Generation of Transgenic Mice Expressing Lfng in the Cranial PSM Stripe

We examined the function of Lfng in the PSM by driving Lfng expression in the cranial PSM stripe via a tandem duplication of the B-block enhancer of the Lfng promoter which drives stripe expression more strongly than a single B-block (BB-cLfng; Figure 1A; data not shown; [15], [16]). We expressed chick Lfng (referred to as cLfng for clarity) so that we could distinguish between transgenic and endogenous Lfng expression by in situ hybridisation. Although mouse and chick Lfng are 70% identical and 87% similar in aminoacid sequence, indicating that they should be functionally equivalent, the genes share less than 60% identity in DNA sequence, sufficient for a cLfng probe to be selective for the chick transcript under stringent hybridisation conditions (Methods). As a control, we used a 5 kb fragment of the mouse Lfng promoter to drive cLfng in both the stripe and the oscillating PSM domains (Lfng5kb-cLfng; Figure 1A; [15], [16]).

We obtained 13 transgenic mouse lines with BB-cLfng and 8 lines with Lfng5kb-cLfng. All 8 Lfng5kb-cLfng lines express cLfng in the expected wildtype expression pattern: cyclically in the caudal PSM and stably in a stripe in the cranial compartment of somitomere s–I. 9 of the 13 BB-cLfng lines express the cranial stripe. Surprisingly, all 9 lines also weakly express cLfng dynamically in the caudal and mid-PSM. This staining indeed represents cLfng expression because no cross-reaction with endogenous Lfng is seen following equivalent staining of wildtype embryos (Figure 1C). Rather, it appears that the duplicated B-block can drive dynamic expression in the PSM, albeit at very low levels (see Discussion).

For further study, we selected two lines (referred to as BBL-1 and BBL-2) in which caudal expression is very weak and visible only after extended staining, and two control Lfng5kb-cLfng lines (referred to as 5kL-1 and 5kL-2) (Figure 1B, 1D–F). All four lines express the transgenes throughout somitogenesis (Figure 1G–G″, and not shown). The 5 kb fragment drives stronger stripe expression than the duplicated B-block (Figure 1B, 1G–I), indicating that the longer promoter includes additional elements that synergise with and enhance stripe expression from the B-block, and BBL-1 expresses more highly than BBL-2. The former is homozygous viable, allowing us to compare hemi- (BBL-1/0) and homozygous (BBL-1/BBL-1) animals. Only hemizygous BBL-2 animals were examined because the insertion is homozygous lethal (genotyping was performed following identification of the transgene insertion site; Methods). Together, the transgene lines form an allelic series of cLfng stripe expression (5kL/0>BBL-1/BBL-1>BBL-1/0>BBL-2/0; Figure 1B).

cLfng Expression in the Cranial Stripe Rescues Embryonic Tail Growth but Not Adult Viability

The various transgenes were crossed with Lfng^+/− mice and the patterning of Lfng^−/−;cLfng⁺ offspring (which we refer to by their transgene names) assayed as foetuses and adults. Lfng^−/− mice usually die before weaning, presumably because the deformed axial trunk skeleton reduces the thoracic space and impairs breathing [17], [18]. In our experiments, only three non-transgenic Lfng^−/− animals survived to adulthood (equivalent to 9% of the expected number of homozygous offspring; Table 1), although a much higher proportion of Lfng^−/− foetuses survived till E18.5 (61%; Table 1).

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Table 1. E18.5 and adult viabilities of transgenic animals in a Lfng^−/− background¹.

https://doi.org/10.1371/journal.pone.0007996.t001

Expressing cLfng in both the stripe and the oscillating PSM domains substantially restores adult viability (33% and 68% for 5kL-1 and 5kL-2, respectively; Table 1; see Methods S1 for a likely reason of the unexpected high lethality of 5kL mice), and also enhances foetal recovery (Table 1). Thus, chick Lfng is indeed functional in the mouse and can largely substitute for the endogenous protein. Rescue of adult viability when cLfng is expressed solely in the cranial stripe is less efficient, (9–24% in BBL-1 and -2; Table 1).

The ability of our transgenes to rescue caudal development, as assayed by tail length, correlates with the strength of transgene expression. Lfng^−/− tails are much shorter than wildtype, both at E18.5 and in adult mice (Table 2). This defect is rescued essentially completely by the 5kL transgenes, and efficiently in mice homozygous for BBL-1 (Figure 2A and Figure S1A; Table 2). Rescue by hemizygous BBL-1 and BBL-2 is intermediate and slight, respectively (Figure 2A; Table 2). Rescued BBL tails are often kinked, indicative of locally disrupted somitogenesis due to partially incomplete Lfng expression (Figure S1A).

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Figure 2. Regional and dose-dependent rescue of the Lfng phenotype by transgenic expression in the stripe domain.

(A) Dosage-dependent rescue of the truncated tail phenotype of Lfng^−/−;cLfng⁺ BBL E18.5 foetuses. Homozygous BBL-1 tails resemble those of wildtype/5kL, and weak hemizygous BBL-2 resemble Lfng^−/−. White arrowheads point at the tip of the tails; scale bar, 5 mm; asterisk, midgut that failed to return to the reduced peritoneal cavity (found in 5/42 Lfng^−/−;cLfng⁺ E18.5 foetuses). (B, C) E18.5 skeletons of 5kL embryos show regular vertebrae all along the length axis like wildtype [B, lateral (upper panel) and dorsal (lower panel) view] whereas Lfng^−/− skeletons (C) exhibit regular vertebrae only in the sacral area. (D, E) BBL skeletons show a regional rescue in the tail area adjacent to the sacral area. The cervical, c; thoracic, t; lumbar, l; sacral, s; and tail regions of the vertebral columns are indicated, as are: ar, vertebral arch; bo, vertebral body; il, ilium. Regular sacral vertebrae in the bottom panels of (C) and (E), magnifications of the middle panels, are labelled with white asterisks. Note the cranial shift of the left side of sacral vertebrae in the Lfng^−/−foetus (C bottom), indicative of homeotic transformation. Scale bar (bottom of D), 5 mm. (F) A schematic quantitative comparison of lengths of cervical/thoracic (c+t, green boxes), lumbar (l, yellow boxes), sacral (s, blue boxes), and tail (orange boxes) sections of vertebral columns of various genotypes indicates a dose-dependent rescue of the tail length in BBL foetuses, but not of the length of the cervical+thoracic area. Boxes for each region are aligned to the left (cranial) end. (G) Skeleton preparations of adult mice (dorsal views) show the truncated tail but normal Lfng^−/− sacral area (top), a partial tail rescue in hemizygous BBL-1 mice (middle) and an overall rescue in 5kL mice (bottom); scale bar, 10 mm.

https://doi.org/10.1371/journal.pone.0007996.g002

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Table 2. Comparison of E18.5 foetuses and adults of the four transgenic lines in the Lfng^−/− background (data for adults are given in brackets and italics).

https://doi.org/10.1371/journal.pone.0007996.t002

Surprisingly, 15% (3/21) of the 5kL foetuses show no rescue of tail length (and of skeletal segmentation; not shown). Most likely, these represent individual embryos in which the transgene has been inactivated (see Methods S1), although we cannot exclude the possibility that sequence differences between mouse and chick Lfng also contribute to differences between the activity of the transgenic and endogenous genes.

Stripe Expression of cLfng Provides Regionally Restricted Rescue of the Lfng Segmentation Phenotype

A single endogenous Lfng⁺ gene is sufficient for normal skeletal development, irrespective of whether the animals also include a cLfng transgene (n = 58). However, Lfng^−/− mice display severely disorganised, truncated axial skeletons with irregular and fused vertebrae and vertebral bodies that rarely align to the midline [17], [18]. They also have an unsegmented tail stump with one or no regular tail vertebrae (Table 2).

Surprisingly, most sacral vertebrae in these animals resemble wildtype [Figure 2C, 2G top; compare sacral (s) to irregular thoracic (t) or lumbar (l) regions] with symmetric bodies and proximal dorsal arches. At least three of the sacral vertebrae form distinct ribs connecting the axial skeleton to the ilium of the pelvic girdle, whereas thoracic ribs and vertebrae undergo multiple fusions in the thorax (Figure 2C, 2G top; Table 2). Relative normality of sacral structures is also evident in previous images of the mutant embryos (e.g. Figure 2B, 2C in [17], Figure 2C in [18]). Thus, the sacral area undergoes considerable segmentation independently of Lfng activity.

The 5kL control transgenes give substantial, albeit sometimes incomplete, rescue of the Lfng^−/− skeleton (Figure 2B, 2G bottom; Table 2). Most 5kL animals have wildtype-like vertebral columns and the same number of tail segments as wildtype (Table 2), confirming that transgenic cLfng is functional in the mouse and showing that the previously described mutant skeletal phenotype is indeed due to disruption of the Lfng gene [17].

The rescued mice also frequently exhibit homeotic transformations of single segments: Out of 32 5kL mice, 14 (44%) have an extra rib-bearing thoracic vertebra, 15 have an extra pair of “true ribs” (connected to the sternum), and 12 miss a lumbar vertebra. This phenotype is likely to be due to subtle alterations of Notch signalling in the PSM that lead to a delay in Hox gene expression in the PSM [22], [23], [24].

Finally, all rescued 5kL males (Lfng^−/−;cLfng⁺) are sterile (7 5kL-1, 10 5kL-2), confirming the reported reduced fertility of Lfng^−/− males [25]. This finding excludes the possibility that skeletal malformations contribute to the reduced fertility, and indicates that a testis-specific Lfng enhancer lies outside the 5 kb promoter used in the 5kL lines.

By contrast with the 5kL transgenes, BBL transgenes only rescue segmentation in the tail areas. The sacral area of BBL mice is always regularly patterned, as in most Lfng^−/− mice. Cervical, thoracic and lumbar vertebrae are only slightly restored (e.g. dorsal arch quality; Figure 2D, 2E, 2G middle), indicating that oscillating Lfng expression is necessary for proper segmentation in these axial regions, and that this requirement cannot be met by the weak dynamic expression in the caudal PSM of these embryos.

However, the stripe domain is sufficient for proper segmentation of the tail [Figure 2D, 2E, 2G middle; compare tail to lumbar area (l) and to the short tail in Figure 2C; Table 2]. The efficiency of tail rescue correlates with the strength of transgene expression (BBL-1/BBL-1>BBL-1/0>BBL-2/0; Figure 2F; Table 2). Even the weakest BBL-2-rescued foetus shows three regular tail vertebrae. These results suggest that stripe expression of Lfng in the cranial PSM helps maintain somitogenesis in the tail (see below).

Lfng Stripe Expression Restores Somite Organisation in the Tail

The rescue of tail vertebrae by Lfng stripe expression could be due to restoration of cranial-caudal compartmentalisation of somites. We tested this idea by visualising expression of Uncx4.1, a marker of caudal somite halves [26].

Segmentation and cranial-caudal differentiation are both greatly disrupted in Lfng^−/− embryos, in which Uncx4.1 is expressed in irregularly spaced and sized stripes and patches [17], [18]. Nevertheless, from about somite 28 onwards, Lfng^−/− embryos form 17±2 (n = 5) somites that are regular in the sacral and adjacent tail area but become increasingly irregular towards the end of the truncated tail (Figure 3A–3C). The Uncx4.1 stripes in these somites look slightly less distinct than in wildtype, but are much more regular than in somites 1–27 of the same Lfng^−/− embryos (n = 9; Figure 3A, B).

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Figure 3. Influence of Lfng PSM domains on segmentation and extension of the tail.

(A–I) Transgenic Lfng stripe expression improves somite compartmentalisation only in the tail. (A–C) Cranial-caudal somite compartmentalisation in Lfng^−/− embryos (visualised by in situ hybridization with Uncx4.1) is irregular in the cranial body half but regular in the sacral (red box) and first tail somites. Lfng^−/− E11.5 tails are shorter than wildtype-like Lfng^+/− tails (compare C and D). The lack of Uncx4.1 stripes close to the PSM suggests that segmentation has ceased in the mutant (red bar in C). (E, F) Transgenic cLfng expression in 5kL lines (5kL-1/0) almost fully rescues compartmentalisation in trunk and tail (arrows in F indicate occasional irregular stripes). (G–I) Transgenic cLfng expression in BBL lines (G, BBL-1/BBL-1; H, BBL-1/0; I, BBL-2/0) rescues cranial-caudal compartmentalisation mainly in the tail (arrows in I indicate irregular stripes). (J–O) Lfng is important for clear separation of sclerotomes (ventral compartments of somites) except for those in the sacrum, and influences dorsal-ventral patterning of the paraxial mesoderm of the tail. Sclerotomes (stained with Pax-1) are regular and segmented in wildtype mice (J, K) but enlarged and undefined in Lfng^−/− embryos except in the sacral area (L, M). 10 µm transverse sections of embryos in K and M at tail region indicated by a dash (N, O; same magnification) counterstained with nuclear fast red (Vector Labs) show an increase in sclerotome size in the mutant (Pax1-positive area encircled with a dashed line on right side of sections). (P–U) Expression of PSM markers Fgf8 (P–R; red arrows point at PSM expression in the tail; expression in Q is weak and restricted, and undetectable in slightly older embryos, not shown) and Tbx6 (S–U) is indistinguishable between Lfng^+/− and Lfng^−/− embryos. The PSM is boxed in blue. Forelimb buds (fl) align to somites ∼8–14, hindlimb buds (hl) to somites ∼25–30; nt, neural tube.

https://doi.org/10.1371/journal.pone.0007996.g003

The cranial somite marker Tbx18 [27] also reveals relatively regular segments in the sacral and tail area of Lfng^−/− embryos (Figure S2A–D), while the myogenic marker MyoD [28] suggests that the regionalisation is less distinct for the myotomal than for the sclerotomal compartment (Figure S2E–H).

Regular segmentation of the sacrum and adjacent tail in Lfng^−/− embryos is not due to compensatory expression of another fringe paralogue. Neither Manic fringe (Mfng) or Radical fringe (Rfng) is expressed in wildtype PSMs between E8.0 and E11.5 [29, our own observation], and Mfng and Rfng mRNA levels are not increased in Lfng^−/− embryos compared to wildtype embryos as determined by quantitative RT-PCR on dissected E10.25 PSMs (data not shown).

These relatively well-patterned somites are incorporated into both regular and the severely disorganised Lfng^−/− sacral/tail regions (Figure 2C, 2G top). The latter observation shows that cranial-caudal compartmentalisation is not sufficient for proper development of the sclerotome, which derives from a ventral portion of the somite [reviewed in 3].

5kL embryos stain almost normally for Uncx4.1 (n = 7; Figure 3E, 3F). Lfng stripe expression in either BBL line also restores orderly Uncx4.1 expression in the tail (n = 10; Figure 3G–I), albeit less regularly in BBL-2 tails (Figure 3I), but not in more cranial regions (Figure 3G). These results argue that Lfng stripe expression is necessary for tail segmentation.

Lfng Regulates Dorsal-Ventral Compartmentalisation in Tail Somites

As Lfng^−/− tail vertebrae were more irregular than expected from their regular cranial-caudal compartmentalisation (compare Figure 3B and 3C with 2C), we examined sclerotome development in Lfng^−/− embryos using the marker Pax1 [30], [31]. We found that lack of Lfng affects dorsal-ventral compartmentalisation during tail development.

In wildtype embryos, sclerotomes are regular and clearly separated all along the length axis (n = 17; Figure 3J, 3K). In Lfng^−/− embryos, sclerotomes cranial to the hindlimb buds are fused, while the more caudal ones appear distinctly separated (n = 10; Figure 3L, 3M). Sclerotome borders in the mutant never become as clearly visible as in wildtype because Pax1 expression is less concentrated at the borders, but a relative reduction of Pax1 in the cranial area is discernible in some sacral sclerotomes (Figure 3L). The Pax1 domain in the truncated Lfng^−/− E11.5 tails is much thicker than in wildtype tails (compare Figures 3K and 3M and corresponding transverse sections in Figures 3N and 3O), indicating that Lfng limits formation of sclerotome. Presumably, over-production of Pax1-expressing cells leads to blurring of the borders between sclerotomes in Lfng^−/− embryos (Figure 3L, 3M). This morphological difference between sacral and tail sclerotomes (which is not prefigured by a difference in cranial-caudal compartmentalisation as visualised by Uncx4.1; compare Figure 3B, 3C to 3L, 3M) is likely to cause the difference between normal sacral vertebrae and the deformed/fused tail vertebrae in Lfng^−/− mice.

Lfng and Axial Extension of the Tail

Lfng plays a role in maintaining axial extension, a process whereby axial progenitor cells which lie at the chordoneural hinge of the tailbud generate caudal growth of the embryo and elongation of the body axis [32], [33], [34]. In Lfng^−/− embryos, axial extension ceases around E10.5, resulting in truncated tails. This phenotype can be rescued by increased Lfng expression in the cranial PSM (cf. tail-lengths in hemi- and homozygous BBL-1 and in BBL-2 embryos; Figure 2A, 2F; Table 2), indicating that Lfng expression in the cranial stripe domain is sufficient for sustained segmentation of the tail. Such a dose-dependent rescue of the length of the vertebral column is not observed in the cranial body half, which is similar in BBL-1/BBL-1, BBL-1/0, BBL-2/0 and Lfng^−/− (Figure 2F).

How might cranial Lfng expression sustain progenitors in the tailbud? Lfng regulates Notch signalling cell-autonomously [reviewed in 35], suggesting that Lfng activity acts indirectly to influence the behaviour of axial progenitor cells. For example, Lfng activity might be needed to maintain the Fgf and Wnt signalling that is required for axial extension.

To test this idea, we analysed PSMs of Lfng^−/− embryos. Lacking known markers for axial progenitor cells, we analysed the expression of Fgf8, a target of Wnt signalling [36], which forms a caudal-cranial concentration gradient and reflects the maturation of the PSM and of the general PSM marker Tbx6 [37], [38]. Fgf8 (n = 6) and Tbx6 (n = 3) expression in Lfng^−/− embryos is indistinguishable from that in control Lfng^+/− embryos (Figure 3P–U) indicating that despite of cessation of progenitor proliferation the Lfng^−/− PSM is still largely intact when segmentation terminates. Thus, Lfng is not required to maintain longrange signals of Fgf8, Wnt and retinoic acid in the caudal PSM (see Discussion). We conclude that continued tail segmentation promoted by the cranial Lfng stripe in BBL mice does not depend on known long-range signals in the PSM.

Further evidence that loss of Fgf8 expression does not cause the short-tail phenotype comes from our finding that Fgf8 becomes downregulated between E10.5 and E11.5 in Lfng^+/− embryos (n = 21; Figure 3Q), i.e. about two days before the end of wildtype tail segmentation. This result indicates that other factors control progenitor maintenance from about E11.

Regular Formation of the Sacral Axial Skeleton Does Not Require Hes7 Oscillation

The dispensability of Lfng for the formation of sacral somites and vertebrae might be due to changing requirements for Notch signalling during axial elongation. To test this idea, we analysed mutants of Hes7, a cycling Notch target gene required for somitogenesis that encodes a Notch effector [39], [40], [41]. Hes7 oscillates in the caudal PSM in phase with Lfng, but is not expressed in the cranial PSM [39], [42]. Lfng and Hes7 oscillations depend on each other's expression, consistent with a regulatory loop in which Hes7 protein represses transcription of Hes7 and Lfng and, in turn, Lfng modulates Notch signalling to regulate Hes7 expression [reviewed in 43].

Hes7^−/− E10.5 and E11.5 embryos lack any regular Uncx4.1 stripes (n = 15; Figure 4A), and Hes7^−/− foetuses do not form regular sacral vertebrae (Figure 4B; [39]). Thus, Hes7 activity (and presumably its activation by Notch signalling) is required throughout somitogenesis.

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Figure 4. Hes7 is required for regular sacral segmentation in Lfng^−/− embryos, but need not oscillate.

(A, B) Segmentation in Hes7^−/− mice is irregular all along the axis (including the sacrum) as visualised by Uncx4.1 staining of embryos (A) and foetal skeleton preparation (B; top, lateral view; bottom, ventral view; scale bars are 5 mm). For annotation, see legend to Figures 3 and 2. (C) In situ hybridisation with a Hes7 intron probe to visualise active transcription (left) shows a clearly dynamic pattern in Lfng^+/− PSMs (top) but a strong background throughout Lfng^−/− PSMs (bottom). A Hes7 cDNA probe (C right) visualises dynamic transcript distribution in Lfng^+/− PSMs (top) but mainly ubiquitous staining in Lfng^−/− PSMs (bottom; embryos showing dynamic expression domain most clearly were selected).

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Cyclic transcription of Hes7 is best revealed by in situ hybridisation to an intron probe that visualises newly synthesised, unspliced pre-mRNA in the nucleus [15], [40]. Control Lfng^+/− embryos show robust oscillation of unspliced transcripts (and of cDNA; Figure 4C top row). In Lfng^−/− embryos, repression is incomplete so that cyclic Hes7 expression is superimposed on uniform expression throughout the PSM (n = 49; Figure 4C bottom row, left; Figure S3A,B).

This uniform expression is more evident when probing with an exonic Hes7 probe. Transcripts are detected throughout the PSM in all Lfng^−/− embryos, and oscillatory transcript accumulation is visible only in few of them (n = 31; Figure 4C bottom row, right; [42], [44]). The largely uniform transcript distribution implies that Lfng^−/− PSMs do not experience dynamic Hes7 protein levels. Presumably, residual dynamic transcription of Hes7 (as visualised by in situ hybridisation with the intron probe) in Lfng^−/− embryos is caused by dynamic transcriptional activation rather than by dynamic transcriptional repression via Hes7 protein. As these mutant embryos form relatively normal sacral segments, Hes7 expression does not always need to oscillate for proper segmentation. This conclusion is corroborated by our analysis of hypomorphic Hes7^BAP/BAP mice (see below).

Differential Requirements for Hes7 Activity During Segmentation in the Sacral Area

As threshold requirements for Lfng activity show axial variations, we hypothesized that the same might be true for Hes7 activity. Thus, we examined embryos expressing a partial loss-of-function Hes7 allele in which a 14-aminoacid biotin acceptor peptide (BAP) was knocked into the fourth exon of Hes7, 17 aminoacids upstream of the C-terminus. Although the tagged protein (Hes7^BAP) retains wildtype-like repressor activity in cultured cells (Figure 5A), it shows greatly impaired activity in vivo. Only 22% of the expected number of homozygous Hes7^BAP/BAP (BAP/BAP) mice survive till weaning (14/254), and the survivors' tails are truncated although not as severely as in Hes7^−/− mice (Figure 5B and Figure S1B, Table 3; [39]).

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Figure 5. Differential requirement for hypomorphic Hes7^BAP during segmentation.

(A, B) BAP-tagged Hes7 (Hes7^BAP) shows full activity in luciferase assays of cultured mouse fibroblast cells (A, relative luciferase activities are shown as mean±s.d. for three experiments) but causes a hypomorphic Hes7 phenotype in vivo (B; note tail lengths of E18.5 foetuses, arrowheads point at the tip of the tails). (C–E) Hes7 (C, in situ hybridisation with an intron probe; D, staining with a Hes7 antibody) and Lfng (E, in situ hybridisation with a cDNA probe) are oscillating in heterozygous (BAP/+; n = 8 Hes7, n = 9 Lfng) but not in homozygous (BAP/BAP; n = 9 Hes7, n = 4 Lfng) E10.5 embryos. (F–H) Skeletons of heterozygous BAP/+ foetuses (F) and adults (H left) are regular and resemble wildtype whereas homozygous (BAP/BAP) foetuses (G) and adults (H right) are regular only in the sacrum and adjacent tail, similar to BBL mice. Foetal skeletons are shown in lateral and dorsal view, adult skeletons in dorsal view; magnification of sacral and adjacent areas of the middle panel in (F,G) are shown in the bottom panel of (F,G); for annotation see Figure 2; arrowheads in (H) point at kinks in tails.

https://doi.org/10.1371/journal.pone.0007996.g005

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Table 3. Comparison of Hes7^BAP/+, Hes7^BAP/BAP, and Hes7^KO/KO E18.5 foetuses and adults (data for adults are given in brackets and italics).

https://doi.org/10.1371/journal.pone.0007996.t003

Hes7^BAP transcript levels do not oscillate in the BAP/BAP knock-in embryos (Figures 5C and S3D,E; n = 27 covering all stages between E9.5 and E11.5). Hes7 protein is expressed uniformly in the whole PSM (Figure 5D, n = 7∼E10.25). Likewise, Lfng is expressed continuously throughout the BAP/BAP PSM (Figure 5E). A likely explanation for these findings is that BAP-tagging Hes7 has disrupted its repressive activity so that it is unable to maintain an auto-inhibitory feedback loop in vivo. This hypothesis is supported by analysis of heterozygous Hes7^BAP (BAP/+) embryos, in which Hes7 and Lfng transcript levels are normal and oscillating (Figure 5C, 5E), and by our observation that Hes7, a target of its own protein, the repressor Hes7, is upregulated in BAP/BAP embryos (Figure 5C,D).

BAP/BAP mice display strongly regionalised segmental phenotypes, indicating that Hes7 activity, like that of Lfng, is required differentially during the course of somitogenesis. The mutant mice show severe segmentation defects in the cervical, thoracic and lumbar area with fused vertebrae and fewer ribs (Figure 5F–5H; Table 3). However, the sacral (and to variable degrees, the adjacent lumbar and tail) vertebrae (and Uncx4.1 stripes; not shown) appear relatively normal, and the tail length is significantly longer than in null mutant mice (cf. Figures 4B and 5F–5H; Table 3). Together, our results indicate that Hes7 activity is required along the whole body axis for proper segmentation, but that Hes7 oscillation is not essential during the segmentation of the sacral and adjacent tail area.

Regional Requirements for Different Modes of Lfng Expression

An important role for Lfng during mouse and chick somitogenesis is indicated by its expression pattern, and by the effects of manipulating its activity [14], [17], [18], [45], [46], [47]. It is less clear if Lfng also acts in somitogenesis of lower vertebrates such as zebrafish and medaka, where it is expressed in one or two stripes in the cranial PSM but does not oscillate [48], [49], [50], [51]. Corn snake embryos express Lfng in up to nine stripes of variable size and spacing in the mid- and cranial PSM, but they too lack a caudal oscillating domain [52].

Thus, the oscillating, caudal domain may be an evolutionary novelty of higher vertebrates, and the more widely conserved stripe domain may fulfil a more fundamental role during segmentation. Nevertheless, both domains play crucial and distinct roles in mouse segmentation. The oscillating, caudal Lfng domain appears essential for the formation of the cranial half of the axial skeleton. Expression of cLfng in the cranial stripe fails to rescue somite formation and cranial-caudal patterning of somites 1 to ∼30, and few BBL pups, in which this is the only domain of Lfng expression, survive to weaning (Figures 2A, 2D–G, 3G–I; Table 1). By contrast, the stripe domain appears to be sufficient and, in the absence of the oscillating domain, necessary for caudal somite formation and patterning, from somite ∼34 onwards (Figures 2A, 2D–G, 3G–I).

Alternatively, the regional phenotypes may reflect different threshold requirements for striped Lfng in different body regions. Transgenic Lfng expression levels are below wildtype levels, and might be too low for regular segmentation cranial to the sacral somites, but sufficiently high for tail somites. In this scenario, high Lfng stripe expression is needed to form somites 1 to ∼30, while weak Lfng stripe expression is sufficient for the formation of tail somites from somite ∼35 on. However, requirement of oscillatory Lfng for normal segmentation of the cranial body has been demonstrated previously [14], [19].

Shifley et al. [53] recently reached similar conclusions to our former model by analysing mice in which Lfng PSM-expression is restricted to the stripe domain. They studied mice (Lfng^ΔFCE1) in which FCE1/A block, the major enhancer element driving caudal oscillating expression, has been deleted from the endogenous Lfng locus. They too found that Lfng stripe expression rescues caudal but not cranial segmentation, indicating that Lfng requirements differ between trunk and tail formation.

Although a single B-block drives expression only in a cranial stripe, our transgenic BBL lines (with tandem B-blocks) also express very low levels of dynamic–presumably oscillating–cLfng in the PSM. Multiple transgenic lines show such expression, indicating that it is indeed due to the duplicated B-block, not the effects of chromosomal flanking sequences on the transgene, i.e. the B-block includes a cryptic oscillator element whose ability to express is enhanced by the tandem duplication. This result is consistent with previous evidence that the Lfng promoter includes multiple cyclic enhancers. In particular, Lfng reporter transgenes lacking the A block still cycle in the caudal PSM, albeit weakly (mlf(1.8); [15]). Such expression is visible only after extended staining, perhaps explaining why such cyclic expression was not observed in the ΔFCE1 embryos [53].

This weak cyclic expression is unlikely to contribute to the rescue of tail segmentation in BBL and ΔFCE1 mice. For this to be the case, segmentation of the trunk would have to require more oscillatory Lfng than tail segmentation, although the intermediate sacral region does not require oscillatory Lfng at all. The oscillatory caudal expression in BBL embryos is insufficient to rescue segmentation in the trunk (Figures 1D, 1E, 2C–G) and to maintain Hes7 oscillation (Figure S3C). Most likely, restoration of the tail is indeed due to the strong, cranial stripe domain of Lfng expression.

Unexpectedly, the sacral region segments almost normally in Lfng^−/− embryos: somites ∼31 to ∼34 are regular and cranial-caudal compartmentalised, and form normal vertebrae (Figures 2C, 2G, 3A–C). The ability of this region to segment independently of Lfng activity has not been noted previously, and is further evidence that the segmentation circuitry varies along the cranial-caudal body axis. Although Hes7 activity is needed for formation of these segments [39], such activity needs not cycle: Hes7 cycling is lost in both Lfng^−/− and Hes7^BAP/BAP embryos (Figures 4C, 5C, 5D, S3B, S3E), yet sacral segments form normally.

Requirements for Hes7 cycling protein show a similar axial variation. Segmentation in Hes7^BAP/BAP embryos is rescued only around the sacral and adjacent tail region (Figure 5G, 5H right), indicating that this region requires less Hes7 activity than elsewhere.

Two lines of evidence argue that Hes7^BAP, in which a peptide tag is inserted near the C-terminus, is almost completely inactive in vivo, even though it retains repressive activity in cultured cells. The homozygous Hes7^BAP/BAP phenotype is almost as strong as that of Hes7^−/− embryos, and is fully recessive. Hes7 is overexpressed in homozygous but not heterozygous BAP embryos, indicative of a recessive failure of repression.

The apparent difference between the ex and in vivo activity of Hes7^BAP could be due to the considerable overexpression in the former context that might allow a greatly weakened Hes7^BAP still to repress target genes. Alternatively, the insertion in Hes7^BAP prevents binding of an accessory factor that is only required in the authentic, in vivo environment. Clearly, because a protein is active in a cultured cell assay, it cannot be assumed to retain activity in vivo.

Temporal Changes of the Segmentation Machinery

Axial changes in the segmentation circuitry were first suggested by the phenotype of Notch signalling mutants. The first 7–9 somites form normally in Notch1/Notch2 double knock-out mutants and in mice lacking the common, downstream transcription factor RBP-Jk/CBF1 [54], [55]. Notch pathway mutations in zebrafish also retain the first ∼7 somites [56], [57]. However, this apparently regional phenotype might be due to gradual desynchronisation of neighbouring oscillators during axial elongation, not to changes in regulatory circuitry [58]. An example of differential regulation of mesoderm development along the length axis is the changing hierarchy of the T-box genes no tail, spadetail and tbx6 in trunk versus tail in zebrafish embryos [59]. Several other genes exert regionalised effects on somite formation by unclear mechanisms, e.g. α5-integrin [60].

Our results define at least three phases of segmentation during the 5 days of mouse somitogenesis (Figure S4). Phases A (somites 1-∼30; E7.75–E10) and C (somites ∼35–65; E10.5–E13.5) rely mainly on oscillating Lfng and the cranial PSM stripe, respectively, and have also been described by Shifley et al [53]. However, we also see clear evidence of a transition phase B (somites ∼31-∼34) that is associated with a drop in the requirement for Lfng activity such that, even in the complete absence of Lfng, mice frequently develop normal sacral vertebrae. This sacral region is also less sensitive to the reduced levels of Hes7 activity in homozygous Hes7^BAP animals (Figure 5G, 5H right). Hes7 requirements may be more stringent for vertebrae in the tail; 43% of adult Hes7^+/− animals have a kinked tail [39].

As our BBL mice express some cyclic Lfng in addition to the cranial PSM stripe that is probably below wildtype levels, we cannot formally exclude the alternative but unlikely model (see discussion above) that stronger, wildtype-like stripe expression would alleviate the Lfng phenotype in the trunk, or that the weak oscillating caudal domain in BBL embryos contributes to the tail rescue. A transgenic line expressing only the cranial PSM stripe at wildtype levels without a caudal PSM domain would allow to unambiguously assign individual expression domains (oscillatory vs. stripe) to phases A and C. However, such a desirable transgenic line cannot be made because the Lfng promoter is more complex than previously thought: the “stripe-specific” enhancer (B-block) also drives a faint caudal domain, and other promoters are unlikely to precisely match timing, position, extent and expression level of the wildtype Lfng stripe.

Requirements for Wnt signalling, which is needed for mouse Lfng to oscillate [61], [62], might also show regional variation. Mice with reduced canonical Wnt signalling due to an in-frame insertion of lacZ in the Lef1 gene form skeletons with severely disorganised vertebrae. These mice exhibit a regularly patterned sacral and cranial tail region similar to that found in BBL animals (Figure 1B in [63]).

It is unclear what positions and triggers the different phases. In wildtype mice, phase B is inconspicuous, and not revealed by any obvious variability in oscillatory modes of expression. The beginning of phase B coincides with the time when mesodermal cells are recruited from the tailbud and no longer by involution and streaming through the primitive streak as for phase A (∼30-somite stage; [64]). The phase B-mode could lead to a partial loss of synchronisation of PSM cells due to changed cell movements, and the segmentation clock circuitry might be modified to take this into account.

The onset of phase B is slightly variable: the site of the first regular vertebra in Lfng^−/− mice varies between the last two lumbar vertebrae and the first two sacral vertebrae. Thus, the lumbosacral boundary, which is positioned by the activity of Hox9 and Hox10 paralogous genes in the PSM [22], [24], does not directly determine the onset of phase B. Nevertheless, the number of vertebrae covered by phase B is relatively invariant (4.1±1.6; details in Table 2), indicating that the duration of the phase is regulated either via the segmentation clock, or by another process independent of axial patterning, e.g. morphological changes associated with the end of gastrulation.

Lfng Is Important for the Maintenance of Somitogenesis

Premature termination of axial extension in Lfng^−/− embryos indicates that progenitor cells become depleted in these embryos at around E10.5. The low variability of Lfng^−/− tail lengths hints at a clear-cut temporal change in the requirement of Lfng for maintenance of axial extension in trunk vs. tail when the segmentation machinery shifts from phase B to C.

Our transgenic BBL lines show an unexpected Lfng expression domain in the caudal PSM. Although this caudal domain could contribute to continued axial extension of the tail by short-range signalling, its expression is very weak and unable to promote segmentation (discussed above). Rather, the rescue of the Lfng^−/− short-tail phenotype by stripe expression of Lfng in BBL mice suggests that Lfng expression in the cranial PSM regulates expression of a component that signals caudally to regulate axial extension.

Our results suggest that this signal is not mediated by retinoic acid, a long-range signal made in newly formed somites; retinoic acid regulates expression of Fgf8 in the caudal PSM and tailbud [65], [66], yet Fgf8 expression is normal in Lfng^−/− E10.5 embryos (Figure 3P, 3R). Either retinoic acid signalling is unaffected in the mutant embryos, or Lfng modifies retinoic acid expression or activity in a manner that does not affect Fgf8 transcription.

Indeed, Fgf8 transcription may not be required to sustain proliferation of axial progenitor cells at late stages of somitogenesis. Fgf8 transcripts are no longer detectable in wildtype PSMs from stage E11.5 (Figure 3Q), yet somitogenesis and Tbx6 and Lfng transcription persists for a further two days (Figure 3T, and not shown). This lack of linkage between Fgf and Lfng expression suggests that other signals from the cranial PSM or somites are involved in maintaining axial elongation.

Mouse Lines

The Lfng null line was obtained from Randy Johnson [17] and the Hes7 null line from Ryoichiro Kageyama [39]. Transgene constructs were injected into F1×F1 (CBA×C57Bl/6J) mouse embryos. Transgenic mice were kept in the C57Bl/6J background. C57Bl/6J also served as wildtype control. All animals were handled in strict accordance with good animal practice as defined by the Animals (Scientific Procedures) Act 1986 and in accordance with the Codes of Practice issued for the use of animals in scientific procedures issued by the UK Home Office. All animal work was approved by the ethical committees for experiments with animals of the London Research Institute.

Preparation of cLfng Transgene Vectors and Hes7^BAP Targeting Construct

The construction of transgene vectors BB-cLfng and Lfng5kb-cLfng (Figure 1A) and of the targeting vector used to generate Hes7^BAP mice is described in Methods S1.

Thermal Asymmetric Interlaced (TAIL) PCR

We mapped the integration sites in the transgenic lines by amplifying the flanking genomic regions using TAIL-PCR with a combination of degenerate and nested primers ([67]; for primers see Table S1).

In 5kL-1 mice, the transgene is inserted into chromosome 19, position 31,973,131 (Build 36), i.e. into the second intron of the APOBEC-1 complementation factor (ACF) locus. We never obtained homozygous transgenics from various hemizygous 5kL-1 parents (100 offspring genotyped for homozygosity), consistent with the pre-implantation lethality of homozygous ACF^−/− embryos [68].

The BBL-1 transgene is inserted into chromosome 8, position 44,990,545. Homozygous BBL-1 transgenics are viable and fertile and appear normal.

In BBL-2 we found three integration sites located within a distance of 500 kb on chromosome 4, all in introns of the “unclassifiable” cDNA AK139518 (positions 23,086,138/23,085,307; 22,619,012; 22,600,785). Integration in position 23,085,307/23,086,135 resulted in a rearrangement of the genomic locus. No homozygous transgenic mice of BBL-2 were identified among the offspring of several hemizygous parents (46 offspring genotyped for homozygosity), indicating that homozygous insertion is embryonic lethal.

We were unable to identify the genomic integration site of the 5kL-2 line, but found tandem insertions of the transgene, as were also present in the other three lines.

In Situ Hybridisation and Immunochemistry

Whole-mount in situ hybridisation was performed by a modification of the method of Henrique et al. ([69]; described in the Supporting Information). RNA probes were made from 2.1 kb cLfng cDNA [70], 0.7 kb Fgf8 cDNA [71], 0.7 kb Hes7 ORF [72], 1.2 kb mouse Lfng cDNA (IMAGE clone 408467), 1.9 kb MyoD cDNA [28], 0.9 kb Pax1 cDNA [30], 0.7 kb Tbx6 cDNA [38], 1.4 kb Tbx18 (PCR-amplified from exon 8) [27], and 0.7 kb Uncx4.1 cDNA [73]. The Hes7 intron probe was synthesised from a 1 kb PCR product of the first intron cloned into pCRII-TOPO vector (Invitrogen) and used as for the cDNA probes except that hybridisation was at 65°C instead of 70°C. Hes7 antibody staining was performed as described [40].

Skeleton Preparation

Adult and E18.5 skeletons were prepared and stained with alcian blue/alizarin red S following standard procedures. All photos were taken with a Leica DC500 digital camera and Leica Firecam version 1.7.1 software. Several photos were assembled for adult skeletons in Figure 2G and 5H.

Luciferase Reporter Assay

C3H10T1/2 cells were plated at a density of 8×10⁴ per well of a 24-well tissue culture plate 24 hours before transfection. 100 ng of the firefly luciferase reporter under the control of six N-boxes and the β-actin promoter [74] was cotransfected with 200 ng pCI (Promega), pCI-Hes7 or pCI-Hes7-BAP using GeneJuice (Novagen) transfection reagent at a ratio of 1∶3, DNA∶GeneJuice. 4 ng of the Renilla luciferase vector pRL-TK (Promega) was used in each sample as reference reading. After incubation for 24 hours at 37°C and 5% CO₂, the assay was analysed using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's guidelines. Firefly and Renilla activities were read using the EnVision Multilabel Reader. The values of the reporter readings were normalised to the values of the Renilla reading. The resulting luciferase activity alone was taken 100%. Each experiment was done in triplicates and repeated at least three times.