SUMMARYA gene expression oscillator called the segmentation clock controls somite segmentation in the vertebrate embryo. In zebrafish, the oscillatory transcriptional repressor genes her1 and her7 are crucial for genesis of the oscillations, which are thought to arise from negative autoregulation of these genes. The period of oscillation is predicted to depend on delays in the negative-feedback loop, including, most importantly, the transcriptional delay -the time taken to make each molecule of her1 or her7 mRNA. her1 and her7 operate in parallel. Loss of both gene functions, or mutation of her1 combined with knockdown of Hes6, which we show to be a binding partner of Her7, disrupts segmentation drastically. However, mutants in which only her1 or her7 is functional show only mild segmentation defects and their oscillations have almost identical periods. This is unexpected because the her1 and her7 genes differ greatly in length. We use transgenic zebrafish to measure the RNA polymerase II elongation rate, for the first time, in the intact embryo. This rate is unexpectedly rapid, at 4.8 kb/minute at 28.5°C, implying that, for both genes, the time taken for transcript elongation is insignificant compared with other sources of delay, explaining why the mutants have similar clock periods. Our computational model shows how loss of her1 or her7 can allow oscillations to continue with unchanged period but with reduced amplitude and impaired synchrony, as manifested in the in situ hybridisation patterns of the single mutants. RESEARCH ARTICLE Gene length and the somite clockThis formula is derived for the idealised case of a single 'her1/7' autoinhibitory gene or, equivalently, of a pair of genes, her1 and her7, that have the same delays, lifetimes and regulation. Computer modelling shows that if her1 and her7 have somewhat different delays and lifetimes but are co-regulated, oscillations will occur with a period that is a compromise between that for a pure her1 oscillator and that for a pure her7 oscillator. Mutants in which her1 remains intact but her7 is functionally null, or vice versa, have recently become available, and in this paper we use them to test this prediction. Because the her1 and her7 genes are very different in length ( Fig. 2A), we anticipated that they should have different transcriptional delays, leading to different periods of oscillation. To our surprise, we found that the difference of period is actually very small. To resolve this paradox, we measured the elongation rate of RNA polymerase II (RNA Pol II), for the first time in vivo in a vertebrate. The value, as measured in the PSM cells of the zebrafish, is 4.8 kb/minute at 28.5°C. This unexpectedly high rate means that the time taken to transcribe the two genes is so short as to be insignificant in comparison with other sources of delay, such as the time required for splicing. These findings reconcile our theory with the experimental observations and remove an important objection to the proposition that her1 and her7 are pacemakers of the seg...

N No ot tc ch h s si ig gn na al li in ng g, , t th he e s se eg gm me en nt ta at ti io on n c cl lo oc ck k, , a an nd d t th he e p pa at tt te er rn ni in ng g o of f v ve er rt te eb br ra at te e s so om mi it te es s In one way or another, at one stage or another, almost every tissue in an animal body depends for its patterning on the Notch cell-cell signaling pathway [1]. The evidence from mutants is clear: disrupted Notch signaling entails disrupted pattern. The challenge is to define precisely what it is that Notch signaling does in any given case, and when it does it. This problem is posed in a particularly striking and curious way by the phenomena of somitogenesis -the process by which the vertebrate embryo lays down the regular sequence of tissue blocks that will give rise to the musculoskeletal segments of the neck, trunk, and tail.These blocks of embryonic tissue, the somites, are arranged symmetrically in a neat, repetitive pattern on either side of the central body axis. Each somite is separated from the next by a cleft -the segment boundary; and each somite has a definite polarity, with an anterior portion and posterior portion expressing different sets of genes [2]. Mutations in components of the Notch signaling pathway play havoc with this whole pattern: although somites may eventually form, the segment boundaries are irregular and randomly positioned, and the regular antero-posterior polarity of individual somites is lost. Genetic screens for mutations that disrupt segmentation in this way chiefly identify Notch pathway components as the critical players. Notch signaling is clearly central to somitogenesis [3][4][5][6]. But precisely how? N No ot tc ch h p pa at th hw wa ay y c co om mp po on ne en nt ts s c ca an n b be e w wi ir re ed d t to og ge et th he er r i in n d di if ff fe er re en nt t w wa ay ys s f fo or r d di if ff fe er re en nt t o ou ut tc co om me es sIn general, the function of the canonical Notch pathway is to coordinate gene expression in contiguous cells. It does this in a particularly direct way. The signal-sending cell expresses a Notch ligand (belonging to either the Delta or the Serrate/Jagged subfamily) on its surface; this binds to the receptor, Notch, in the membrane of the signal-receiving cell and thereby triggers cleavage of Notch, releasing an intracellular fragment, the Notch intracellular domain (NICD); NICD translocates to the nucleus, where it acts as a transcriptional regulator [1,7] (Figure 1). The main -or at least, the best-studied -targets of direct regulation by NICD are the members of the Hairy/E(spl) family (Hes genes in mammals, her genes in zebrafish) [8,9]; these code for inhibitory basic helix-loop-helix (bHLH) transcriptional A Ab bs st tr ra ac ct tThe Notch signaling pathway has multifarious functions in the organization of the developing vertebrate embryo. One of its most fundamental roles is in the emergence of the regular pattern of somites that will give rise to the musculoskeletal structures of the trunk. The parts it plays in t...

FGFs and Wnts are important morphogens during midbrain development, but their importance and potential interactions during neurogenesis are poorly understood. We have employed a combination of genetic and pharmacological manipulations in zebrafish to show that during neurogenesis FGF activity occurs as a gradient along the anterior-posterior axis of the dorsal midbrain and directs spatially dynamic expression of the Hairy gene her5. As FGF activity diminishes during development, Her5 is lost and differentiation of neuronal progenitors occurs in an anterior-posterior manner. We generated mathematical models to explain how Wnt and FGFs direct the spatial differentiation of neurons in the midbrain through Wnt regulation of FGF signalling. These models suggested that a negative-feedback loop controlled by Wnt is crucial for regulating FGF activity. We tested Sprouty genes as mediators of this regulatory loop using conditional mouse knockouts and pharmacological manipulations in zebrafish. These reveal that Sprouty genes direct the positioning of early midbrain neurons and are Wnt responsive in the midbrain. We propose a model in which Wnt regulates FGF activity at the isthmus by driving both FGF and Sprouty gene expression. This controls a dynamic, posteriorly retracting expression of her5 that directs neuronal differentiation in a precise spatiotemporal manner in the midbrain.

SUMMARYWe describe the production and characterisation of two monoclonal antibodies, zdc2 and zdd2, directed against the zebrafish Notch ligands DeltaC and DeltaD, respectively. We use our antibodies to show that these Delta proteins can bind to one another homo-and heterophilically, and to study the localisation of DeltaC and DeltaD in the zebrafish nervous system and presomitic mesoderm (PSM). Our findings in the nervous system largely confirm expectations from previous studies, but in the PSM we see an unexpected pattern in which the localisation of DeltaD varies according to the level of expression of DeltaC: in the anterior PSM, where DeltaC is plentiful, the two proteins are colocalised in intracellular puncta, but in the posterior PSM, where DeltaC is at a lower level, DeltaD is seen mainly on the cell surface. Forced overexpression of DeltaC reduces the amount of DeltaD on the cell surface in the posterior PSM; conversely, loss-of-function mutation of DeltaC increases the amount of DeltaD on the cell surface in the anterior PSM. These findings suggest an explanation for a long-standing puzzle regarding the functions of the two Delta proteins in the somite segmentation clock -an explanation that is based on the proposition that they associate heterophilically to activate Notch.

The somite segmentation clock is a robust oscillator used to generate regularly-sized segments during early vertebrate embryogenesis. It has been proposed that the clocks of neighbouring cells are synchronised via inter-cellular Notch signalling, in order to overcome the effects of noisy gene expression. When Notch-dependent communication between cells fails, the clocks of individual cells operate erratically and lose synchrony over a period of about 5 to 8 segmentation clock cycles (2–3 hours in the zebrafish). Here, we quantitatively investigate the effects of stochasticity on cell synchrony, using mathematical modelling, to investigate the likely source of such noise. We find that variations in the transcription, translation and degradation rate of key Notch signalling regulators do not explain the in vivo kinetics of desynchronisation. Rather, the analysis predicts that clock desynchronisation, in the absence of Notch signalling, is due to the stochastic dissociation of Her1/7 repressor proteins from the oscillating her1/7 autorepressed target genes. Using in situ hybridisation to visualise sites of active her1 transcription, we measure an average delay of approximately three minutes between the times of activation of the two her1 alleles in a cell. Our model shows that such a delay is sufficient to explain the in vivo rate of clock desynchronisation in Notch pathway mutant embryos and also that Notch-mediated synchronisation is sufficient to overcome this stochastic variation. This suggests that the stochastic nature of repressor/DNA dissociation is the major source of noise in the segmentation clock.

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E El lu uc ci id da at ti in ng g d de ev ve el lo op pm me en nt ta al l g ge en ne e n ne et tw wo or rk ks s This year's annual meeting of the British Societies for Cell and Developmental Biology focused on several aspects of signaling mechanisms and gene and protein networks in relation to cell architecture, animal and plant development and evolution. Here, we summarize some highlights on morphogen gradients, mouse embryo genetic manipulations, stem-cell biology and evolution and gene networks during evolution and development.

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