Organization of<Emphasis Type="Italic"> Iroquois</Emphasis> genes in fish

In mammals, a total of six iroquois (Irx) genes exist, which are organized into two clusters. Here we report on the organization of all iroquois genes present in fish, using zebrafish (Danio rerio) and pufferfish (Fugu rubripes and Tetraodon nigroviridis) as examples. A total of 10 Irx genes were found in pufferfish, and 11 in zebrafish; all but one of these genes are organized into clusters (four clusters plus one isolated gene locus). The extra fish clusters result from chromosome duplication in the fish lineage, after its divergence from tetrapod vertebrates. Two of the four fish clusters are highly conserved to the ones in mammals, with regard to similarity of genes and cluster architecture. Irx genes within the other two clusters have diverged in sequence and cluster organization, suggesting functional divergence. These results will allow us to use the zebrafish system for functional and comparative studies of iroquois genes in vertebrate development.Electronic Supplementary Material Supplementary material is available in the online version of this article at Edited by D. Tautz     

<Emphasis Type="Italic">her11</Emphasis> is involved in the somitogenesis clock in zebrafish

Somitogenesis requires an intricate process of pre-patterning, which is driven by an oscillator mechanism consisting of the Delta-Notch pathway and hairy- (h) and Enhancer of split- [E(spl)] related genes. With the aim of unravelling the complex mechanism of somite pre-patterning, we have conducted an extensive search for h/E(spl)-related genes in the third release of the Danio rerio genomic sequence. We identified 14 new h/E(spl) genes and analysed them by in situ hybridisation for their potential role in the somitogenesis process. We describe here the functional analysis of one of these genes, which we have named her11. her11 is a paralogue of her1 and, similar to her1, is arranged in a head to head fashion with another her gene, namely the previously described her5. It shares an expression in the midbrain-hindbrain boundary with her5, but is in addition cyclically expressed in patterns overlapping those of her1 and her7 and complementary to those of hey1. Furthermore it is expressed in the anterior half of the most caudally formed somites. We show that Delta-Notch pathway genes and fused somites (fss) are necessary for the control of her11 expression. However, some aspects of the her11 regulation suggest that at least one additional as yet unknown gene of the Delta-Notch cascade is required to explain its expression. Morpholino-oligonucleotide-mediated knockdown of her11 shows that it is involved in the zebrafish somitogenesis clock via an interaction with her1 and her7. We have also studied the role of hey1 by morpholino injection, but could not find a direct function for this gene, suggesting that it reflects the output of the clock rather than being a core component of the mechanism.Edited by B. Herrmann     

Oscillations of the snail genes in the presomitic mesoderm coordinate segmental patterning and morphogenesis in vertebrate somitogenesis

The segmented body plan of vertebrate embryos arises through segmentation of the paraxial mesoderm to form somites. The tight temporal and spatial control underlying this process of somitogenesis is regulated by the segmentation clock and the FGF signaling wavefront. Here, we report the cyclic mRNA expression of Snail 1 and Snail 2 in the mouse and chick presomitic mesoderm (PSM), respectively. Whereas Snail genes' oscillations are independent of NOTCH signaling, we show that they require WNT and FGF signaling. Overexpressing Snail 2 in the chick embryo prevents cyclic Lfng and Meso 1 expression in the PSM and disrupts somite formation. Moreover, cells mis-expressing Snail 2 fail to express Paraxis, remain mesenchymal, and are thereby inhibited from undergoing the epithelialization event that culminates in the formation of the epithelial somite. Thus, Snail genes define a class of cyclic genes that coordinate segmentation and PSM morphogenesis.     

Comparative analysis of her genes during fish somitogenesis suggests a mouse/chick-like mode of oscillation in medaka

Somitogenesis is the key developmental step, which divides the vertebrate body axis into segmentally repeated structures. It requires an intricate process of pre-patterning, which is driven by an oscillator mechanism consisting of the Delta–Notch pathway and various hairy- and Enhancer of split-related (her) genes. The subset of her genes, which are necessary to set up the segmentation clock, reveal a complex scenario of interactions. To understand which her genes are essential core players in this process, we compared the expression patterns of somitogenesis-relevant her genes in zebrafish and medaka (Oryzias latipes). Most of the respective medaka genes (Ol-her) are duplicated like what has been shown for zebrafish (Dr-her) and pufferfish genes (Fr-her). However, zebrafish genes show some additional copies and significant differences in expression patterns. For the paralogues Dr-her1 and Dr-her11, only one copy exists in the medaka (Ol-her1/11), which combines the expression patterns found for both zebrafish genes. In contrast to Dr-her5, the medaka orthologue appears to play a role in somitogenesis because it is expressed in the presomitic mesoderm (PSM). PSM expression also suggests a role for both Ol-her13 genes, homologues of mouse Hes6 (mHes6), in this process, which would be consistent with a conserved mHes6 homologue gear in the segmentation clock exclusively in lower vertebrates. Members of the mHes5 homologue group seem to be involved in somite formation in all vertebrates (e.g. Dr- and Ol-her12), although different paralogues are additionally recruited in zebrafish (e.g. Dr-her15) and medaka (e.g. Ol-her4). We found that the linkage between duplicates is strongly conserved between pufferfish and medaka and less well conserved in zebrafish. Nevertheless, linkage and orientation of several her duplicates are identical in all three species. Therefore, small-scale duplications must have happened before whole genome duplication occurred in a fish ancestor. Expression of multiple stripes in the intermediate PSM, characteristic for the zebrafish orthologues, is absent in all somitogenesis-related her genes of the medaka. In fact, the expression mode of Ol-her1/11 and Ol-her5 indicates dynamism similar to the hairy clock genes in chicken and mouse. This suggests that Danio rerio shows a rather derived clock mode when compared to other fish species and amniotes or that, alternatively, the clock mode evolved independently in zebrafish, medaka and mouse or chicken.An erratum to this article can be found at     

The zebrafish Hairy/Enhancer-of-split-related gene her6 is segmentally expressed during the early development of hindbrain and somites

Several vertebrate genes of the Hairy/Enhancer-of-split (HES) family are involved in paraxial mesoderm segmentation and intersomitic boundary establishment/maintenance. Here, we show that the zebrafish hairy-related gene, her6, highly homologous to the mammalian and chicken HES-1 genes, is expressed in the posterior part of each segmented somite and in stripes in the anterior presomitic mesoderm (PSM), and also in a dynamic, segmentally restricted pattern during hindbrain segmentation, with all rhombomeres expressing her6 at different time points and at different levels.     

Intron loss in the SART1 genes of Fugu rubripes and Tetraodon nigroviridis.

The human SART1 gene was initially identified in a screen for proteins recognised by IgE, which may be implicated in atopic disease. We have examined the genomic structure and cDNA sequence of the SART1 gene in the compact genomes of the pufferfish Fugu rubripes and Tetraodon nigroviridis. The entire coding regions of both the Fugu and Tetraodon SART1 genes are contained within single exons. The Fugu gene contains only one intron located in the 5' untranslated region. Southern blot hybridisation of Fugu genomic DNA confirmed the SART1 gene to be single copy. Partial genomic structures were also determined for the human, mouse, Drosophila and C. elegans SART1 homologues. The human and mouse genes both contain many introns in the coding region, the human gene possessing at least 20 exons. The Drosophila and C. elegans homologues contain 6 and 12 exons, respectively. This is only the second time such a difference in the organization of homologous Fugu and human genes has been reported. The Fugu and Tetraodon SART1 genes encode putative proteins of 772 and 774 aa, respectively, each having 65% amino acid identity to human SART1. Leucine zipper and basic motifs are conserved in the predicted Fugu and Tetraodon proteins.     

Developmentally regulated expression of two members of the Nrarp family in zebrafish

Delta-Notch signaling is essential for somitogenesis in vertebrate embryos. In a search for genes that control somite formation in zebrafish we have identified two paralogues encoding proteins related to Nrarp (Notch regulated ankyrin repeat protein). Zebrafish nrarp-a and-b encode small proteins with two ankyrin repeat domains. Here, we report the expression patterns of both genes in normal and mutant embryos.     

The structure and evolution of the melanocortin and MCH receptors in fish and mammals

Zebrafish are an excellent genetic model system for studying developmental and physiological processes. Pigment patterns in zebrafish are affected by mutations in three types of chromatophores. The behavior of these cells is influenced by alpha-melanocyte-stimulating hormone (alphaMSH) and melanin-concentrating hormone (MCH). Mammals have five alphaMSH receptors (melanocortin receptors) and one or two MCH receptors. We have identified the full complement of melanocortin and MCH receptors in both zebrafish and the pufferfish, Fugu. Zebrafish have six melanocortin receptors, including two MC5R orthologues, while Fugu, lacking MC3R, has only four. We also demonstrate that Fugu and zebrafish have two and three MCHR genes, respectively. MC2R and MC5R are physically linked in all species examined. Unlike other species, we find the Fugu genes contain introns, one of which is in a conserved location and is probably ancestral. We also detail the differential expression of the zebrafish genes throughout development.     

Analysis of her1 and her7 mutants reveals a spatio temporal separation of the somite clock module

Somitogenesis is controlled by a genetic network consisting of an oscillator (clock) and a gradient (wavefront). The "hairy and Enhancer of Split"- related (her) genes act downstream of the Delta/Notch (D/N) signaling pathway, and are crucial components of the segmentation clock. Due to genome duplication events, the zebrafish genome, possesses two gene copies of the mouse Hes7 homologue: her1 and her7. To better understand the functional consequences of this gene duplication, and to determine possible independent roles for these two genes during segmentation, two zebrafish mutants her1(hu2124) and her7(hu2526) were analyzed. In the course of embryonic development, her1(hu2124) mutants exhibit disruption of the three anterior-most somite borders, whereas her7(hu2526) mutants display somite border defects restricted to somites 8 (+/-3) to 17 (+/-3) along the anterior-posterior axis. Analysis of the molecular defects in her1(hu2124) mutants reveals a her1 auto regulatory feedback loop during early somitogenesis that is crucial for correct patterning and independent of her7 oscillation. This feedback loop appears to be restricted to early segmentation, as cyclic her1 expression is restored in her1(hu2124) embryos at later stages of development. Moreover, only the anterior deltaC expression pattern is disrupted in the presomitic mesoderm of her1(hu2124) mutants, while the posterior expression pattern of deltaC remains unaltered. Together, this data indicates the existence of an independent and genetically separable anterior and posterior deltaC clock modules in the presomitic mesdorm (PSM).     

Interaction between Notch signalling and Lunatic fringe during somite boundary formation in the mouse.

BACKGROUND: The process of somitogenesis can be divided into three major events: the prepatterning of the mesoderm; the formation of boundaries between the prospective somites; and the cellular differentiation of the somites. Expression and functional studies have demonstrated the involvement of the murine Notch pathway in somitogenesis, although its precise role in this process is not yet well understood. We examined the effect of mutations in the Notch pathway elements Delta like 1 (Dll1), Notch1 and RBPJkappa on genes expressed in the presomitic mesoderm (PSM) and have defined the spatial relationships of Notch pathway gene expression in this region. RESULTS: We have shown that expression of Notch pathway genes in the PSM overlaps in the region where the boundary between the posterior and anterior halves of two consecutive somites will form. The Dll1, Notch1 and RBPJkappa mutations disrupt the expression of Lunatic fringe (L-fng), Jagged1, Mesp1, Mesp2 and Hes5 in the PSM. Furthermore, expression of EphA4, mCer 1 and uncx4.1, markers for the anterior-posterior subdivisions of the somites, is down-regulated to different extents in Notch pathway mutants, indicating a global alteration of pattern in the PSM. CONCLUSIONS: We propose a model for the mechanism of somite border formation in which the activity of Notch in the PSM is restricted by L-fng to a boundary-forming territory in the posterior half of the prospective somite. In this region, Notch function activates a set of genes that are involved in boundary formation and anterior-posterior somite identity.     

Fgf/MAPK signalling is a crucial positional cue in somite boundary formation.

The temporal and spatial regulation of somitogenesis requires a molecular oscillator, the segmentation clock. Through Notch signalling, the oscillation in cells is coordinated and translated into a cyclic wave of expression of hairy-related and other genes. The wave sweeps caudorostrally through the presomitic mesoderm (PSM) and finally arrests at the future segmentation point in the anterior PSM. By experimental manipulation and analyses in zebrafish somitogenesis mutants, we have found a novel component involved in this process. We report that the level of Fgf/MAPK activation (highest in the posterior PSM) serves as a positional cue within the PSM that regulates progression of the cyclic wave and thereby governs the positions of somite boundary formation.     

Disruption of the somitic molecular clock causes abnormal vertebral segmentation

Somites are the precursors of the vertebral column. They segment from the presomitic mesoderm (PSM) that is caudally located and newly generated from the tailbud. Somites form in synchrony on either side of the embryonic midline in a reiterative manner. A molecular clock that operates in the PSM drives this reiterative process. Genetic manipulation in mouse, chick and zebrafish has revealed that the molecular clock controls the activity of the Notch and WNT signaling pathways in the PSM. Disruption of the molecular clock impacts on somite formation causing abnormal vertebral segmentation (AVS). A number of dysmorphic syndromes manifest AVS defects. Interaction between developmental biologists and clinicians has lead to groundbreaking research in this area with the identification that spondylocostal dysostosis (SCD) is caused by mutation in Delta-like 3 (DLL3), Mesoderm posterior 2 (MESP2), and Lunatic fringe (LFNG); three genes that are components of the Notch signaling pathway. This review describes our current understanding of the somitic molecular clock and highlights how key findings in developmental biology can impact on clinical practice.