Abstract
Rhythmic production of vertebral precursors, the somites, causes bilateral columns of embryonic segments to form. This process involves a molecular oscillator — the segmentation clock — whose signal is translated into a spatial, periodic pattern by a complex signalling gradient system within the presomitic mesoderm (PSM). In mouse embryos, Wnt signalling has been implicated in both the clock and gradient mechanisms, but how the Wnt pathway can perform these two functions simultaneously remains unclear. Here, we use a yellow fluorescent protein (YFP)-based, real-time imaging system in mouse embryos to demonstrate that clock oscillations are independent of β-catenin protein levels. In contrast, we show that the Wnt-signalling gradient is established through a nuclear β-catenin protein gradient in the posterior PSM. This gradient of nuclear β-catenin defines the size of the oscillatory field and controls key aspects of PSM maturation and segment formation, emphasizing the central role of Wnt signalling in this process.
Somites are transient epithelial structures that give rise to the axial skeleton, and are the first overt sign of a metameric body plan in vertebrate embryos. They are produced by segmentation of the paraxial mesoderm, a periodic process that has been associated with a molecular oscillator or segmentation clock identified in studies demonstrating periodic transcription of cyclic genes in the PSM1. A large network of signalling genes that are involved in the Notch, Wnt and fibroblast growth factor (FGF) pathways was shown previously to be rhythmically expressed in the posterior PSM with a periodicity matching that of somite production1–3. Although the molecular nature of the oscillator’s pacemaker remains unclear, it has been shown in mouse embryos that intact Wnt signalling is required for oscillations to occur2,4,5. In addition, gradients of Wnt, FGF and retinoic acid signalling translate the signalling pulse generated by the clock into the spatial periodic pattern of segments2,6,7. Activity of the Wnt/FGF gradient is highest in the posterior embryo; however, the antagonistic retinoic acid-signalling gradient peaks towards the anterior part of the embryo. The Wnt/FGF gradient has been shown to set up a threshold defining a PSM level — called the determination front — at which cells become responsive to the clock signal. At the determination front, mRNA oscillations cease and cells exhibit striped expression of key factors, such as Mesoderm posterior 2 (Mesp2), which specify somite polarity and future segment boundaries8,9. Here, we examine the role of the Wnt gradient in somitogenesis and determine how it interconnects with the function of the Wnt pathway in clock oscillations of the PSM.
Using immunohistochemistry, we observed a clear posterior-to-anterior nuclear gradient of β-catenin (a key intracellular mediator of the Wnt pathway) in the PSM of mouse embryos (Fig. 1a–c). A posterior gradient for β-catenin was observed in all embryos analysed (n = 19), irrespective of their segmentation clock phase, as determined by the mRNA expression pattern of the cyclic Wnt target gene Axin2 (refs 2, 10, 11; Supplementary Information, Fig. S1). To examine the role of the nuclear β-catenin gradient in somitogenesis, we used a conditional strategy to selectively delete or stabilize β-catenin in the PSM of mouse embryos. First, to selectively delete β-catenin in the PSM, mice carrying a conditional β-cateninfloxed allele12 were crossed with the T–Cre mouse line in which Cre recombinase is driven by the T (Brachyury) promoter. In this transgenic mouse line, Cre is expressed in precursors of the paraxial mesoderm located in the primitive streak. In embryos homozygous for the conditional-null β-cateninfloxdel allele in primitive streak descendents, a few abnormal somites formed anteriorly and a severe axial truncation was observed (Supplementary Information, Fig. S2 and data not shown). This confirms the requirement of Wnt/β-catenin signalling in antero-posterior axis formation and gastrulation; however, further analysis of the role of β-catenin during somitogenesis was not feasible with this phenotype.
Figure 1.
Conditional stabilization of β-catenin in mouse PSM disrupts somite formation. Fluorescence immunodetection of β-catenin (green) in saggital sections through the PSM of control (a–c) and mutant β-catenindel(ex3)/+–T–Cre embryo (d–f). Nuclei were counterstained with DAPI (blue) and anterior is to the left. (a) A graded distribution of β-catenin protein along the antero-posterior axis showed predominant cytoplasmic localization in the anterior PSM (shown at higher magnification in b), and nuclear localization in the posterior PSM (shown at higher magnification in c). (d) Mutant embryo showing elevated, uniform levels of β-catenin throughout the PSM. Nuclear localization was found both in the anterior PSM (shown at higher magnification in e), as well as in the posterior PSM (shown at higher magnification in f). Scanning electron microscopy of control (g) and mutant β-catenindel(ex3)/+–T–Cre embryo (h). The ectoderm was partially removed during embryo processing. Nine somites were formed in the control embryo (g); however, no somites were visible in the mutant embryo (h). Note expanded, unsegmented PSM in mutant embryo. Scale bars in a and d represent 50 μm; scale bars in b, c, e and f represent 10 μm.
Next, we used T–Cre mice carrying the conditional gain-of-function allele β-cateninlox(ex3) to disrupt the β-catenin gradient by constitutively stabilizing β-catenin in the PSM. β-cateninlox(ex3) contains loxP sites flanking exon 3, which codes for a crucial sequence recognized by the destruction complex that targets β-catenin for degradation13. This strategy resulted in accumulation of mutant β-catenindel(ex3) protein specifically in mesodermal lineages, including the PSM. Nuclear staining of β-catenin was elevated throughout the entire PSM of β-catenindel(ex3) mutants (Fig. 1d–f), without an appreciable posterior-to-anterior protein gradient, as seen in wild-type embryos (Fig. 1a–c). No significant difference in the proliferative state of the posterior PSM or the tail bud was observed between mutant and wild-type embryos (Supplementary Information, Fig. S1). In mutant embryos carrying the β-catenindel(ex3) allele, no sign of morphological boundary formation or somites could be seen along the body axis (Fig. 1g, h), except for up to four irregular somites seen occasionally in the most anterior aspect of mutant paraxial mesoderm. Mutant embryos could not complete the turning process during gastrulation, exhibited a rounded allantois that failed to fuse with the chorion and died at approximately day 10.5 of development. The accumulation of nuclear β-catenin throughout the PSM was accompanied by upregulation of Wnt signalling, as determined by using the Wnt-reporter mice BAT–Gal14. In β-catenindel(ex3)–BAT–Gal embryos, significant anterior extension of lacZ expression was observed in the PSM (Supplementary Information, Fig. S3; n = 8). The Wnt-responsive gene Axin2 (refs 10, 11) was markedly upregulated in the PSM (Fig. 2a, á; n = 4), consistent with sustained activation of canonical Wnt signalling. Other direct targets of the Wnt pathway involved in PSM patterning were upregulated and shifted anteriorly in mutant β-catenindel(ex3)/+ embryos, compared with control littermates. These include the genes T-box6 (Tbx6; ref. 15), mesogenin1 (Msgn1; ref. 16) and Delta-like 1 (Dll1; refs 17, 18; Fig. 2b, b´; n = 5; c, c´; n = 5; d, d´; n = 2). The expression domain of Fgf8 was slightly extended anteriorly (Fig. 2e, é; n = 4). In addition, the FGF-signalling target Pea3 (ref. 19) was clearly upregulated and shifted anteriorly in the PSM, suggesting that the strength of the FGF-signalling gradient is increased (Fig. 2f, f´; n = 5). The anterior PSM of β-catenindel(ex3)/+ mutants, nevertheless, showed signs of maturation. Thus, markers for the anterior PSM and somites, such as Paraxis20 (Fig. 2g, g´; n = 2), and Raldh2 (Aldh1a2; ref. 21; Fig. 2h, h´; n = 2) were present, but their expression was only transient or reduced, respectively, compared with wild-type embryos. We found that Mesp2 expression, which correlates with the level of the determination front in wild-type embryos (Fig. 3a), was significantly shifted anteriorly in mutant embryos (Fig. 3b, n = 16 and Supplementary Information, Fig. S3). Downstream targets of Mesp2 (Epha4 and Cer1; ref. 22) were expressed in stripes in the anterior PSM of controls but absent from anterior PSM in mutant littermates (Fig. 3c–f; n = 5 for both). Furthermore, the expression of a posterior somite marker Uncx4.1 was clearly downregulated in mutant embryos (Fig. 3g, h; n = 3). Thus, overexpression of β-catenin causes an anterior shift of the determination front and delays the activation of Mesp2 expression (Fig. 3i). In wild-type embryos, the position of the Mesp2 stripe was found to lie outside the β-catenin gradient (Supplementary Information, Fig. S1). These data indicate that downregulation of nuclear β-catenin is required for activation of Mesp2 downstream targets, and explains the absence of morphological boundary formation in mutant embryos.
Figure 2.
Expansion of posterior PSM identity in β-catenindel(ex3)/+ mutant embryos. In situ hybridization of embryonic day 9 control ((a–h), β-catenindel(ex3)/+–T–Cre negative) and corresponding mutant littermates ((á–h´), β-catenindel(ex3)/+–T–Cre). Axin2 (a, á), Tbx6 (b, b´), Msgn1 (c, ć), Dll1 (d, d´), Fgf8 (e, é) and Pea3 (f, f´) showed an expanded expression domain in mutant embryos. Note that this expansion of expression in mutants is both absolute and relative to the total axis length when compared with control littermates. (g, g´) In contrast to the control embryo (g), Paraxis was only expressed transiently in the mutant embryo (g´). (h, h´) A shortened Raldh2 expression domain was found in mutant embryos (h´), compared with control littermates (h).
Figure 3.
Anterior shift of determination front in β-catenindel(ex3)/+ mutant embryos. (a, b) Mesp2 expression in control (a) and mutant (b) embryo littermates. Mesp2 expression was shifted anteriorly in mutants. (c, d) Cer1 in control (c) and mutant (d) embryo littermates. (e, f) Epha4 expression in control (e) and mutant (f) embryo littermates. Epha4 and Cer1 were expressed as stripes in the anterior PSM of control embryos, but this expression domain was absent in mutant embryos. (g, h) Uncx4.1, which marks the posterior aspect of formed somites in control embryos (g), was markedly downregulated in mutant embryos (h). (i) Scheme of Mesp2 expression in control (left side) and mutant (right side) embryos. Note the anterior shift of Mesp2 expression in mutants based on measurements (Supplementary Information, Fig. S3). Lfng showed up to five additional stripes in anterior mutant PSM, of which the most anterior two stripes overlap with Mesp2 as judged from double in situ hybridizations (data not shown). In mutants, the posterior broad expression domain of Lfng was weaker but of a comparable size to control embryos.
Oscillations of the Wnt pathway have been postulated to rely on the function of a group of negative feedback inhibitors of the Wnt pathway, such as Axin2, dickkopf 1 (Dkk1) or Dact1 (refs 2, 3). Periodic expression of these direct Wnt targets is expected to rhythmically alter β-catenin levels in PSM cells. We did not detect visible oscillations of β-catenin protein in the PSM (Supplementary Information, Fig. S1), but fluctuations of small amplitude may, in principle, account for the oscillatory transcription of bona fide targets of the canonical Wnt pathway, such as c-myc (ref. 3) or Axin2 (ref. 2) in the PSM. If this were the case, then oscillations of the Wnt pathway should be disrupted in β-catenindel(ex3)/+ embryos in which a high level of nuclear β-catenin is constantly maintained in PSM cells. To test this hypothesis, we analysed the expression of a cyclic direct Wnt target, Dkk1, using an intronic probe to detect only nascent, pre-mRNA3 (Fig.4a–d). In β-catenindel(ex3)/+mutant embryos, Dkk1 expression levels were higher overall when compared with wild-type embryos, and expression was always detected in the posterior PSM (Fig.4c, d). When mutant littermates were compared after identical staining procedures, the intensity of Dkk1 pre-mRNA expression varied in the posterior PSM, ranging from faint (16 out of 32 embryos) to strong (16 out of 32 embryos) (Fig. 4c, d; arrows). In addition, a clear, striped pattern of active transcription was observed in the PSM of mutant embryos (Fig. 4c, d; arrowheads). Therefore, constitutive expression of high nuclear β-catenin levels does not block dynamic expression of the Wnt cyclic gene, Dkk1, suggesting that Wnt-signalling oscillations still occur in β-catenindel(ex3)/+ mutants.
Figure 4.
Dynamic expression of Wnt and Notch cyclic genes is maintained in β-catenindel(ex3)/+ mutant PSM. (a–d) Intronic Dkk1 pre-mRNA expression in control (a, b) and mutant PSM (c, d). PSM expression was highly variable in control embryos (a, b, arrows). The posterior expression domain in mutant littermates was highly variable, ranging from strong expression (c, arrow; 16 out of 32 embryos) to weak expression (d, arrow; 16 out of 32 embryos). In addition, stripes of expression in the middle PSM were observed in mutant embryos (c, d, arrowheads) but not in controls (a, b). (e–h) In situ hybridization of Lfng in control (e, f) and mutant (g, h) embryos. Control embryos showed a highly dynamic posterior expression domain (e, f, arrows). Supernumerary stripes of Lfng expression were visible in the expanded anterior PSM of mutant embryos (g, h, arrowheads). In the posterior PSM of mutant littermates, expression ranged from weak (g, arrow; 8 out of 22 embryos) to strong (h, arrow; 14 out of 22 embryos). (i–q) Analysis of compound β-cateninlox(ex3)/+–Fgfr1f/KO–T–Cre mutant embryos. Control embryos (i, l, o), β-cateninlox(ex3)/+–Fgfr1f/+–T–Cre single mutant embryos (j, m, p) and compound β-cateninlox(ex3)/+–Fgfr1f/KO–T–Cre double mutants (k, n, q) were hybridized for Dusp4 (i–k), Mesp2 (l–n) and Lfng (o–q). The embryos in (j, k), (m, n), (p, q) were littermates, and were processed and stained together. Note that in compound mutants, the FGF signalling target Dusp4 was downregulated in the PSM (k, n = 8), whereas under these conditions Mesp2 still showed an anterior shift in the enlarged PSM (n, n = 5) compared with wild-type embryos (l); however, this anterior shift was less pronounced compared with the β-cateninlox(ex3)/+–Fgfr f/+–T–Cre single mutants (m). In the anterior PSM of compound mutants, we found several stripes of Lfng expression (q, arrowheads; n = 5), similar to that in the β-cateninlox(ex3)/+–Fgfr1f/+–T–Cre single mutants (p, arrowheads).
To confirm the status of Notch oscillations in β-catenindel(ex3)/+ mutants, expression of the Notch cyclic genes lunatic fringe (Lfng; Fig. 4e–h) and Hairy and enhancer of split 7 (Hes7; ref. 23) was examined. Posterior expression of Lfng in mutant embryos varied considerably in intensity between littermates (Fig. 4g, h; arrows; 14/22 strong expression, 8/22 weak expression), suggesting that in the posterior PSM, Notch oscillations still occur. Up to five stripes of variable width and inter-stripe distance were found in the middle and anterior PSM, both for Lfng (Fig. 4g, h; arrowheads) and for Hes7 mRNA (Supplementary Information, Fig. S4). During development, the total number of stripes increased slowly in the PSM of mutant embryos, paralleling the progressive increase in size of the PSM (Supplementary Information, Fig. S5). To rule out the possibility that the remaining wild-type allele in β-catenindel(ex3)/+ mutant embryos controls the dynamic expression of Lfng, we generated β-catenindel(ex3)/floxdel mutants in which PSM cells express only the gain-of-function allele. The phenotype of these mutants was very similar to that of β-catenindel(ex3)/+ mutants, with several stripes of Lfng in the PSM (Supplementary Information, Fig. S4), indicating that the remaining wild-type β-catenin allele does not contribute to this phenotype.
To test directly whether the multiple stripes of Lfng expression in the mutant PSM correspond to oscillating transcription domains, we developed a real-time imaging strategy to visualize the activity of the segmentation clock in living mouse embryos (Fig. 5 a–f). To this end, we generated transgenic animals that expressed a highly destabilized Venus reporter (a variant of yellow fluorescent protein, YFP) under the control of the cyclic Lfng promoter24,25. Real-time imaging of control reporter mouse embryos with two-photon, time-lapse microscopy revealed periodic waves of Lfng expression, which are initiated in the posterior PSM approximately every 2 h (Fig. 5a, c; Supplementary Information, Movie S1; n = 3). These waves of transcriptional activity traversed the PSM from posterior to anterior and stopped in the anterior PSM where they briefly stabilized as a striped expression domain before disappearing (Fig. 5a, arrows; Fig. 5c; Supplementary Information, Movie S1). Cells in the anterior PSM stopped oscillating once they reached a certain maturation state and due to somite formation, there was a posterior regression of the domain showing oscillations (Fig. 5c, e). To examine Lfng oscillations in the mutant background, mice carrying the β-catenindel(ex3) allele were crossed with the Lfng reporter line. Analysis of the movies generated from these mutant embryos showed that the striped expression of Lfng corresponds to multiple, oscillating expression domains that sweep through the expanded anterior PSM (Fig. 5b, arrows; 5d, f; Supplementary Information, Movie S2; n = 4). According to their axial location, these cells should have stopped oscillating, but instead they continued to do so for an extended period of time under the influence of elevated β-catenin levels (Fig. 5b, d, f). Consistently, no regression of the oscillatory domain was observed during time-lapse recordings (Fig. 5d). The signal intensity and the amplitude of the oscillations were, however, reduced in mutants, compared with wild-type embryos (Fig. 5). These results demonstrate that oscillations of the segmentation clock occur even in the presence of high and steady nuclear β-catenin levels. In addition, this shows that β-catenin is able to maintain clock oscillations and suggests that the arrest of oscillations under normal conditions is linked to the level of β-catenin protein in the PSM.
Figure 5.
Real-time imaging of Lfng oscillations in control and β-catenindel(ex3)/+ mutant embryos. (a, b) Representative time series of control (a) and β-catenindel(ex3)/+–T–Cre–LuVeLu (b) embryos, reporting oscillations (green) of Venus–YFP fluorescence driven by the Lfng promoter. Only the posterior part of the embryo is shown. Arrows of different colours indicate successive Venus–YFP waves sweeping through the PSM. The corresponding time within the original time-lapse recording (Supplementary Information, Movies S1 and S2) is indicated in the upper right corner. The vertical dashed line (blue) represents a fixed point in the embryo for orientation. (c, d) Quantification of minimally processed fluorescence data. Fluorescence intensity is colour-coded (see colour code to the right of graphs) and plotted along PSM length (x-axis) and time (y-axis). The intensities were measured along the posterior line, shown in the first frame for each series in panels and centred in the PSM (a) and (b). Peaks of intensity in control (c) and mutant (d) traversed the embryos from posterior (right) to anterior (left) over time. The regression of the oscillatory field from anterior to posterior seen in control embryos was not observed in the mutant embryo during the recording time. (e, f) Fluorescence intensity (y-axis) at one given position within the PSM, as indicated by a vertical black line (c, d) shown over time (x-axis) of development. Note that at this fixed axial position, cells in the control embryo (e) showed three pronounced oscillations before oscillations stopped, caused by posterior regression of the oscillatory field. In contrast, cells at a similar position in the mutant embryo (f) continued to oscillate (five times) during the entire recording time (12 h), with lower overall intensity and lower amplitude.
In this study, we have shown that by deleting β-catenin in mesoderm precursors in the primitive streak, formation of the paraxial mesoderm is blocked, causing axis truncation. In contrast, expression of a stable form of β-catenin throughout the paraxial mesoderm led to a Wnt-signalling gain of function, causing a delayed and defective maturation process and a subsequent increase in the size of the PSM along the antero-posterior axis. This suggests that the function of the Wnt-signalling gradient in the PSM is carried out by the β-catenin protein gradient.
The delay in maturation was accompanied by sustained oscillations of the Wnt and Notch pathway in PSM cells. Thus, Wnt activation in the posterior PSM provides a permissive environment for oscillations of the segmentation clock to occur. This also suggests that the decision to stop oscillations in the anterior PSM under normal conditions is not based on a limited ability of cells to oscillate (that is, a counting mechanism) but requires the downregulation of Wnt signalling.
However, the PSM of mutant embryos retained signs of maturation and Mesp2 became consistently activated at the level where oscillations eventually stopped in the extended anterior PSM of the β-catenindel(ex3) mutants. Thus, maintaining a high level of nuclear β-catenin was not sufficient to maintain the posterior PSM identity indefinitely, indicating that β-catenin interacts with other signalling pathways to perform this function. The Wnt/nuclear β-catenin gradient overlaps with an FGF-signalling gradient, which has also been shown to have an important role in the maintenance of posterior PSM identity6,26,27. Wnt3a/β-catenin was shown in loss-of-function experiments to function upstream of Fgf8 (refs 2, 28). Here, we show that in a gain-of-function situation, β-catenin accumulation can increase Fgf8 expression, and FGF signalling extends more anteriorly in the expanded PSM. Thus, some of the observed effects in β-catenindel(ex3) mutants could be mediated indirectly by the FGF gradient. To test this possibility directly, we generated compound β-cateninlox(ex3)/+–Fgfr1f/ko–T–Cre mutant embryos to block FGF signalling in the PSM (Fig. 4i–q). In the PSM of compound mutants, expression of the FGF target Dusp4 (ref. 29) was downregulated (Fig. 4k; n = 8). The anterior shift of the Mesp2 stripe was still observed in the compound mutants (Fig. 4n; n = 5), but was not as pronounced as that in the β-cateninlox(ex3)/+–Fgfr1f/+–T–Cre single mutants (Fig. 4m). Therefore, part of the effect of β-catenin stabilization in the PSM seems to be mediated indirectly through FGF signalling. This also suggests that the β-catenin gradient can influence PSM patterning and maturation directly, possibly through the activation of targets such as Msgn1 (ref. 16). In addition, although no oscillations of Lfng were detected in the PSM of conditional homozygous Fgfr1–T–Cre single mutants30, the presence of a β-catenindel(ex3) allele led to the formation of several Lfng stripes in the extended PSM of compound mutants (Fig. 4q; n = 5), indicating that Lfng oscillations were rescued. This is consistent with the proposed role of Wnt signalling downstream of FGF signalling in the control of Lfng oscillations30. Thus, these experiments demonstrate the lack of a simple epistatic relationship between Wnt and FGF signalling in the PSM. Rather, these pathways are tightly interconnected and seem to synergize in controlling PSM maturation, thereby defining the permissive environment for oscillations of the segmentation clock.
Although accumulation of nuclear β-catenin markedly altered PSM maturation, activity of the segmentation clock was not disrupted by these experimental conditions. We conclude that oscillations of Wnt and Notch targets are not achieved by oscillating (nuclear) β-catenin protein levels. Regulation of β-catenin protein levels has normally been regarded as essential in defining canonical Wnt-signalling activity. However, there is growing evidence that nuclear β-catenin is not the sole determinant of transcriptional activity downstream of canonical Wnt signalling, but relies also on the regulated interaction with additional (nuclear) cofactors31. Our data suggest that oscillations of Wnt targets in the PSM are probably caused by the cyclic activity of a β-catenin cofactor. Interestingly, cyclic recruitment of several nuclear cofactors of β-catenin to the genomic enhancer/promoter locus of Wnt targets (such as c-myc, a Wnt cyclic gene in mice)3 has indeed been shown to occur32. This cyclic recruitment occurred even in the presence of a steady and high nuclear β-catenin level. The present study illustrates two distinct strategies of Wnt-signalling regulation that operate simultaneously in embryonic cells. Clarifying the molecular details of this versatile regulation in the developing embryo might reveal some underlying principles of signalling regulation in biological systems. □
METHODS
Mice breeding and embryo production.
Mice with either the conditional gain-of-function allele β-cateninlox(ex3) (see Supplementary Information), the loss-of-function allele β-cateninfloxed, described previously12 (obtained from Jackson Laboratory, Bar Harbor, ME) or the conditional Fgfr1f allele, described previously (see Supplementary Information), were kept on a Bl6 background. Transgenic animals for T–Cre (see Supplementary Information) and BAT–Gal14 were described previously and kept on a Bl6 or CD1 background, respectively. To generate gain-of-function mutant embryos, β-cateninlox(ex3)/lox(ex3) animals were mated with T–Cre heterozygous animals (these embryos are referred to as β-catenindel(ex3) mutants). To obtain β-cateninlox(ex3)/floxed embryos, β-cateninfloxed/floxed animals were first crossed with T–Cre animals. Double heterozygote animals were then mated with β-cateninlox(ex3)/lox(ex3) animals. To generate conditional deletion of β-catenin, we crossed β-cateninfloxed/floxed with β-cateninfloxed/+–T–Cre positive animals. To generate compound β-cateninlox(ex3)/+–Fgfr1f/ko–T–Cre mutants, males heterozygous for a germline deletion of the conditional Fgfr1 allele (see Supplementary Information) and T–Cre positive (Fgfrko/+–T–Cre) were mated with β-cateninlox(ex3)/lox(ex3))–Fgfr1f/f females. For real-time imaging experiments, LuVeLu transgenic animals were mated with β-cateninlox(ex3)/lox(ex3) animals and double heterozygote animals were mated with T–Cre homozygote transgenic animals. No significant phenotypic difference was observed in this mixed genetic background.
Generation of transgenic Lfng reporter mice (LuVeLu).
A reporter allowing detection of luciferase cyclic activity driven by the Hairy and enhancer of split 1 (Hes1) promoter was recently described in mice33. To image Lfng oscillations in vivo we used a fluorescent reporter (Venus–YFP), which has the potential to achieve high cellular resolution in vivo. A 2-kb fragment of the cyclic Lfng promoter (a gift from D. Ish-Horowicz)24,25 was used to drive the expression of Venus–YFP (a gift from A. Miyawaki)34 and fused with a modified PEST domain (see Supplementary Information) to increase protein instability. In addition, this construct was fused with the Lfng 3ÚTR to destabilize Venus–YFP mRNA. Transgenic animals were produced by pronuclear injection using standard procedures. Five out of seven transgenic lines showed correct expression and two were further bred on a CD1 outbreed background. Experiments were performed using F4 generation mice.
Genotyping.
Embryos were harvested at day 9.0 of development and genotyped with PCR using the yolk sac (see Supplementary Information). For LuVeLu, the following primers were used: Ala 1: tgctgctgcccgacaaccact and Ala 3: tgaagaacacgactgcccagc. Detailed protocols are available in the Supplementary Information.
Culture conditions during real-time imaging.
Embryos at the 7- to 12-somite stage were dissected in pre-warmed DMEM (Invitrogen), with 10% FBS, 100 mg dl−1 D-glucose and 20 mM HEPES. Embryos were first incubated for 1.5 h in DMEM containing 50% rat serum (produced as described previously35) in a rotating culture apparatus (BTC Engineering). Subsequently, embryos were transferred to a glass bottom Petri dish (MatTek) containing 1.1 ml of DMEM/50% rat serum. Embryos were then transferred to and imaged inside a microscope incubation chamber (37°C, 65% O2 and 5% CO2; Solent Scientific).
Two-photon microscopy.
Images were acquired with a Zeiss LSM510 laser-scanning microscope (Carl Zeiss MicroImaging). Samples were excited using a Ti:Sapphire Laser (Chameleon-Ultra, Coherent) at a wavelength of 960 nm through a 20× Plan Apo Objective (numerical aperture (NA) 0.8). Emission was collected using a 500–550 nm band-pass filter. A Z-stack of 6–10 planes at 12–16 μm distance was scanned every 8.5 min. Power settings were kept constant between all experiments.
Data visualization in time-lapse movies.
Carl Zeiss AIM software (Carl Zeiss MicroImaging) was used to record all data and for basic image processing. Representative z-planes were added and the mean fluorescence intensity for combined z-planes was calculated using the AIM software. For further image processing, we developed software in IDL (ITT Visual Information Solutions).
All images are recorded in 12 bit, 512 × 512 square pixels with a size of 2.5 μm × 2.5 μm. The images were scaled to 8 bit, maximum intensity was set to 255 and the minimum to zero. To smooth the fluorescent images, a boxcar filter (30 × 30 pixels) was used. We developed an algorithm based on the transmitted light image that allowed automatic definition of the embryo border within each image. Only the region covering the embryo was used to process the fluorescent signal. To emphasize small differences in intensity, the fluorescent signal was taken to the power of three. For display, the maximum number was scaled to 255 and data below 30 was clipped. We applied a boosted Laplace filter with a centre value of 19 to the raw data of the transmitted light image.
Fluorescence quantification.
Data were only minimally processed for graphical representation (Fig. 5c–f), and a linear relationship of the fluorescence intensities was maintained. We developed an algorithm that automatically defined a centreline through the PSM of the embryo based on the transmitted light image (detailed Methods are available in the Supplementary Information). This included a correction for slight embryonic movement during recordings. To that end, we defined a common reference point (vertical blue dashed line in Fig. 5a, b) and then cross-correlated the bright-field intensities of the 400 most anterior pixels (red lines in PSM, Fig. 5a, b). Fluorescence intensity was sampled along the centreline (blue portion of line in PSM in Fig. 5a, b), and the mean of 30 pixels calculated by applying a boxcar filter with a kernel size of 30 (30 pixels correspond approximately to the width of the PSM). This algorithm was applied to all time-points of the experiment. These data are shown as a false-colour intensity plot, with the x-axis representing the position along the embryo axis and the y-axis representing the time during development.
In situ hybridization.
Single and double in situ hybridizations were performed as described2 (see Supplementary Information for details of the probes). Littermates were used for comparisons and were processed identically and in parallel.
Fluorescence immunodetection.
Embryos were fixed in zinc formaldehyde for 1 h at room temperature and processed for paraffin embedding and sectioning (4 μm). Immunofluorescence detection was performed according to the following protocol: antigen retrieval was carried out in citrate buffer (pH 6) placed in a microwave at 95°C for 10 min. After blocking for 10 min (Power Block Universal Blocking Reagent, Biogenex HK085–5K), slides were incubated with primary antibody (mouse monoclonal anti-β-catenin, BD Transduction Laboratories Number 610153, dilution at 1:500, anti-phospho histone 3, Upstate 06–570, 1:1000) for 1 h at room temperature, followed by an overnight incubation at 4°C. Slides were washed three times in PBS Tween (PBST) and incubated with a secondary antibody (goat anti-mouse Alexa-488 or Alexa-568, Invitrogen) for 1 h at room temperature. Slides were washed three times in PBST and nuclei were counterstained with DAPI (InnoGenex CS-2010–06) for 5 min. Slides were mounted using an anti-fade reagent (ProLong Gold, Invitrogen).
Images were acquired on a Zeiss LSM510 laser scanning microscope (Carl Zeiss MicroImaging). Alexa-488 or Alexa-568 was excited by confocal microscopy, DAPI was visualized by two-photon excitation at 720 nm and a 20× Plan Apo objective (NA 0.8) was used.
Scanning electron microscopy.
After in situ hybridization, fixed embryos were dehydrated in an ethanol series, incubated in hexamethyldisilazane for 30 min and then dried. Samples were imaged using a Hitachi TM-1000 unit.
Measurement of anterior shift of Mesp2 expression.
Images from age-matched embryos (day 9.0 of development) were taken as lateral views and imported into ImageJ software (open source from the National Institutes of Health, USA). Using the line and measure tool, the distance from tail end to Mesp2 expression was measured and compared with known standard scales for conversion to mm. The mean was calculated from control (n = 11) and β-catenindel(ex3)/+ mutant embryos (n = 10).
Supplementary Material
ACKNOWLEDGMENTS
We thank members of the Pourquié laboratory for discussions and comments on the manuscript, S. Esteban for artwork and J. Chatfield for manuscript editing. We thank the Stowers Institute Core Facilities, especially D. Dukes and M. Durnin in the Laboratory Animal Service and S. Beckham in the Histology Facility for their excellent technical assistance. A.A was funded by the Swiss Foundation for medical-biological grants, Swiss National Science Foundation. This research was supported by Stowers Institute for Medical Research. O.P. is a Howard Hughes Medical Institute Investigator.
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