative to mammals could reflect some functional divergence in the development of these tissues. For the zebrafish Pax7 protein we report the first full-length amino acid sequences in vertebrates and show the existence of three additional isoforms which have truncations in the homeodomain and/or the C-terminal region. These novel variants provide evidence for additional isoform diversity of vertebrate Pax proteins.     

Archetypal organization of the amphioxus Hox gene cluster

Organization into gene clusters is an essential and diagnostic feature of Hox genes. Insect and nematode genomes possess single Hox gene clusters (split in Drosophila); in mammals, there are 38 Hox genes in four clusters on different chromosomes. A collinear relationship between chromosomal position, activation time and anterior expression limit of vertebrate Hox genes suggests that clustering may be important for precise spatiotemporal gene regulation and hence embryonic patterning. Hox genes have a wide phylogenetic distribution within the metazoa, and are implicated in the control of regionalization along the anteroposterior body axis. It has been suggested that changes in Hox gene number and genomic organization played a role in metazoan body-plan evolution, but identifying significant changes is difficult because Hox gene organization is known from only very few and widely divergent taxa (principally insects, nematodes and vertebrates). Here we analyse the complexity and organization of Hox genes in a cephalochordate, amphioxus, the taxon thought to be the sister group of the vertebrates. We find that the amphioxus genome has only one Hox gene cluster. It has similar genomic organization to the four mammalian Hox clusters, and contains homologues of at least the first ten paralogous groups of vertebrate Hox genes in a collinear array. Remarkably, this organization is compatible with that inferred for a direct ancestor of the vertebrates; we conclude that amphioxus is a living representative of a critical intermediate stage in Hox cluster evolution.     

Ectopic expression of hoxb2 after retinoic acid treatment or mRNA injection: disruption of hindbrain and craniofacial morphogenesis in zebrafish embryos

To investigate pattern formation in the vertebrate hindbrain, we isolated a full length hoxb2 cDNA clone from zebrafish. In a gene phylogeny, zebrafish hoxb2 clusters with human HOXB2, and it maps on linkage group 3 along with several other loci whose orthologues are syntenic with human HOXB2. In the hindbrain, hoxb2 is expressed at high levels in rhombomere 3 (r3), lower levels in r4, still lower in r5, and at undetectable levels in r6. In r7, r8, and the rostral spinal cord, hoxb2 is expressed at a lower level than in r5. Lateral cells appearing to emanate from r4 express both hoxb2 and dlx2, suggesting that they are neural crest. Overexpression of hoxb2 by mRNA injections into early cleavage stage embryos resulted in abnormal morphogenesis of the midbrain and rostral hindbrain, abnormal patterning in r4, fusion of cartilage elements arising from pharyngeal arches 1 and 2, and ectopic expression of krx20 and valentino (but not pax2, rtk1, or hoxb1) in the rostral hindbrain, midbrain, and, surprisingly, the eye. Treatments with retinoic acid produced a phenotype similar to that of ectopic hoxb2 expression, including ectopic krx20 (but not valentino) expression in the eye, and fusion of cartilages from pharyngeal arches 1 and 2. The results suggest that hoxb2 plays an important role in the patterning of hindbrain and pharyngeal arches in the zebrafish.     

Evolution of Emx genes and brain development in vertebrates

Emx1 and Emx2 genes are known to be involved in mammalian forebrain development. In order to investigate the evolution of the Emx gene family in vertebrates, a phylogenetic analysis was carried out on the Emx genes sequenced in man, mice, frogs, coelacanths and zebrafish. The results demonstrated the existence of two clades (Emx1 and Emx2), each grouping one of the two genes of the investigated taxa. The only exception was the zebrafish Emx1-like gene which turned out to be a sister group to both the Emx1 and Emx2 clusters. Such striking sequence divergence observed for the zebrafish Emx1-like gene could indicate that it is not orthologous to the other Emx1 genes, and therefore, in vertebrates there must be three Emx genes. Alternatively, if the zebrafish emx1 gene is orthologous to the tetrapod one, it must have undergone to strong diversifying selection.     

Isolation of a Drosophila homolog of the vertebrate homeobox gene Rx and its possible role in brain and eye development

Vertebrate and invertebrate eye development require the activity of several evolutionarily conserved genes. Among these the Pax-6 genes play a major role in the genetic control of eye development. Mutations in Pax-6 genes affect eye development in humans, mice, and Drosophila, and misexpression of Pax-6 genes in Drosophila can induce ectopic eyes. Here we report the identification of a paired-like homeobox gene, DRx, which is also conserved from flies to vertebrates. Highly conserved domains in the Drosophila protein are the octapeptide, the identical homeodomain, the carboxyl-terminal OAR domain, and a newly identified Rx domain. DRx is expressed in the embryo in the procephalic region and in the clypeolabrum from stage 8 on and later in the brain and the central nervous system. Compared with eyeless, the DRx expression in the embryo starts earlier, similar to the pattern in vertebrates, where Rx expression precedes Pax-6 expression. Because the vertebrate Rx genes have a function during brain and eye development, it was proposed that DRx has a similar function. The DRx expression pattern argues for a conserved function at least during brain development, but we could not detect any expression in the embryonic eye primordia or in the larval eye imaginal discs. Therefore DRx could be considered as a homolog of vertebrate Rx genes. The Rx genes might be involved in brain patterning processes and specify eye fields in different phyla.     

Rescue of Caenorhabditis elegans pharyngeal development by a vertebrate heart specification gene

Development of pharyngeal muscle in nematodes and cardiac muscle in vertebrates and insects involves the related homeobox genes ceh-22, nkx2.5, and tinman, respectively. To determine whether the nematode and vertebrate genes perform similar functions, we examined activity of the zebrafish nkx2.5 gene in transgenic Caenorhabditis elegans. Here, we report that ectopic expression of nkx2.5 in C. elegans body wall muscle can directly activate expression of both the endogenous myo-2 gene, a ceh-22 target normally expressed only in pharyngeal muscle, and a synthetic reporter construct controlled by a multimerized CEH-22 binding site. nkx2.5 also efficiently rescues a ceh-22 mutant when expressed in pharyngeal muscle. Together, these results indicate that nkx2.5 and ceh-22 provide a single conserved molecular function. Further, they suggest that an evolutionarily conserved mechanism underlies heart development in vertebrates and insects and pharyngeal development in nematodes.     

From zebra stripes to postal zones: deciphering patterns of gene expression in the cerebellum

The analysis of patterned gene expression has been an important tool for dissecting the molecular and developmental bases of functional compartmentalization in the mammalian cerebellum. In particular, sagittally-oriented cellular aggregates arranged along the mediolateral axis are the patterning element most commonly invoked to illustrate cerebellar compartmentalization, and these are revealed both by patterns of afferent projection and by a number of classical biochemical markers that are distributed in a pattern of'zebra stripes'. Compartmentation along both the mediolateral and rostrocaudal axes might be linked mechanistically to segmentation in the fruit fly, since early cerebellar development is especially dependent upon the expression of mammalian homologs of Drosophila segmentation genes. In addition, as has been demonstrated in the retinotectal system, some of these genes are likely to control positional information required for the sagittal organization of cerebellar afferent projections. However, in contrast to these global or macro zones, the cerebellum is also compartmentalized at the subcellular or micro level. This can be visualized by differential patterns of mRNA distribution within the sole cerebellar efferent system, the Purkinje cell, defining within such cells a number of distinct subcellular domains or 'postal zones'. The global versus subcellular levels of cerebellar compartmentalization are related since they both appear to be linked to patterns of afferent innervation.A major goal of cerebellar research will be to unravel the true nature of such a relationship, and its relevance to function and behavior.     

Laminar development of the mouse barrel cortex: effects of neurotoxins against monoamines

To investigate the inductive activities of the vertebrate organizer, we transplanted the chicken organizer (Hensen's node) into zebrafish gastrula and analyzed resulting secondary axes. Grafted Hensen's node did not differentiate or participate in the secondary axis. It also did not induce a secondary notochord or expression of the genes normally expressed by the fish organizer including no tail, axial, goosecoid. Nevertheless, it recruited fish cells to organize a variety of tissues: the dorsal portion of the central nervous system including Rohon-Beard sensory neurons, otic vesicles, dorsal pigment stripe, dorsal fin, somites, heart, and pronephric ducts. Enlarged neural plate induced by the organizer was shown by the expression pattern of dlx3 and msxB genes, which demarcates the early presumptive neural tissue. In addition, Hensen's node of an earlier stage chicken embryo displayed differential movement in zebrafish from that of a later stage. This might reflect unknown differences in properties between the organizer at two different developmental stages related to its normal organizer activity. To create a model system to study the molecular mechanisms of the organizer, we next transplanted genetically modified mouse cells into zebrafish embryos. We found that Wnt3A-transfected NIH3T3 cells are much more potent in inducing a secondary axis than NIH3T3 cells alone. These results suggest that formation of a variety of tissues are controlled by signalling from the organizer itself with no requirement of participation of the organizer-derived tissues. Additionally, the activities of the organizer may involve a function of Wnt-family genes.     

Syntenic organization of the mouse distal chromosome 7 imprinting cluster and the Beckwith-Wiedemann syndrome region in chromosome 11p15.5

In human and mouse, most imprinted genes are arranged in chromosomal clusters. Their linked organization suggests co-ordinated mechanisms controlling imprinting and gene expression. The identification of local and regional elements responsible for the epigenetic control of imprinted gene expression will be important in understanding the molecular basis of diseases associated with imprinting such as Beckwith-Wiedemann syndrome. We have established a complete contig of clones along the murine imprinting cluster on distal chromosome 7 syntenic with the human imprinting region at 11p15.5 associated with Beckwith-Wiedemann syndrome. The cluster comprises approximately 1 Mb of DNA, contains at least eight imprinted genes and is demarcated by the two maternally expressed genes Tssc3 (Ipl) and H19 which are directly flanked by the non-imprinted genes Nap1l4 (Nap2) and Rpl23l (L23mrp), respectively. We also localized Kcnq1 (Kvlqt1) and Cd81 (Tapa-1) between Cdkn1c (p57(Kip2)) and Mash2. The mouse Kcnq1 gene is maternally expressed in most fetal but biallelically transcribed in most neonatal tissues, suggesting relaxation of imprinting during development. Our findings indicate conserved control mechanisms between mouse and human, but also reveal some structural and functional differences. Our study opens the way for a systematic analysis of the cluster by genetic manipulation in the mouse which will lead to animal models of Beckwith-Wiedemann syndrome and childhood tumours.     

Fibroblast growth factor 2 increases Otx2 expression in precursor cells from mammalian telencephalon

Dissociated primary cultures from rat telencephalon at different developmental stages were used to study the effect of basic fibroblast growth factor (FGF2) on Otx2, Dlx1, and Emx1, three homeobox genes expressed in different regions of the developing mammalian forebrain. At embryonic day (E)13.5. the regional pattern of expression of Otx1, Otx2, Dlx1, Dlx2, Dlx5, and Emx1 is maintained in primary culture, suggesting that cells are already committed to a regional identity at this stage. In these cultures, Otx2 is expressed by precursor cells, whereas Dlx1 and Emx1 are predominantly expressed by postmitotic cells. We found that FGF2 increased Otx2 expression within precursor cells and the total number of Otx2-expressing cells. This effect was gene-specific, dose-dependent, and temporally regulated, with larger effects at earlier stages of development (E11.5). At E13.5, the effect of FGF2 on Otx2 expression was restricted to the basal telencephalon. Our results suggest that a restricted population of neuroblasts respond to FGF2 in a temporally regulated fashion by proliferating and increasing Otx2 expression. This interaction between FGF2 and Otx2 may be important for the regulation of neurogenesis in the forebrain.