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Most cave-obligate species (troglobionts) have small ranges due to limited dispersal ability and the isolated nature of cave habitats. The troglobiontic linyphiid spider Phanetta subterranea (Emerton, 1875), the only member of its genus, is a notable exception to this pattern; it has been reported from more counties and caves than any other troglobiont in North America. As many troglobionts exhibit significant genetic differentiation between populations over even small geographic distances, it has been hypothesized that Phanetta may comprise multiple, genetically distinct lineages. To test this hypothesis, we examined genetic diversity in Phanetta across its range at the mitochondrial cytochrome c oxidase subunit I gene for 47 individuals from 40 caves, distributed across seven states and 37 counties. We found limited genetic differentiation across the species’ range with haplotypes shared by individuals collected up to 600 km apart. Intraspecific nucleotide diversity was 0.006 +/- 0.005 (mean +/- SD), and the maximum genetic p-distance observed between any two individuals was 0.022. These values are within the typical range observed for other spider species. Thus, we found no evidence of cryptic genetic diversity in Phanetta. Our observation of low genetic diversity across such a broad distribution raises the question of how these troglobiontic spiders have managed to disperse so widely.

The copepod crustacean fauna collected from subterranean habitats, including caves, wells, and the hyporheos of streams in and near the Interior Low Plateaus of the United States is dominated by Cyclopoida, with 39 species, followed by Harpacticoida with 9, and Calanoida with 2. Nearly all of the harpacticoid and calanoid species are widespread, primarily surface-dwelling generalists. Fourteen of the cyclopoids, members of the genera Diacyclops, Itocyclops, Megacyclops, and Rheocyclops, are apparently obligate stygobionts or hyporheic. Several of the species that are more strongly modified for subterranean existence occur only in the more southern, unglaciated areas. Our sampling data support the hypothesis that the more specialized, groundwater-interstitial species have been unable to disperse into previously glaciated regions; whereas some, less-specialized species may have invaded groundwaters from surface habitats as the glaciers receded.

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.

The Notch signaling pathway controls differentiation of hair cells and supporting cells in the vertebrate inner ear. Here, we have investigated whether Numb, a known regulator of Notch activity in Drosophila, is involved in this process in the embryonic chick. The chicken homolog of Numb is expressed throughout the otocyst at early stages of development and is concentrated at the basal pole of the cells. It is asymmetrically allocated at some cell divisions, as in Drosophila, suggesting that it could act as a determinant inherited by one of the two daughter cells and favoring adoption of a hair-cell fate. To test the implication of Numb in hair cell fate decisions and the regulation of Notch signaling, we used different methods to overexpress Numb at different stages of inner ear development. We found that sustained or late Numb overexpression does not promote hair cell differentiation, and Numb does not prevent the reception of Notch signaling. Surprisingly, none of the Numb-overexpressing cells differentiated into hair cells, suggesting that high levels of Numb protein could interfere with intracellular processes essential for hair cell survival. However, when Numb was overexpressed early and more transiently during ear development, no effect on hair cell formation was seen. These results suggest that in the inner ear at least, Numb does not significantly repress Notch activity and that its asymmetric distribution in dividing precursor cells does not govern the choice between hair cell and supporting cell fates.

Formation of somites, the rudiments of vertebrate body segments, is an oscillatory process governed by a gene-expression oscillator, the segmentation clock. This operates in each cell of the presomitic mesoderm (PSM), but the individual cells drift out of synchrony when Delta/Notch signalling fails, causing gross anatomical defects. We and others have suggested that this is because synchrony is maintained by pulses of Notch activation, delivered cyclically by each cell to its neighbours, that serve to adjust or reset the phase of the intracellular oscillator. This, however, has never been proved. Here, we provide direct experimental evidence, using zebrafish containing a heat-shock-driven transgene that lets us deliver artificial pulses of expression of the Notch ligand DeltaC. In DeltaC-defective embryos, in which endogenous Notch signalling fails, the artificial pulses restore synchrony, thereby rescuing somite formation. The spacing of segment boundaries produced by repetitive heat-shocking varies according to the time interval between one heat-shock and the next. The induced synchrony is manifest both morphologically and at the level of the oscillations of her1, a core component of the intracellular oscillator. Thus, entrainment of intracellular clocks by periodic activation of the Notch pathway is indeed the mechanism maintaining cell synchrony during somitogenesis.

A 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.

FBW7 is a crucial component of an SCF-type E3 ubiquitin ligase, which mediates degradation of an array of different target proteins. The Fbw7 locus comprises three different isoforms, each with its own promoter and each suspected to have a distinct set of substrates. Most FBW7 targets have important functions in developmental processes and oncogenesis, including Notch proteins, which are functionally important substrates of SCF(Fbw7). Notch signalling controls a plethora of cell differentiation decisions in a wide range of species. A prominent role of this signalling pathway is that of mediating lateral inhibition, a process where exchange of signals that repress Notch ligand production amplifies initial differences in Notch activation levels between neighbouring cells, resulting in unequal cell differentiation decisions. Here we show that the downstream Notch signalling effector HES5 directly represses transcription of the E3 ligase Fbw7β, thereby directly bearing on the process of lateral inhibition. Fbw7(Δ/+) heterozygous mice showed haploinsufficiency for Notch degradation causing impaired intestinal progenitor cell and neural stem cell differentiation. Notably, concomitant inactivation of Hes5 rescued both phenotypes and restored normal stem cell differentiation potential. In silico modelling suggests that the NICD/HES5/FBW7β positive feedback loop underlies Fbw7 haploinsufficiency. Thus repression of Fbw7β transcription by Notch signalling is an essential mechanism that is coupled to and required for the correct specification of cell fates induced by lateral inhibition., Competing Interests: The authors have declared that no competing interests exist.

Somites are formed from the presomitic mesoderm (PSM) and give rise to the axial skeleton and skeletal muscles. The PSM is dynamic; somites are generated at the anterior end, while the posterior end is continually renewed with new cells entering from the tailbud progenitor region. Which genes control the conversion of tailbud progenitors into PSM and how is this process coordinated with cell movement? Using loss- and gain-of-function experiments and heat-shock transgenics we show in zebrafish that the transcription factor Mesogenin 1 (Msgn1), acting with Spadetail (Spt), has a central role. Msgn1 allows progression of the PSM differentiation program by switching off the progenitor maintenance genes ntl, wnt3a, wnt8 and fgf8 in the future PSM cells as they exit from the tailbud, and subsequently induces expression of PSM markers such as tbx24. msgn1 is itself positively regulated by Ntl/Wnt/Fgf, creating a negative-feedback loop that might be crucial to regulate homeostasis of the progenitor population until somitogenesis ends. Msgn1 drives not only the changes in gene expression in the nascent PSM cells but also the movements by which they stream out of the tailbud into the PSM. Loss of Msgn1 reduces the flux of cells out of the tailbud, producing smaller somites and an enlarged tailbud, and, by delaying exhaustion of the progenitor population, results in supernumerary tail somites. Through its combined effects on gene expression and cell movement, Msgn1 (with Spt) plays a key role both in genesis of the paraxial mesoderm and in maintenance of the progenitor population from which it derives.

We 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.

Background & Aims: Ablation of Notch signaling within the intestinal epithelium results in loss of proliferating crypt progenitors due to their conversion into postmitotic secretory cells. We aimed to confirm that Notch was active in stem cells (SCs), investigate consequences of loss of Notch signaling within the intestinal SC compartment, and identify the physiologic ligands of Notch in mouse intestine. Furthermore, we investigated whether the induction of goblet cell differentiation that results from loss of Notch requires the transcription factor Krüppel-like factor 4 (Klf4)., Methods: Transgenic mice that carried a reporter of Notch1 activation were used for lineage tracing experiments. The in vivo functions of the Notch ligands Jagged1 (Jag1), Delta-like1 (Dll1), Delta-like4 (Dll4), and the transcription factor Klf4 were assessed in mice with inducible, gut-specific gene targeting (Vil-Cre-ER(T2))., Results: Notch1 signaling was found to be activated in intestinal SCs. Although deletion of Jag1 or Dll4 did not perturb the intestinal epithelium, inactivation of Dll1 resulted in a moderate increase in number of goblet cells without noticeable effects of progenitor proliferation. However, simultaneous inactivation of Dll1 and Dll4 resulted in the complete conversion of proliferating progenitors into postmitotic goblet cells, concomitant with loss of SCs (Olfm4(+), Lgr5(+), and Ascl2(+)). Klf4 inactivation did not interfere with goblet cell differentiation in adult wild-type or in Notch pathway-deficient gut., Conclusions: Notch signaling in SCs and progenitors is activated by Dll1 and Dll4 ligands and is required for maintenance of intestinal progenitor and SCs. Klf4 is dispensable for goblet cell differentiation in intestines of adult Notch-deficient mice., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The stem cells of the small intestine are multipotent: they give rise, via transit-amplifying cell divisions, to large numbers of columnar absorptive cells mixed with much smaller numbers of three different classes of secretory cells--mucus-secreting goblet cells, hormone-secreting enteroendocrine cells, and bactericide-secreting Paneth cells. Notch signaling is known to control commitment to a secretory fate, but why are the secretory cells such a small fraction of the population, and how does the diversity of secretory cell types arise? Using the mouse as our model organism, we find that secretory cells, and only secretory cells, pass through a phase of strong expression of the Notch ligand Delta1 (Dll1). Onset of this Dll1 expression coincides with a block to further cell division and is followed in much less than a cell cycle time by expression of Neurog3--a marker of enteroendocrine fate--or Gfi1--a marker of goblet or Paneth cell fate. By conditional knock-out of Dll1, we confirm that Delta-Notch signaling controls secretory commitment through lateral inhibition. We infer that cells stop dividing as they become committed to a secretory fate, while their neighbors continue dividing, explaining the final excess of absorptive over secretory cells. Our data rule out schemes in which cells first become committed to be secretory, and then diversify through subsequent cell divisions. A simple mathematical model shows how, instead, Notch signaling may simultaneously govern the commitment to be secretory and the choice between alternative modes of secretory differentiation.

To trace cell lineages in a developing vertebrate and to observe, in vivo, how behaviors of individual cells are affected by the genes they express, we created a zebrafish line containing a transgene called mosaic analysis in zebrafish (MAZe), built around a self-excising hsp70:Cre cassette. Heat shock triggers Cre recombinase-mediated recombination in a random subset of cells, bringing the transcriptional activator Gal4:VP16 under control of the EF1alpha promoter. Gal4-VP16 then activates expression of a fluorescent protein from an upstream activating sequence (UAS) promoter. Marked clones of cells expressing any desired gene product can be generated by crossing MAZe fish with other lines containing UAS-driven transgenes. The number of clones induced, and their time of origin, could be varied by adjusting heat-shock timing and duration. As an alternative to heat shock, we introduced Cre under a tissue-specific promoter in MAZe fish to generate clones in a designated tissue.

Unlike mammals, birds regenerate auditory hair cells (HCs) after injury. During regeneration, mature non-sensory supporting cells (SCs) leave quiescence and convert into HCs, through non-mitotic or mitotic mechanisms. During embryogenesis, Notch ligands from nascent HCs exert lateral inhibition, restricting HC production. Here, we examined whether Notch signaling (1) is needed in mature birds to maintain the HC/SC pattern in the undamaged auditory epithelium or (2) governs SC behavior once HCs are injured. We show that Notch pathway genes are transcribed in the mature undamaged epithelium, and after HC injury, their transcription is upregulated in the region of highest mitotic activity. In vitro treatment with DAPT, an inhibitor of Notch activity, had no effect on SCs in the undamaged epithelium. Following HC damage, DAPT had no direct effect on SC division. However, after damage, DAPT caused excessive regeneration of HCs at the expense of SCs, through both mitotic and non-mitotic mechanisms. Conversely, overexpression of activated Notch in SCs after damage caused them to maintain their phenotype and inhibited HC regeneration. Therefore, signaling through Notch is not required for SC quiescence in the healthy epithelium or to initiate HC regeneration after damage. Rather, Notch prevents SCs from regenerating excessive HCs after damage.

The 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 the early operation of the segmentation clock and the later definition and differentiation of the somites are beginning to be understood.

We now have a wealth of information about the molecular signals that act on cells in embryos, but how do the control systems based on these signals generate pattern and govern the timing of developmental events? Here, I discuss four examples to show how mathematical modeling and quantitative experimentation can give some useful answers. The examples concern the Bicoid gradient in the early Drosophila embryo, the dorsoventral patterning of a frog embryo by bone morphogenetic protein signals, the auxin-mediated patterning of plant meristems, and the Notch-dependent somite segmentation clock.

The vertebrate body axis is subdivided into repeated segments, best exemplified by the vertebrae that derive from embryonic somites. The number of somites is precisely defined for any given species but varies widely from one species to another. To determine the mechanism controlling somite number, we have compared somitogenesis in zebrafish, chicken, mouse and corn snake embryos. Here we present evidence that in all of these species a similar 'clock-and-wavefront' mechanism operates to control somitogenesis; in all of them, somitogenesis is brought to an end through a process in which the presomitic mesoderm, having first increased in size, gradually shrinks until it is exhausted, terminating somite formation. In snake embryos, however, the segmentation clock rate is much faster relative to developmental rate than in other amniotes, leading to a greatly increased number of smaller-sized somites.

Somite segmentation depends on a gene expression oscillator or clock in the posterior presomitic mesoderm (PSM) and on read-out machinery in the anterior PSM to convert the pattern of clock phases into a somite pattern. Notch pathway mutations disrupt somitogenesis, and previous studies have suggested that Notch signalling is required both for the oscillations and for the read-out mechanism. By blocking or overactivating the Notch pathway abruptly at different times, we show that Notch signalling has no essential function in the anterior PSM and is required only in the posterior PSM, where it keeps the oscillations of neighbouring cells synchronized. Using a GFP reporter for the oscillator gene her1, we measure the influence of Notch signalling on her1 expression and show by mathematical modelling that this is sufficient for synchronization. Our model, in which intracellular oscillations are generated by delayed autoinhibition of her1 and her7 and synchronized by Notch signalling, explains the observations fully, showing that there are no grounds to invoke any additional role for the Notch pathway in the patterning of somite boundaries in zebrafish., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

The regular pattern of somite segmentation depends on a clock, the somite segmentation clock, in the form of a gene expression oscillator, operating in the presomitic mesoderm (the PSM) at the tail end of the vertebrate embryo. Genetic screens and other approaches have identified a variety of genes, including components and targets of the Notch signalling pathway, that show transcriptional oscillations in this region and appear to be necessary for correct segmentation. Mathematical modelling shows that the oscillations could plausibly be generated by a simple mechanism of delayed negative feedback, based on autoinhibition of Notch target genes of the Hes/her family by their own protein products. To move beyond plausible models to an experimentally validated theory, however, it is necessary to measure the parameters on which the proposed model is based and to devise ways of probing the dynamics of the system by means of timed disturbances so as to compare with the model's predictions. Some progress is being made in these directions., (Copyright 2007 Wiley-Liss, Inc.)

Notch signalling is well-known to mediate lateral inhibition in inner ear sensory patches, so as to generate a balanced mixture of sensory hair cells and supporting cells. Recently, however, we have found that ectopic Notch activity at an early stage can induce the formation of ectopic sensory patches. This suggests that Notch activity may have two different functions in normal ear development, acting first to promote the formation of the prosensory patches, and then later to regulate hair-cell production within the patches. The Notch ligand Serrate1 (Jag1 in mouse and humans) is expressed in the patches from an early stage and may provide Notch activation during the prosensory phase. Here, we test whether Notch signalling is actually required for prosensory patch development. When we block Notch activation in the chick embryo using the gamma-secretase inhibitor DAPT, we see a complete loss of prosensory epithelial cells in the anterior otocyst, where they are diverted into a neuroblast fate via failure of Delta1-dependent lateral inhibition. The cells of the posterior prosensory patch remain epithelial, but expression of Sox2 and Bmp4 is drastically reduced. Expression of Serrate1 here is initially almost normal, but subsequently regresses. The patches of sensory hair cells that eventually develop are few and small. We suggest that, in normal development, factors other than Notch activity initiate Serrate1 expression. Serrate1, by activating Notch, then drives the expression of Sox2 and Bmp4, as well as expression of the Serrate1 gene itself. The positive feedback maintains Notch activation and thereby preserves and perhaps extends the prosensory state, leading eventually to the development of normal sensory patches.

The somites of the vertebrate embryo are clocked out sequentially from the presomitic mesoderm (PSM) at the tail end of the embryo. Formation of each somite corresponds to one cycle of oscillation of the somite segmentation clock--a system of genes whose expression switches on and off periodically in the cells of the PSM. We have previously proposed a simple mathematical model explaining how the oscillations, in zebrafish at least, may be generated by a delayed negative feedback loop in which the products of two Notch target genes, her1 and her7, directly inhibit their own transcription, as well as that of the gene for the Notch ligand DeltaC; Notch signalling via DeltaC keeps the oscillations of neighbouring cells in synchrony. Here we subject the model to quantitative tests. We show how to read temporal information from the spatial pattern of stripes of gene expression in the anterior PSM and in this way obtain values for the biosynthetic delays and molecular lifetimes on which the model critically depends. Using transgenic lines of zebrafish expressing her1 or her7 under heat-shock control, we confirm the regulatory relationships postulated by the model. From the timing of somite segmentation disturbances following a pulse of her7 misexpression, we deduce that although her7 continues to oscillate in the anterior half of the PSM, it governs the future somite segmentation behaviour of the cells only while they are in the posterior half. In general, the findings strongly support the mathematical model of how the somite clock works, but they do not exclude the possibility that other oscillator mechanisms may operate upstream from the her7/her1 oscillator or in parallel with it.

Notch signalling by the ligand Delta-like 4 (Dll4) is essential for normal vascular remodelling, yet the precise way in which the pathway influences the behaviour of endothelial cells remains a mystery. Using the embryonic zebrafish, we show that, when Dll4-Notch signalling is defective, endothelial cells continue to migrate and proliferate when they should normally stop these processes. Artificial overactivation of the Notch pathway has opposite consequences. When vascular endothelial growth factor (Vegf) signalling and Dll4-Notch signalling are both blocked, the endothelial cells remain quiescent. Thus, Dll4-Notch signalling acts as an angiogenic ;off' switch by making endothelial cells unresponsive to Vegf.

The lining of the intestine is renewed at an extraordinary rate, outpacing all other tissues in the vertebrate body. The renewal process is neatly organized in space, so that the whole production line, from the ever-youthful stem cells to their dying, terminally differentiated progeny, is laid out to view in histological sections. A flurry of recent papers has clarified the key regulatory signals and brought us to the point where we can begin to give a coherent account, for at least one tissue, of how these signals collaborate to organize the architecture and behaviour of a stem-cell system.

Each of the sensory patches in the epithelium of the inner ear is a mosaic of hair cells and supporting cells. Notch signalling is thought to govern this pattern of differentiation through lateral inhibition. Recent experiments in the chick suggest, however, that Notch signalling also has a prior function - inductive rather than inhibitory - in defining the prosensory patches from which the differentiated cells arise. Several Notch ligands are expressed in each patch, but their individual roles in relation to the two functions of Notch signalling are unclear. We have used a Cre-LoxP approach to knock out two of these ligands, Delta1 (Dll1) and Jagged1 (Jag1), in the mouse ear. In the absence of Dll1, auditory hair cells develop early and in excess, in agreement with the lateral inhibition hypothesis. In the absence of Jag1, by contrast, the total number of these cells is strongly reduced, with complete loss of cochlear outer hair cells and some groups of vestibular hair cells, indicating that Jag1 is required for the prosensory inductive function of Notch. The number of cochlear inner hair cells, however, is almost doubled. This correlates with loss of expression of the cell cycle inhibitor p27(Kip1) (Cdkn1b), suggesting that signalling by Jag1 is also needed to limit proliferation of prosensory cells, and that there is a core part of this population whose prosensory character is established independently of Jag1-Notch signalling. Our findings confirm that Notch signalling in the ear has distinct prosensory and lateral-inhibitory functions, for which different ligands are primarily responsible.

The Tübingen large-scale zebrafish genetic screen completed in 1996 identified a set of five genes required for orderly somite segmentation. Four of them have been molecularly identified and three were found to code for components of the Notch pathway, which are required for the coordinated oscillation of gene expression, known as the segmentation clock, in the presomitic mesoderm (PSM). Here, we show that the final member of the group, beamter (bea), codes for the Notch ligand DeltaC, and we present and characterize two new alleles, including one allele encoding for a protein truncated in the 7th EGF repeat and an allele deleting only the DSL domain which was previously shown to be necessary for ligand function. Interestingly however, when we over-express any of the mutant deltaC mRNAs, we observe antimorphic effects on both hindbrain neurogenesis and hypochord formation. Expression of bea/deltaC oscillates in the PSM, and a triple fluorescent in situ analysis of its oscillation in relation to that of other oscillating genes in the PSM reveals differences in subcellular localization of the oscillating mRNAs in individual cells in different oscillation phases. Mutations in aei/deltaD and bea/deltaC differ in the way they disrupt the oscillating expression of her1 and deltaC. Furthermore, we find that the double mutants have significantly stronger defects in hypochord formation but not in somitogenesis or hindbrain neurogenesis, indicating genetically that the two delta's may function either semi-redundantly or distinctly, depending upon context.

Hair cells of the vertebrate inner ear are directional mechanosensors: they have a polarity, defined by a vector in the plane of the sensory epithelium. It has been suggested that this polarity might be controlled by genes homologous to those that control planar cell polarity (PCP) in Drosophila, and vertebrate homologues of the Drosophila PCP genes Van Gogh/strabismus and flamingo/starry night are indeed essential for normal hair cell PCP. The underlying molecular mechanism is unclear, however. Although the PCP protein Flamingo shows a polarized intracellular distribution in the fly, it is unknown whether this is necessary for its function. Here, we describe the expression pattern of a flamingo homologue, c-flamingo-1 (c-fmi-1), in the developing chick ear and show that its protein product, like that of flamingo in the fly, has a polarized distribution in each hair cell, defining an axis that corresponds to the structural PCP axis. This conservation between fly and vertebrate suggests that the polarized protein localization is functionally important. In the basilar papilla, the same localization is seen in supporting cells also, suggesting that supporting cells are cryptically polarized, despite having no overt structural polarity; they may thus participate in PCP signal transmission across the sensory patch.

The transparency of the juvenile zebrafish and its genetic advantages make it an attractive model for study of cell turnover in the gut. BrdU labelling shows that the gut epithelium is renewed in essentially the same way as in mammals: the villi are lined with non-dividing differentiated cells, while cell division is confined to the intervillus pockets. New cells produced in the pockets take about 4 days to migrate out to the tips of the villi, where they die. We have generated monoclonal antibodies to identify the absorptive and secretory cells in the epithelium, and we have used these antibodies to examine the part that Delta-Notch signalling plays in producing the diversity of intestinal cell types. Several Notch receptors and ligands are expressed in the gut. In particular, the Notch ligand DeltaD (Delta1 in the mouse) is expressed in cells of the secretory lineage. In an aei mutant, where DeltaD is defective, secretory cells are overproduced. In mind bomb (mib), where all Delta-Notch signalling is believed to be blocked, almost all the cells in the 3-day gut epithelium adopt a secretory character. Thus, secretory differentiation appears to be the default in the absence of Notch activation, and lateral inhibition mediated by Delta-Notch signalling is required to generate a balanced mixture of absorptive and secretory cells. These findings demonstrate the central role of Notch signalling in the gut stem-cell system and establish the zebrafish as a model for study of the mechanisms controlling renewal of gut epithelium.

Lateral inhibition mediated by Notch is thought to generate the mosaic of hair cells and supporting cells in the inner ear, but the effects of the activated Notch protein itself have never been directly tested. We have explored the role of Notch signalling by transiently overexpressing activated Notch (NICD) in the chick otocyst. We saw two contrasting consequences, depending on the time and site of gene misexpression: (1) inhibition of hair-cell differentiation within a sensory patch; and (2) induction of ectopic sensory patches. We infer that Notch signalling has at least two functions during inner ear development. Initially, Notch activity can drive cells to adopt a prosensory character, defining future sensory patches. Subsequently, Notch signalling within each such patch mediates lateral inhibition, restricting the proportion of cells that differentiate as hair cells so as to generate the fine-grained mixture of hair cells and supporting cells.

Drosophila fringe and its homologues in vertebrates code for glycosyltransferases that modify Notch, altering the sensitivity of this receptor protein to its ligands Delta and Serrate and, thereby, playing an essential part in the demarcation of tissue boundaries. We describe the isolation and characterization of three zebrafish (Danio rerio) fringe homologues: lunatic fringe (lfng), radical fringe (rfng), and manic fringe (mfng). In addition to the sites previously described (Prince et al. [2001] Mech. Dev. 105:175-180; Leve et al. [ 2001] Dev. Genes Evol. 211:493-500), lfng is also expressed in the sensory patches of the inner ear. The newly described rfng is expressed in adaxial cells, tectum, rhombomere boundaries, and formed somites, but the expression of mfng is only detectable by reverse transcription-polymerase chain reaction and not by whole-mount in situ hybridization (WISH) during early embryonic development; later, it is expressed in the sensory patches of the ear. In mib mutants, where Notch signaling is defective and rhombomere boundaries fail to form, the rfng expression in hindbrain is almost completely lost. None of the three zebrafish fringe genes is detectably expressed in the posterior presomitic mesoderm, suggesting that, in contrast with chick and mouse, the somitogenesis oscillator in this tissue in the zebrafish does not depend on Fringe activity., ((c) 2004 Wiley-Liss, Inc.)

Delta proteins activate Notch through a binding reaction that depends on their extracellular domains; but the intracellular (C-terminal) domains of the Deltas also have significant functions. All classes of vertebrates possess a subset of Delta proteins with a conserved ATEV* motif at their C termini. These ATEV Deltas include Delta1 and Delta4 in mammals and DeltaD and DeltaC in the zebrafish. We show that these Deltas associate with the membrane-associated scaffolding proteins MAGI1, MAGI2 and MAGI3, through a direct interaction between the C termini of the Deltas and a specific PDZ domain (PDZ4) of the MAGIs. In cultured cells and in subsets of cells in the intact zebrafish embryo, DeltaD and MAGI1 are co-localized at the plasma membrane. The interaction and the co-localization can be abolished by injection of a morpholino that blocks the mRNA splicing reaction that gives DeltaD its terminal valine, on which the interaction depends. Embryos treated in this way appear normal with respect to some known functions of DeltaD as a Notch ligand, including the control of somite segmentation, neurogenesis, and hypochord formation. They do, however, show an anomalous distribution of Rohon-Beard neurons in the dorsal neural tube, suggesting that the Delta-MAGI interaction may play some part in the control of neuron migration.