Synchronized oscillation of the segmentation clock gene in vertebrate development

In vertebrate somitogenesis, “segmentation clock” genes (her in zebrafish, hes in mouse, and hairy in chick) show oscillation, synchronized over nearby cells through intercellular interaction. In zebrafish, neighboring cells interact by Delta-Notch signaling to realize synchronization. Under Delta-Notch, however, a cell with a high expression of the segmentation clock gene tends to suppress its expression in adjacent cells, which might produce spatial heterogeneity instead of synchronized oscillation. Here we studied the conditions under which pre-somitic mesoderm cells show synchronized oscillation of gene expression mathematically. We adopted a model that explicitly considers the kinetics of the mRNA and proteins of the segmentation clock gene and cell–cell interaction via Delta-Notch signaling. From statistical study of a model with randomly generated parameters, we revealed how the likelihood that the system generates stable synchronized oscillation depends on the rate of each reaction in the gene–protein kinetics.     

<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     

FGF receptor gene expression and its regulation by FGF signaling during early zebrafish development

The expression of all four fgfr genes was extensively examined throughout early embryogenesis of the zebrafish (Danio rerio). fgfr1 alone was expressed maternally throughout the blastoderm, and then zygotically in the anterior neural plate and presomitic mesoderm. fgfr4 expression was first detected in late blastulae and was gradually restricted to the brain. fgfr2 and fgfr3 expression were initiated in early and late gastrulae, respectively; fgfr2 was expressed in the anterior neural plate and somitic mesoderm, whereas fgfr3 was activated in the axial mesoderm and then in the midbrain and somitic mesoderm. During somitogenesis, each of these fgfr genes was expressed in a characteristic manner in the brain. Using an FGF signal inhibitor, dominant-negative FGF receptors and fgf8.1/fgf8a mutants, we found that fgfr expression is directly or indirectly regulated by FGF signaling during epiboly and at the end of somitogenesis, revealing the presence of an autoregulatory mechanism.     

The role of Suppressor of Hairless in Notch mediated signalling during zebrafish somitogenesis

Suppressor of Hairless (Su(H)) codes for a protein that interacts with the intracellular domain of Notch to activate the target genes of the Delta-Notch signalling pathway. We have cloned the zebrafish homologue of Su(H) and have analysed its function by morpholino mediated knockdown. While there are at least four notch and four delta homologues in zebrafish, there appears to be only one complete Su(H) homologue. We have analysed the function of Su(H) in the somitogenesis process and its influence on the expression of notch pathway genes, in particular her1, her7, deltaC and deltaD. The cyclic expression of her1, her7 and deltaC in the presomitic mesoderm is disrupted by the Su(H) knockdown mimicking the expression of these genes in the notch1a mutant deadly seven. deltaD expression is similarly affected by Su(H) knockdown like deltaC but shows in addition an ectopic expression in the developing neural tube. The inactivation of Su(H) in a fss/tbx24 mutant background leads furthermore to a clear breakdown of cyclic her1 and her7 expression, indicating that the Delta-Notch pathway is required for the creation of oscillation and not only for the synchronisation between neighbouring cells. The strongest phenotypes in the Su(H) knockdown embryos show a loss of all somites posterior to the first five to seven ones. This phenotype is stronger than the known amorphic phenotypes for notch1 (des) or deltaD (aei) in zebrafish, but mimicks the knockout phenotype of RBP-Jkappa gene in the mouse, which is the homologue of Su(H). This suggests that there is some functional redundancy among the Notch and Delta genes. This fact that the first five to seven somites are only weakly affected by Su(H) knockdown indicates that additional genetic pathways may be active in the specification of the most anterior somites.     

Two linked hairy/Enhancer of split-related zebrafish genes,her1 and her7, function together to refine alternating somite boundaries

The formation of somites, reiterated structures that will give rise to vertebrae and muscles, is thought to be dependent upon a molecular oscillator that may involve the Notch pathway. hairy/Enhancer of split related [E(spl)]-related (her or hes) genes, potential targets of Notch signaling, have been implicated as an output of the molecular oscillator. We have isolated a zebrafish deficiency, b567, that deletes two linked her genes, her1 and her7. Homozygous b567 mutants have defective somites along the entire embryonic axis. Injection of a combination of her1 and her7 (her1+7) morpholino modified antisense oligonucleotides (MOs) phenocopies the b567 mutant somitic phenotype, indicating that her1 and her7 are necessary for normal somite formation and that defective somitogenesis in b567 mutant embryos is due to deletion of her1 and her7. Analysis at the cellular level indicates that somites in her1+7-deficient embryos are enlarged in the anterior-posterior dimension. Weak somite boundaries are often found within these enlarged somites which are delineated by stronger, but imperfect, boundaries. In addition, the anterior-posterior polarity of these enlarged somites is disorganized. Analysis of her1 MO-injected embryos and her7 MO-injected embryos indicates that although these genes have partially redundant functions in most of the trunk region, her1 is necessary for proper formation of the anteriormost somites and her7 is necessary for proper formation of somites posterior to somite 11. By following somite development over time, we demonstrate that her genes are necessary for the formation of alternating strong somite boundaries. Thus, even though two potential downstream components of Notch signaling are lacking in her1+7-deficient embryos, somite boundaries form, but do so with a one and a half to two segment periodicity.     

The Role of her4 in Inner Ear Development and Its Relationship with Proneural Genes and Notch Signalling

The generation of sensory neurons and hair cells of the inner ear is under tight control. Different members of the Hairy and Enhancer of Split genes (HES) are expressed in the inner ear, their full array of functions still not being disclosed. We have previously shown that zebrafish her9 acts as a patterning gene to restrict otic neurogenesis to an anterior domain. Here, we disclose the role of another her gene, her4, a zebrafish ortholog of Hes5 that is expressed in the neurogenic and sensory domains of the inner ear. The expression of her4 is highly dynamic and spatiotemporally regulated. We demonstrate by loss of function experiments that in the neurogenic domain her4 expression is under the regulation of neurogenin1 (neurog1) and the Notch pathway. Moreover, her4 participates in lateral inhibition during otic neurogenesis since her4 knockdown results in overproduction of the number of neurog1 and deltaB-positive otic neurons. In contrast, during sensorigenesis her4 is initially Notch-independent and induced by atoh1b in a broad prosensory domain. At later stages her4 expression becomes Notch-dependent in the future sensory domains but loss of her4 does not result in hair cell overproduction, suggesting that there other her genes can compensate its function.     

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     

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.     

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).     

Retinoic Acid Signaling Organizes Endodermal Organ Specification along the Entire Antero-Posterior Axis

Background

Endoderm organ primordia become specified between gastrulation and gut tube folding in Amniotes. Although the requirement for RA signaling for the development of a few individual endoderm organs has been established a systematic assessment of its activity along the entire antero-posterior axis has not been performed in this germ layer.

Methodology/Principal Findings

RA is synthesized from gastrulation to somitogenesis in the mesoderm that is close to the developing gut tube. In the branchial arch region specific levels of RA signaling control organ boundaries. The most anterior endoderm forming the thyroid gland is specified in the absence of RA signaling. Increasing RA in anterior branchial arches results in thyroid primordium repression and the induction of more posterior markers such as branchial arch Hox genes. Conversely reducing RA signaling shifts Hox genes posteriorly in endoderm. These results imply that RA acts as a caudalizing factor in a graded manner in pharyngeal endoderm. Posterior foregut and midgut organ primordia also require RA, but exposing endoderm to additional RA is not sufficient to expand these primordia anteriorly. We show that in chick, in contrast to non-Amniotes, RA signaling is not only necessary during gastrulation, but also throughout gut tube folding during somitogenesis. Our results show that the induction of CdxA, a midgut marker, and pancreas induction require direct RA signaling in endoderm. Moreover, communication between CdxA ⁺ cells is necessary to maintain CdxA expression, therefore synchronizing the cells of the midgut primordium. We further show that the RA pathway acts synergistically with FGF4 in endoderm patterning rather than mediating FGF4 activity.

Conclusions/Significance

Our work establishes that retinoic acid (RA) signaling coordinates the position of different endoderm organs along the antero-posterior axis in chick embryos and could serve as a basis for the differentiation of specific endodermal organs from ES cells.     

Cyclical expression of the Notch/Wnt regulator Nrarp requires modulation by Dll3 in somitogenesis

Delta-like 3 (Dll3) is a divergent ligand and modulator of the Notch signaling pathway only identified so far in mammals. Null mutations of Dll3 disrupt cycling expression of Notch targets Hes1, Hes5, and Lfng, but not of Hes7. Compared with Dll1 or Notch1, the effects of Dll3 mutations are less severe for gene expression in the presomitic mesoderm, yet severe segmentation phenotypes and vertebral defects result in both human and mouse. Reasoning that Dll3 specifically disrupts key regulators of somite cycling, we carried out functional analysis to identify targets accounting for the segmental phenotype. Using microdissected embryonic tissue from somitic and presomitic mesodermal tissue, we identified new genes enriched in these tissues, including Limch1, Rhpn2, and A130022J15Rik. Surprisingly, we only identified a small number of genes disrupted by the Dll3 mutation. These include Uncx, a somite gene required for rib and vertebral patterning, and Nrarp, a regulator of Notch/Wnt signaling in zebrafish and a cycling gene in mouse. To determine the effects of Dll3 mutation on Nrarp, we characterized the cycling expression of this gene from early (8.5 dpc) to late (10.5 dpc) somitogenesis. Nrarp displays a distinct pattern of cycling phases when compared to Lfng and Axin2 (a Wnt pathway gene) at 9.5 dpc but appears to be in phase with Lfng by 10.5 dpc. Nrarp cycling appears to require Dll3 but not Lfng modulation. In Dll3 null embryos, Nrarp displayed static patterns. However, in Lfng null embryos, Nrarp appeared static at 8.5 dpc but resumed cycling expression by 9.5 and dynamic expression at 10.5 dpc stages. By contrast, in Wnt3a null embryos, Nrarp expression was completely absent in the presomitic mesoderm. Towards identifying the role of Dll3 in regulating somitogenesis, Nrarp emerges as a potentially important regulator that requires Dll3 but not Lfng for normal function.     

Interaction of Wnt and caudal-related genes in zebrafish posterior body formation

Although Wnt signaling plays an important role in body patterning during early vertebrate embryogenesis, the mechanisms by which Wnts control the individual processes of body patterning are largely unknown. In zebrafish, wnt3a and wnt8 are expressed in overlapping domains in the blastoderm margin and later in the tailbud. The combined inhibition of Wnt3a and Wnt8 by antisense morpholino oligonucleotides led to anteriorization of the neuroectoderm, expansion of the dorsal organizer, and loss of the posterior body structure-a more severe phenotype than with inhibition of each Wnt alone-indicating a redundant role for Wnt3a and Wnt8. The ventrally expressed homeobox genes vox, vent, and ved mediated Wnt3a/Wnt8 signaling to restrict the organizer domain. Of posterior body-formation genes, expression of the caudal-related cdx1a and cdx4/kugelig, but not bmps or cyclops, was strongly reduced in the wnt3a/wnt8 morphant embryos. Like the wnt3a/wnt8 morphant embryos, cdx1a/cdx4 morphant embryos displayed complete loss of the tail structure, suggesting that Cdx1a and Cdx4 mediate Wnt-dependent posterior body formation. We also found that cdx1a and cdx4 expression is dependent on Fgf signaling. hoxa9a and hoxb7a expression was down-regulated in the wnt3a/wnt8 and cdx1a/cdx4 morphant embryos, and in embryos with defects in Fgf signaling. Fgf signaling was required for Cdx-mediated hoxa9a expression. Both the wnt3a/wnt8 and cdx1a/cdx4 morphant embryos failed to promote somitogenesis during mid-segmentation. These data indicate that the cdx genes mediate Wnt signaling and play essential roles in the morphogenesis of the posterior body in zebrafish.