Trevor Jowett

Insights into early vasculogenesis revealed by expression of the ETS-domain transcription factor Fli-1 in wild-type and mutant zebrafish embryos

Fli-1 is an ETS-domain transcription factor whose locus is disrupted in Ewings Sarcoma and F-MuLV induced erythroleukaemia. To gain a better understanding of its normal function, we have isolated the zebrafish homologue. Similarities with other vertebrates, in the amino acid sequence and DNA binding properties of Fli-1 from zebrafish, suggest that its function has been conserved during vertebrate evolution. The initial expression of zebrafish fli-1 in the posterior lateral mesoderm overlaps with that of gata2 in a potential haemangioblast population which likely contains precursors of blood and endothelium. Subsequently, fli-1 and gata2 expression patterns diverge, with separate fli-1 and gata2 expression domains arising in the developing vasculature and in sites of blood formation respectively. Elsewhere in the embryo, fli-1 is expressed in sites of vasculogenesis. The expression of fli-1 was investigated in a number of zebrafish mutants, which affect the circulatory system. In cloche, endothelium is absent and blood is drastically reduced. In contrast to the blood and endothelial markers that have been studied previously, fli-1 expression was initiated normally in cloche embryos, indicating that induction of fli-1 is one of the earliest indicators of haemangioblast formation. Furthermore, although fli-1 expression in the trunk was not maintained, the normal expression pattern in the anterior half of the embryo was retained. These anterior cells did not, however, condense to form blood vessels. These data indicate that cloche has previously unsuspected roles at multiple stages in the formation of the vasculature. Analysis of fli-1 expression in midline patterning mutants floating head and squint, confirms a requirement for the notochord in the formation of the dorsal-aorta. The formation of endothelium in one-eyed pinhead, cyclops and squint embryos indicates a novel role for the endoderm in the formation of the axial vein. The phenotype of sonic-you mutants implies a likely role for Sonic Hedgehog in mediating these processes.

Analysis of protein and gene expression.

Publisher Summary There are well-established methods for determining the temporal and spatial expression of RNA and proteins, and these can be successfully applied to zebrafish embryos and tissues. The mRNA transcribed by a specific gene is best detected in situ by hybridization with an antisense RNA probe labeled so that it can be detected either by chromogenic stains or fluorescence. Modifications to this procedure can allow the identification of more than one mRNA. This chapter describes several alternative approaches for identifying multiple transcripts. These methods allow visualization of the transcripts either with chromogenic substrates or different fluorochromes. These methods are particularly appropriate when coupled with the use of confocal microscopy. Localization of proteins can be performed using an antibody raised against the protein. The signal is visualized using a secondary antibody, either labeled with a fluorochrome or conjugated with an enzyme, for which there are chromogenic substrates. Protein localization can also be combined with in situ hybridization to mRNA. However, this may not always be possible, because it requires that the protein antigen not be destroyed by the treatments given to the tissue during in situ hybridization.

Exogenous retinoic acid causes specific alterations in the development of the midbrain and hindbrain of the zebrafish embryo including positional respecification of the Mauthner neuron

Exogenously applied retinoic acid given at the early stages of gastrulation causes abnormal development of the caudal midbrain and anterior hindbrain in vertebrate embryos. We describe the limits of the brain regions that are affected using neuroanatomical criteria in the zebrafish embryo. Analysis of the reticulospinal complex shows that the Mauthner cell, which normally differentiates in rhombomere 4, is duplicated either in this rhombomere or in rhombomere 2. Using probes for zebrafish krx20 and pax2, it is demonstrated that retinoic acid affects the expression domains of these regulatory genes in a manner that is consistent with the neuroanatomical data. Expression of the goosecoid gene, which expressed in the prospective anterior mesoderm from the onset of gastrulation, is unaffected by the doses of retinoic acid used in this study, reflecting the normal development of the anterior end of the embryo.

Expression of sox11 gene duplicates in zebrafish suggests the reciprocal loss of ancestral gene expression patterns in development

To investigate the role of sox genes in vertebrate development, we have isolated sox11 from zebrafish (Danio rerio). Two distinct classes of sox11‐related cDNAs were identified, sox11a and sox11b. The predicted protein sequences shared 75% identity. In a gene phylogeny, both sox11a and sox11b cluster with human, mouse, chick, and Xenopus Sox11, indicating that zebrafish, like Xenopus, has two orthologues of tetrapod Sox11. The work reported here investigates the evolutionary origin of these two gene duplicates and the consequences of their duplication for development. The sox11a and sox11b genes map to linkage groups 17 and 20, respectively, together with other loci whose orthologues are syntenic with human SOX11, suggesting that during the fish lineage, a large chromosome region sharing conserved syntenies with mammals has become duplicated. Studies in mouse and chick have shown that Sox11 is expressed in the central nervous system during development. Expression patterns of zebrafish sox11a and sox11b confirm that they are expressed in the developing nervous system, including the forebrain, midbrain, hindbrain, eyes, and ears from an early stage. Other sites of expression include the fin buds and somites. The two sox genes, sox11a and sox11b, are expressed in both overlapping and distinct sites. Their expression patterns suggest that sox11a and sox11b may share the developmental domainsof the single Sox11 gene present in mouse and chick. For example, zebrafish sox11a is expressed in the anterior somites, and zebrafish sox11b is expressed in the posterior somites, but the single Sox11 gene of mouse is expressed in all the somites. Thus, the zebrafish duplicate genes appear to have reciprocally lost expression domains present in the sox11 gene of the last common ancestor of tetrapods and zebrafish. This splitting of the roles of Sox11 between two paralogues suggests that regulatory elements governing the expression of the sox11 gene in the common ancestor of zebrafish and tetrapods may have been reciprocally mutated in the zebrafish gene duplicates. This is consistent with duplicate gene evolution via a duplication‐degeneration‐complementation process. Dev Den;217:279–292.

Molecular characterization of the zebrafish PEA3 ETS-domain transcription factor

The PEA3 subfamily of ETS-domain proteins play important roles in regulating transcriptional activation and have been implicated in several tumorigenic processes. Here we describe the identification of a further member of this family from zebrafish which most likely represents a homologue of PEA3. A high degree of sequence conservation is observed in the ETS DNA-binding domain and acidic transcriptional activation domain. The DNA binding specificity of zebrafish PEA3 is virtually identical to that exhibited by mammalian family members and is autoregulated by cisacting inhibitory domains. Transcriptional activation by zebrafish PEA3 is potentiated by the ERK MAP kinase and protein kinase A pathways. During embryogenesis, PEA3 is expressed in complex spatial and temporal patterns in both mesodermal somites and ectodermal tissues including the brain, dorsal spinal chord and neural crest. Our characterisation of zebrafish PEA3 furthers our understanding of its molecular function and its expression profile suggests a novel role in cell patterning in the early vertebrate embryo.

The pattern of segment formation, as revealed by engrailed expression, in a centipede with a variable number of segments.

SUMMARY Arthropods vary enormously in segment number, from less than 20 to more than 200. This between‐species variation must have originated, in evolution, through divergent selection operating in ancestral arthropod species with variable segment numbers. Although most present‐day arthropod species are invariant in this respect, some are variable and so can serve as model systems. Here, we describe a study based on one such species, the coastal geophilomorph centipede Strigamia maritima. We investigate the way in which segments are formed using in situ hybridization to demonstrate the expression pattern of the engrailed gene during embryogenesis. We also analyze segment number data in mother–offspring broods and thereby demonstrate a significant heritable component of the variation. We consider how natural selection might act on this intraspecific developmental variation, and we discuss the similarities and differences in segment formation between the geophilomorphs and their phylogenetic sister‐group.

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. Dev. Dyn. 1998;213:370–385.

Homeotic transformation in a centipede

The suggested involvement of homeotic mutations in evolution1xHomeotic mutatants and evolution. Goldschmidt, R. Acta Biotheoretica. 1952; 10: 87–104Crossref | Scopus (15)See all References1 is controversial, and is rejected by most evolutionary biologists. However, an acknowledged difficulty is that these mutations are best known in Drosophila, which is a highly derived arthropod. Perhaps the severe fitness depression accompanying homeotic transformation would have been less pronounced, even absent, in a primitive arthropod with many similar segments. Centipedes provide a model for studying the effects of homeotic transformation as they might have been manifested in an ancient arthropod stem species. This is not to say that their particular body plan is not highly derived – it clearly is – but, rather, that it retains the feature of many broadly similar segments that characterized the first arthropods. The key question, then, is whether homeotic transformation in a system of this kind would be radically different in effect to its Drosophila counterpart.Despite more than a century of study of the morphology of centipedes, and the existence of at least 3000 species, there has until now been no observed case of homeotic transformation of a segment in any centipede. However, we have recently found a case of this in an adult male Strigamia maritima – a geophilomorph species of intermediate length – collected at Whitburn on the coast of NE England (grid ref. NZ 412615). This specimen (see Fig. 1Fig. 1) exhibits transformation of the most anterior of the normally legless terminal segments (the ‘intermediate’ segment) into a repeat of the posteriormost leg-bearing segment, giving it two pairs of specialized rear legs (male sexual characters) instead of one. This makes it the first centipede ever discovered with an even number of leg-bearing segments (48; normally only uneven numbers between 15 and 191 are observed).FIGURE 1Homeotic transformation in a centipede. (a) Normal and (b) transformed morphologies of the centipede Strigamia maritima. In the normal arrangement, there is a single segment with multiple coxal pores and with special rearward-pointing legs that are swollen in the male (pictured) but slender in the female. In the mutant centipede there are two such segments, the second deriving from the transformation of the normally legless ‘intermediate segment’.View Large Image | Download PowerPoint SlideThe symmetry of the transformed segment suggests a hereditary origin, perhaps involving a mutation of the Abdominal-B gene. [A homologue of this Drosophila gene was detected in one recent centipede study (M.L. Smith, PhD thesis, University of Cambridge, 1998) but not in another2xEvolution of the entire arthropod Hox gene set predated the origin and radiation of the onychophoran/arthropod clade. Grenier, J.K. et al. Curr. Biol. 1997; 7: 547–553Abstract | Full Text | Full Text PDF | PubMedSee all References2.] The mutant individual was viable under natural conditions: it had reached adulthood, and so must have been at least two years old3xThe life history and ecology of the littoral centipede Strigamia (= Scolioplanes) maritima (Leach). Lewis, J.G.E. Proc. Zool. Soc. London. 1961; 137: 221–248CrossrefSee all References3. Its fertility is unknown, although the genital segments, immediately posterior to the transformed one, appear normal. Although these observations appear to argue for an evolutionary role of homeotic mutation in this kind of body plan, there is a more pressing counter-argument. The homeotic centipede shares with the four-winged fly a morphological shift that appears never to have happened in evolution. Relative to the realized direction of evolutionary change, homeotic transformations seem to be always either ‘backwards’ (two-wings-to-four as opposed to four-wings-to-two) or ‘dead-ends’ (our centipede). So, even if the fitness problem is overcome, morphology itself argues against an evolutionary role for these mutations. However, in a final twist to the story, there is now evidence4xA role of Ultrabithorax in morphological differences between Drosophila species. Stern, D.L. Nature. 1998; 396: 463–466Crossref | PubMed | Scopus (174)See all References, 5xHox genes, homeosis and the evolution of segment identity: no need for hopeless monsters. Akam, M. Int. J. Dev. Biol. 1998; 42: 445–451PubMedSee all References that other (lesser) mutations of the Hox genes are involved in arthropod morphological evolution.

sox30: a novel zebrafish sox gene expressed in a restricted manner at the midbrain-hindbrain boundary during neurogenesis.

Abstract The Sox family of proteins is thought to act to regulate gene expression in a wide variety of developmental processes. Here we describe the cloning of sox30, a novel sox gene from the zebrafish (Danio rerio). In situ hybridization shows that sox30 is expressed in a restricted manner at the boundary between the midbrain and hindbrain during nervous system development. This expression pattern is in direct contrast to that of most other neuronally expressed Sox genes which are expressed throughout the nervous system.

Cloning of fish zinc-finger genes related to Krox-20 and Krox-24.

Southern hybridization suggests that the zebrafish genome contains multiple zinc-finger genes related to the putative mouse developmental genes, Krox-20 and Krox-24. The polymerase chain reaction was employed to amplify and clone the zinc-finger regions of genes related to Krox-20, from two fish species and, for comparison, mouse, hamster and fox. DNA sequence analyses suggest that the genes cloned include the guppy homologue of Krox-20 and the zebrafish homologue of Krox-24, and that these genes diverged prior to the separation of the lineages leading to teleosts and to mammals.