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Home , Axial mesoderm, Paraxial mesoderm

Development 121, 877-886 (1995) 877 Printed in Great Britain © The Company of Biologists Limited 1995

The T gene is necessary for normal mesodermal morphogenetic cell movements during

Valerie Wilson1, Linda Manson2, William C. Skarnes3 and Rosa S. P. Beddington1 1Laboratory of Mammalian Development, National Institute for Medical Research, London NW7 1AA, UK 2Biomedical Research Centre, University of Dundee, Ninewells Hospital, Dundee DD1 9SY, UK 3Centre for Genome Research, Edinburgh EH9 3JQ, UK

SUMMARY

The T (Brachyury) deletion in mouse is responsible for parison of T expression in the developing tailbud with the defective and morphogenesis, sites of accumulation of T/T cells in chimeras shows that leading to a failure of the axis to elongate properly T/T cells collect in sites where T would normally be posterior to the forelimb bud. T/T embryonic stem (ES) expressed. T expression becomes internalised in the tailbud cells colonise wild-type , but in chimeras at 10.5 following posterior neuropore closure while, in abnormal days post coitum (dpc) onwards they are found predomi- chimeric tails, T/T cells remain on the surface of the distal nantly in the distal tail, while trunk paraxial and lateral tail. We conclude that prevention of posterior neuropore are deficient in T/T cells (Wilson, V., Rashbass, closure by the wedge of T/T cells remaining in the primitive P. and Beddington, R. S. P. (1992) Development 117, 1321- streak after gastrulation is one source of the abnormal tail 1331). To determine the origin of this abnormal tissue dis- phenotypes observed. tribution, we have isolated T/T and control T/+ ES cell Accumulation of T/T cells in the node and anterior streak clones which express lacZ constitutively using a gene trap during gastrulation results in the preferential incorpora- strategy. Visualisation of T/T cell distribution in chimeric tion of T/T cells into the ventral portion of the embryos throughout gastrulation up to 10.5 dpc shows that and . The latter forms compact blocks a progressive buildup of T/T cells in the primitive streak which are often fused with the ventral neural tube, remi- during gastrulation leads to their incorporation into the niscent of the notochordal defects seen in intact mutants. tailbud. These observations make it likely that one role of Such fusions may be attributed to cell-autonomous changes the T gene product is to act during gastrulation to alter cell in cell adhesion, possibly related to those observed at surface (probably adhesion) properties as cells pass earlier stages in the primitive streak. through the primitive streak. As the chimeric tail elongates at 10.5 dpc, abnormal mor- Key words: T (Brachyury), ES cell, chimera, mouse , phology in the most distal portion becomes apparent. Com- gastrulation

INTRODUCTION during the early stages of gastrulation, since development rostral to the forelimb bud appears grossly normal. Only in more caudal The T gene is deleted in mouse Brachyury mutants (Herrmann trunk regions and in later embryos is the notochord missing and et al., 1990) causing a reduction in tail length in hemizygotes other mesodermal derivatives deficient or defective (Herrmann, and death of homozygous embryos on the 11th day of gestation 1992; Beddington et al., 1992; Rashbass et al., 1994). One expla- (Chesley, 1935; Gluecksohn-Schonheimer, 1944; Gruneberg, nation for this would be that T is only required once all tissues 1958). The T gene encodes a putative transcription factor rostral to the forelimb have been laid down (i.e. after the 1- to (Kispert and Herrmann, 1993) and both its mRNA and protein 2- stage). The demonstration that wild-type T protein is are present in the primitive streak from the onset of gastrulation only required from the 9th day of gestation for continuation of (Wilkinson et al., 1990; Herrmann, 1991; Kispert and Herrmann, its own expression (Herrmann, 1991) and that of certain other 1994). Expression persists in the streak and later in the tailbud genes (Wnt-3a, Wnt-5a (McMahon, 1992), Evx-1 (Dush and for the entire period of axis formation and axis elongation (6.5- Martin, 1992)) transcribed in the streak (Rashbass et al., 1994) 12.5 dpc; Kispert and Herrmann, 1994). It is also expressed in is consistent with such an hypothesis. However, the chimeric the node and notochord. The pattern of T expression appears to studies reported in this paper indicate that T is necessary for be essentially identical in vertebrates where its homologue has normal morphogenetic movements during earlier stages of gas- been studied (Smith et al., 1991; Schulte-Merker et al., 1992). trulation. However, the phenotype of homozygous mutant mouse embryos We have shown previously that abnormalities characteristic does not obviously correlate with an essential function for T of T/T embryos occur in chimeras made between T/T mutant

878 V. Wilson and others cells and wild-type ones (Rashbass et al., 1991). Using a 6% CO2 in air. Subsequently the dishes were inverted, the drops glucose phosphate isomerase isozyme variant as a genetic covered with paraffin oil (Boots, UK, Ltd) and incubated overnight. marker to distinguish mutant cells, we found that T/T cells in Embryos that had developed to the stage were transferred 9.5-11.5 dpc chimeras were unevenly distributed along the to pseudopregnant recipients. embryonic craniocaudal axis (Wilson et al., 1993; Beddington Recovery and staining of chimeras et al., 1992). Mutant cells predominated in caudal regions and Potential chimeras were dissected from the uterus 5-8 days following were relatively sparse in mesodermal derivatives compared to transfer. Fixation and X-gal staining of embryos was as described else- non-ingressing ectodermal tissues. This led to the hypothesis where (Beddington et al., 1989). Embryos up to 8.5 dpc were fixed for that mutant cells ingressing through the streak were defective 10-20 minutes, whereas later stages were fixed for 20-30 minutes. The compared to wild-type cells (with which they were in compe- duration of staining varied according to the ES cell clone used and tition) in their ability to move away from the midline and to ranged from 1 hour to 24 hours. Embryos showing positive staining populate the mesoderm at a normal rate. As a result, mutant were processed for wax histology as described in Beddington (1994), cells accumulate in the region responsible for axial elongation, and 7 µm serial sections were cut (Bright 6030 Microtome). Once and eventually inhibit it. By virtue of a transgenic single cell dewaxed, sections were mounted in DPX mountant (BDH, Ltd.) and marker introduced into mutant cells, we can now verify that photographed (Kodak Ektachrome 64T film) in a Zeiss Axiophot T/T cells start to amass in the primitive streak during gastrula- microscope using differential interference contrast optics. tion. These results demonstrate that wild-type T protein does Whole-mount in situ hybridisation have a function during the earlier stages of gastrulation. They The T mRNA probe was synthesised as described (Wilson et al., 1993) suggest that at least one of its early roles is to regulate the mor- and in situ hybridisation was performed according to Wilkinson phogenetic behaviour of nascent mesoderm by altering cell (1992). autonomous properties, probably related to cell adhesion. RESULTS MATERIALS AND METHODS Isolation and validation of ubiquitously expressing Vectors lacZ ES cell clones The gene trap vectors pGT1,2 and 3 contain the βgeo reporter Following electroporation and drug selection, 210 T/T ES cell (Friedrich and Soriano, 1991), flanked by the En-2 splice acceptor and β SV40 polyadenylation signal from pGT4.5 (Gossler et al., 1989). The clones and 229 T/+ clones were tested for bacterial -galac- En-2 exon sequence was modified to generate three derivatives which tosidase activity in 96-well plates. Approximately half of the can form fusion proteins in one of each of the three reading frames. clones of each genotype were positive (β-gal+) and of these, 21 Equal quantities of each vector were mixed together for electropora- (17.8%) T/T clones and 22 (17.7%) T/+ clones showed blue tion. staining in all cells in the well (Table 1). Four T/T and 5 T/+ strongly staining clones were selected for testing in chimeras Isolation of gene trap ES cell lines ubiquitously expressing lacZ (Table 2). Like the parent cell line (Wilson et al., 1993), the four T/T clones gave rise to chimeras showing a range of abnor- ES cells were maintained as described previously (Wilson et al., 1993). 108 ES cells (either ES line BTBR6 (T/T) or BTBR7 (T/+)) malities (Table 3) depending on the extent of mutant cell con- were electroporated with a mixture of gene trap vectors pGT1,2 and tribution (Fig. 1A,B). A high contribution of T/T cells (greater 3 using standard conditions (Hill and Wurst, 1993) and selected in than about 60%) produced detectable abnormalities by early medium containing G418 (200 µg/ml, Gibco). After 10 days of somite stages reminiscent of the intact T/T phenotype. Chimeras selection, 200-250 macroscopic colonies from each electroporation with a low T/T cell contribution exhibited tail and allantoic were picked into 96-well plates (Nunc) and grown for 3 days in the deformities, most evident from approximately 10.0 dpc. absence of drug selection. Each clone was then passaged into Although carrying independent gene trap insertions, all four T/T duplicate wells in separate 96-well plates and grown overnight. One clones behaved similarly in chimeras (Table 3), indicating that plate was stained with X-gal (Sigma) as described (Beddington et al., the hemizygous transgene was developmentally neutral. Having 1989), except that fixation was performed for 5-10 minutes at 4¡C, and clones showing staining in all cells, including peripheral differ- demonstrated that lacZ was expressed in all embryonic tissues entiated cells, identified. Fifteen clones from each of the two electro- (see below), three of these clones (GM 6.6, 6.12 and 6.15) were porated cell lines, exhibiting apparently ubiquitous staining, were used for subsequent phenotypic analysis. Clone GM7.3 which expanded from the duplicate 96-well plates. After expansion, four T/T exhibited high levels of β-galactosidase activity was selected as and five T/+ clones were tested in chimeras for ubiquitous reporter a heterozygous control. Like its parent (BTBR7), which gave gene expression. rise to more normal chimeras which had only minor tail defects Construction of chimeras ES cells were injected into C57BL/6 or C57BL/10 × CBA Table 1. Generation of β-galactosidase positive (β-gal+) as described previously (Rashbass et al., 1990) and the embryos trans- ES cell clones by electroporation of GT1,2 and 3 ferred to pseudopregnant recipient C57BL/10 × CBA F1 females. Aggregation of ES cells with 8-cell embryos were performed in 5 cm No. resistant No. colonies No. clones β + bacteriological dishes (Sterilin) in hanging drops of M16 medium Cell line colonies/ -gal / where all 8 β + (Hogan et al., 1986) containing 10% FCS. 3-4 clumps, each com- (Genotype) 10 cells No. picked cells -gal prising 3-4 ES cells, were added to each drop containing a single 8- BTRB6 460 118/210 21 cell MF1 embryo, denuded of its zona pellucida using Acid Tyrodes (T/T) (56.1%) (17.8%) solution (Hogan et al., 1986). ES cells and embryos were co-cultured BTBR7 324 124/229 22 in hanging drops for 4 hours at 37°C in a humidified atmosphere with (T/+) (54.1%) (17.7%)

T in morphogenetic movement 879

Table 2. Characteristics of lacZ expressing ES cell clones marked T/T cells in the appears random and there is No. chimeras/ Staining profile of no evidence for specific exclusion of mutant cells from partic- Independent No. embryos Speed of β-gal+ cells in ular regions. Likewise, no epiblast or ectodermal domains are Gentoype clone recovered staining embryonic tissues consistently devoid of mutant cells at head fold and early T/T GM6.6* 12/14 >12 hours strong/ubiquitous somite stages (Fig. 2A). However, by these stages, accumula- T/T GM6.11 1/1 >12 hours strong/ubiquitous tion of T/T cells along the length of the primitive streak is T/T GM6.12* 17/27 1 hour strong/ubiquitous apparent (12/16 chimeras; Fig. 2B,C). Large numbers of T/T T/T GM6.15* 22/37 15 min strong/ubiquitous cells are particularly evident in the node region (5/16 T/+ GM7.1 14/23 >12 hours† weak/ubiquitous chimeras), whereas lateral and are rela- T/+ GM7.2 2/6 1 hour strong/ubiquitous tively devoid of mutant cells (Fig. 2C,D). In sections, T/T cells T/+ GM7.3* 12/14 1 hour strong/ubiquitous appear to congegrate in the midline immediately ventral to the T/+ GM7.4 5/9 15 min strong/ubiquitous primitive streak (mesodermal layer) but there are also concen- T/+ GM7.12 4/6 1 hour strong/ubiquitous trations of mutant cells in the epiblast constituent of the streak *Lines used for subsequent phenotypic analysis. (Fig. 2E). †The notochord stained strongly within 1 hour. Accumulation of T/T cells also occurs at the base of the allantois (Fig. 2C; 11/16 chimeras) and, in some chimeras, abnormal clumps of blue cells are seen on the surface of the at later (10.5-11.5 dpc) stages, GM7.3↔+/+ chimeras at 9.5 dpc allantois, a feature reminiscent of intact homozygous Brachyury were normal in phenotype (Fig. 1C, Table 3). embryos. Sections from 3 embryos show some isolated stained 7.5-10.5 dpc chimeras containing the greatest ES cell contri- cells in the distal portion of the allantois but these are relatively bution were sectioned transversely so that any tissue or site rare. There was no evidence of aberrant morphology within the failing to express lacZ could be identified. Serial sections of wild-type component of chimeric allantoides. GM6.6, GM6.12, GM6.15 and GM7.3 chimeras demonstrated T/+↔+/+ chimeras show no sign of tissue- or region- that bacterial β-galactosidase activity was present in all tissues specific differences in levels of chimerism up to early somite of the embryo (Fig. 1D,E; Table 2). All clones also produced stages (data not shown). stained descendants in extraembryonic mesoderm derivatives. Trophectoderm and primitive derivatives were not Disruption of tail formation in chimeras included in the analysis since they are seldom colonised by ES In our previous electrophoretic analysis of 9.5-11.5 dpc cell descendants (Beddington and Robertson, 1989), and the T chimeras, we demonstrated an increase in T/T cell contribution gene is never expressed in these tissues (Wilkinson et al., 1990). to caudal regions compared with more rostral domains (Wilson Within the embryo, no difference was observed in the staining et al., 1993). Seeing the precise distribution of all ES cell intensity of positive cells located in cranial as opposed to caudal progeny, by virtue of a single cell marker, makes this bias even regions. Therefore, the gene trap strategy has proved an more evident, both at the gross morphological level (Fig. efficient means of generating ES cell clones containing a con- 1A,B) and in histological preparations (Fig. 3). This is partic- stitutive, ubiquitous and neutral single cell marker with which ularly true in chimeras with an otherwise low T/T contribution. to compare the behaviour of T/T and control T/+ cells in The following observations are based on the analysis of serial midgestation chimeras. While embryos containing a high pro- sections of 7 T/T↔+/+ chimeras at 9.5 dpc. From 9.0-10.5 portion of ES cell descendants validate the marker system those dpc, mutant cells are particularly prevalent in the tailbud itself with a contribution of 40% or less are the most informative and in the midline ‘mesoderm’ immediately rostral to it (Table regarding the aberrant behaviour of mutant cells. The subse- 4; Fig. 3C-E), i.e. in the region normally occupied by the quent phenotypic analysis is based on results from at least 2 of notochord, or immediately adjacent to it. Mutant cells in this the independent T/T marked clones for any given stage and is region appear to adhere closely to one another indicating that derived from chimeras containing 40% or less donor cells. accumulation may be due to inappropriate cell:cell adhesion. When wild-type notochord is present only very occasionally T/T cells show abnormal behaviour during the latter are blue cells observed within it. Usually, the T/T midline stages of gastrulation mesodermal cells do not mix with wild-type notochord (Fig. Up to full-length primitive streak stages the distribution of 3F-H) but instead form short stretches of compacted mutant

Table 3. Gross morphology of chimeras made with marked ES cells compared to those derived from parent cell line No. Equivalent No. No. tail and/or No. multiple Genotype Cell line chimeras age (dpc) normal† allantois defects defects T/T GM6.12 13 9.0 4 (30.8%) 5 (38.4%) 4 (30.8%) T/T GM6.15 20 9.5-10.5 7 (35%) 11 (55%) 2 (10%) T/T GM6.6 11 10.5 1 (9.1%) 7 (63.6%) 3 (27.3%) T/T(parent) BTBR6‡ 16 10.5-11.5 0 9 (56.3%) 7 (43.7%) T/+ GM7.3 12 9.5 10 (83.3%) 0 2 (16.7%)* T/+(parent) BTBR7‡ 10 11.5 3 (30%) 4 (40%) 3 (30%)

†The number of normal embryos decreases at later developmental stages due to the inception of tail defects. *Both embryos developmentally retarded by approximately 1 day but morphologically normal. ‡Data from Wilson, Rashbass and Beddington, 1993. 880 V. Wilson and others tissue which often appear to fuse and become incorporated into Mutant cells accumulate in sites of T expression areas of mutant ventral neurectoderm (Fig. 3F-H). The distri- To examine how the sites of accumulation of T/T cells in bution of T/T cells in the neural tube is not uniform. Mutant chimeras correspond to the normal domains of T expression cells predominate in the ventral region (including the future during tail development, the distribution of T transcripts was floorplate) and in the midline of the caudalmost neurectoderm. re-examined in the cell population responsible for axial In addition, a strip of mutant cells is seen extending from the elongation from the start of tailbud formation (~8.75 dpc) to origin of the allantois (on the ventral surface of the tail) back the stage at which the posterior neuropore closes (10.0 dpc). to the distal tip of the tail (Figs 3C-H, 4D,F). In contrast, the At 8.75 dpc, T expression is restricted to a caudal domain, com- paraxial mesoderm and hindgut contain very few T/T cells prising both and mesoderm layers and flanked by the (Figs 3D-H, 4D,F). neural folds (Fig. 4A). Thus, part of the population of cells

Fig. 1. Chimeras from marked T/T and T/+ ES cell clones. GM6.15↔+/+(A), GM6.6↔+/+(B) and GM7.3↔+/+(C) chimeras dissected at an equivalent age of 10.5 dpc (A,B) and 9.5 dpc (C). Arrowheads in A and B indicate abnormally truncated or divided tail. (D,E). Transverse sections through a GM6.15↔+/+ chimera with high level ES cell contribution, showing βgal+ cells in all tissues. (D) Hindbrain level. (E) Forelimb level. N, neural tube; OV, ; S, somite; G, gut; F, forelimb bud. Scale bar: 1.67 mm in A and B; 1.0 mm in C; 330 µm in D and E. T in morphogenetic movement 881 expressing T remains on the dorsal surface of the embryo and tion in chimeras correspond to the normal sites of T gene not until after posterior neuropore closure, which occurs at expression (Fig. 4A,C,E). As a result posterior neuropore about the 30-somite stage (Copp, 1982), does the entire popu- closure is often prevented due to the mass of mutant cells inter- lation of cells expressing T become internalised. (Fig. 4C,E). posed between the posterior lateral neural folds (Fig. 4B,D,F). Presumably, internalisation is normally achieved by rostro- Such a wedge of mutant cells may be responsible for the forked caudal closure of the lateral neural folds and adjoining caudal tail tips frequently observed in T/T↔+/+ chimeras (eg. Fig.1B, ectoderm. Consequently, the posterior streak is internalised second row, second embryo from left). While the distribution first and the node last (Fig. 5A). of ES cell descendants in T/+↔+/+ chimeras is much more It is striking that the sites of maximum T/T cell accumula- uniform along the rostrocaudal axis (Figs 1C, 2F), several 9.5

Fig. 2. T/T cells accumulate in the primitive streak. (A) Uniform distribution of T/T cells in epiblast of an early headfold stage high level chimera, dorsal view; anterior faces right. (B) Accumulation of T/T cells in primitive streak of a second headfold stage embryo, viewed from lateral side; anterior faces right. Regions other than primitive streak contain a lower proportion of T/T cells. Vertical line indicates the extent of the primitive streak. (C) Posterior view of an 8.5 dpc embryo in which T/T cells preferentially colonise primitive streak, node (white arrowhead) and the base of the allantois (black arrowhead). (D) Dorsal view of a 10-somite embryo (9.0 dpc) with T/T cells clustered in the prospective tailbud. (E) Transverse section through primitive streak of embryo shown in D. T/T cells are clustered in the midline of both the ectodermal and mesodermal component of the primitive streak. (F) 9.5 dpc GM7.3↔+/+ embryo showing random distribution of T/+ cells, except for some accumulation in the tailbud. (G) Transverse section through embryo shown in F. T/+ cells are seen in cranial (upper left) and caudal (lower right) ectoderm and mesoderm. Paired horizontal lines in D and F indicate the planes of section in E and G, respectively. Scale bar: 180 µm in A; 170 µm in B and C; 620 µm in D; 210 µm in E; 400 µm in G. 882 V. Wilson and others dpc chimeras show elevated numbers of T/+ cells in the same that independently marked clones behave identically in the parts of the distal tail as those populated by T/T cells (Table embryo. Here we show that the progeny of three independent 4). This is also associated with tail defects although less severe β-gal+ T/T clones exhibit the same overall colonisation profile than those observed in T/T chimeras (Wilson et al., 1993). in chimeras as their unmarked parent (Wilson et al., 1993) and that this differs from the colonisation pattern of a β-gal+ T/+ DISCUSSION cell line and its parent (Wilson et al., 1993). Consequently, hemizygosity for these insertions of lacZ into ubiquitously Validation of the gene trap marker expressed endogenous genes does not seem to have perturbed The frequency of ubiquitously staining ES cell clones in vitro their developmental or morphogenetic potential. (approximately 20% of all β-gal+ clones; Table 1), which cor- responded to ubiquitous enzyme activity in mid-gestation Nascent mesodermal T/T cells exhibit a cell chimeras for all clones tested (Table 2), indicates, as do other autonomous morphogenetic defect gene trap screens (Friedrich and Soriano, 1991; Korn et al., In this section, the abnormal craniocaudal distribution of T/T 1993) that the frequency of ubiquitously expressed genes cells, as opposed to defective notochord differentiation, will be accessible to the gene trap vector may be relatively high. While discussed. Our results show that the characteristic bias of T/T this makes a gene trap strategy attractive for introducing a cell cells to colonise the caudal tail is not due to exclusion of marker the mutagenic nature of the vector insertion has to be mutant cells from regions of the epiblast destined to give rise considered. Although potential dominant lethal mutations to cranial and trunk structures (Fig. 2A). Mutant cells can (identified by germ line transmission of ES cell genotype populate all regions of the epiblast and thus will be represented without gene trap vector sequence) appear to be rare (e.g. 1/28 in all prospective tissue domains defined by fate maps (Bed- lines tested; Friedrich and Soriano, 1991), it is important to dington, 1981, 1982; Lawson and Pedersen, 1992; Tam and confirm neutrality of hemizygous insertions by demonstrating Beddington, 1987).

Fig. 3. Successively more rostral sections through a 10.5 dpc GM6.15↔+/+ chimeric tail. (A) T/T cells located both internally and on the surface of the distal tail. (B-D) T/T cells predominate in axial ectoderm and mesoderm. White arrowhead in D marks the distal end of the hindgut. (E-H) Collections of T/T cells are found in midline , mesoderm and ventral . More proximally, T/T cells are located in the ventral third of the closing neural folds (F-H). (F) Compacted blocks of T/T tissue (arrows) beneath the neural tube fuse proximally (G, left hand block; H, right hand block) with the chimeric ventral neural tube. A normal wild-type notochord is present in F-H (empty arrowheads). Black arrowheads mark the position of a ventral strip of midline ectoderm T/T cells extending from the base of the allantois to the distal tip of the tail. Distally (C,D), these form an abnormal furrow along the ventral edge of the tail. Scale bar: 200 µm. T in morphogenetic movement 883

However, the chimeras make it clear that the imbalance convergent extension characteristic of more caudal mesoderm between rostral and caudal chimerism has its inception during (Niehrs et al., 1993). gastrulation and is rooted in the abnormal accumulation of T/T The accumulation of mutant cells in the site of mesoderm pro- cells in the node, primitive streak and regions immediately duction, and hence the region responsible for axial elongation, ventral to the streak (Fig. 2B-E). Accumulation is first clearly continues throughout late gastrulation and persists in the tailbud discernible at the headfold stage but probably results from when this structure becomes the source of additional caudal abnormal cell behaviour somewhat earlier. However, this mesoderm. As a consequence, there is a deficit of mutant cells in chimeric analysis did not identify abnormal mesoderm definitive mesodermal derivatives. Given the apparently normal movement during the earliest phase of gastrulation. Therefore, differentiation of mutant cells in the gut and all rostral embry- either the most rostral mesoderm cells do not require T for their onic mesodermal derivatives (with the exception of the noto- normal exit from the primitive streak, or this assay is not suf- chord) in chimeras containing a high percentage of T/T cells, and ficiently sensitive to detect earlier abnormalities. The most the demonstration that T/T tissue can give rise to mature endo- rostral mesoderm may be derived from those cells that express derm and mesoderm derivatives when grafted to an ectopic site goosecoid and downregulate T (Schulte-Merker et al., 1994) (Ephrussi, 1935; Fujimoto and Yanigasawa, 1979; Beddington and in lower vertebrates move by active migration rather than et al., 1992), the accumulation of mutant cells in the streak and

Fig. 4. Comparison of T expression with sites of accumulation of T/T cells as primitive streak is replaced by tailbud. (A,C,E). T expression detected by in situ hybridisation to T mRNA. (A). 8.5 dpc embryo, dorsal view; T is expressed on the surface of the primitive streak. (C). 9.5 dpc embryo, lateral view. T expression is enclosed ventrally by surface ectoderm but remains exposed dorsally by the posterior neuropore. (E). Lateral view showing internal T expression once the posterior neuropore has closed at 10.5 dpc. (B,D,F). GM6.15↔+/+ chimeras. (B) 8.5 dpc chimera at an equivalent stage to that of the embryo in A. (D) 10.0 dpc chimeric tail (ventrolateral view) showing ventral midline ectoderm cells and divided tail. (F) 10.5 dpc chimera at equivalent stage to that in E. T/T cells remain located externally in the distal tail. Empty arrowheads: posterior neuropore. Black arrowhead: open posterior neuropore with bifurcation of T/T cell aggregation. Empty arrow: T/T cells located on surface. Scale bar: 210 µm in A-C and E; 310 µm in D; 290 µm in F. 884 V. Wilson and others

allantois amnion dorsal visceral yolk sac anterior posterior ventral primitive streak

head 7.5 d.p.c. process node

somite 8.5 d.p.c. neural tube notochord hindgut

posterior neuropore

somite neural tube notochord 9.5 d.p.c. gut

amnion visceral

yolk sac allantois

neural tube 10.5 d.p.c. notochord gut

A. T expression B. T/+ < > +/+ chimera C. T/T < > +/+ chimera

Fig. 5. Model of progressive accumulation of T/T cells in distal tail. (A) T expression in primitive streak, tailbud, node and notochord. (B) Random distribution of T/+ cells in chimeras. (C) Accumulation of T/T cells in the primitive streak and allantois between 7.5 and 8.5 dpc leads to a mass of T/T cells in the ectodermal and mesodermal components of the tailbud at 9.5 dpc. This results in a physical block to posterior neuropore closure, which would normally occur between 9.5 dpc and 10.5 dpc. Consequently, at 10.5 dpc T/T cells are present on the outer surface of the distal tail, having been enclosed only partly by surface ectoderm ventrally and neuroectoderm dorsally. tailbud cannot be due to an inability to undergo appropriate more, the obvious aggregation of mutant cells in the streak mesodermal or endodermal terminal cytodifferentiation. Instead demarcated by a rather sharp boundary also points to altered cell the defect seems to stem from a morphogenetic failure prevent- adhesion being the primary defect (Fig. 2B-D). The presence of ing nascent mesoderm from moving away from the streak or relatively large numbers of mutant cells in the ectodermal layer tailbud. The tendency of internal T/T cells to clump together of the streak would argue that adhesive changes regulated by T (Fig. 3), instead of intermixing with wild-type cells as their T/+ occur during the epithelial to mesenchymal transition. Since counterparts do (Fig. 2G), suggests that the abnormality in wild-type cells are deployed normally the lack of T protein mesoderm is one of adhesion rather than migration. Further- appears to have a cell autonomous effect, probably mediated by

Table 4. Sites of T/T and T/+ cell accumulation during tail formation Caudal ectoderm* Caudal Ventral Cell Equivalent No. and tailbud axial ectoderm Base of Genotype line age (dpc) chimeras mesoderm mesoderm strip† allantois T/T GM6.15 9.0 5 4 2 4 3 T/T GM6.12 9.0 9 8 7 7 2 14 12 (85.7%) 9 (64.3%) 11 (78.6%) 5 (35.7%) T/T GM6.15 10.0 10 8 6 8 6 T/T GM6.15 10.5 8 4 6 4 6 T/T GM6.6 10.5 8 7 7 7 7 26 19 (73.1%) 19 (73.1%) 19 (73.1%) 19 (73.1%) T/+ GM7.3 9.5 11 4 (36.4%) 0 2 (18.2%) 1 (9.1%)

*Surface and deep cells interposed between posterior lateral neural folds and in older embryos preventing posterior neuropore closure. †Midline ventral strip of surface cells joining origin of the allantois to distal tip of the tail. T in morphogenetic movement 885 cell surface proteins involved in cell adhesion. This would Chesley (1935) and Grüneberg (1958) both report explain why mutant cells accumulate precisely where the T gene notochord/neural tube fusions in intact heterozygous and is normally expressed (e.g. Fig. 4). homozygous Brachyury embryos and attribute this behaviour Adhesive and migratory changes in cells isolated from T/T to altered cell surface properties. Such an affinity between embryos in vitro have been reported by Yanagisawa and mutant axial mesoderm and ventral neural tube may derive Fujimoto (1977), and Hashimoto et al. (1987). Aggregates of from the common origin of these tissues. In both zebrafish midgestation embryonic T/T cells in rotation culture were (Kimmel and Warga, 1986) and Xenopus (Dale and Slack, smaller than those formed by wild-type cells (Yanagisawa and 1987), single from the 128-cell and 32-cell stage Fujimoto, 1977), although the significance of these results is respectively give rise to progeny in both the notochord and unclear since a range of both morphologically normal and floorplate. More impressively, single cells in Hensen’s node of abnormal T/T tissues were present in the assay. Mesodermal the chick (Selleck and Stern, 1991) and in the anterior streak cells from 8-9 dpc T/T embryos cultured on an extracellular of the mouse (Lawson et al., 1991) can contribute descendants matrix substratum migrate slightly more slowly than their wild- to both floorplate and notochord. In the chimeras described type counterparts and, although the number of embryos used in here, it is clear that within the neurectoderm T/T cells prefer- the analysis was small (Hashimoto et al., 1987), these results are entially colonise the ventral neural tube (Fig. 3 and data not consistent with altered adhesion or migration of T/T mesoder- shown). This is presumably because mutant cells accumulate mal cells. In addition, Yanagisawa et al. (1981) have proposed in the node and, consequently, will predominate in the region that the reduced mesoderm/ectoderm ratio of T/T mutants stems from which the ventral neural tube is derived (Lawson et al., from a failure of morphogenetic movement during gastrulation. 1991). Mutant cells that find themselves in the axial mesoderm The chimeric assay described here extends these studies and but are blocked in their differentiation may, therefore, closely identifies the affected population as those cells moving through resemble ventral neurectoderm cells resulting in fusions the primitive streak from midgastrulation stages. between the two cell populations. It is interesting that embryos lacking the protein integrin α5 (Yang et al., 1993) closely resemble homozygous Brachyury Insights into tail development embryos. This raises the possibility that α5 integrin regulation The progressive accumulation of T/T cells in the caudal aspect may be either directly or indirectly downstream of T and, at of chimeras demonstrates that there is a physical continuity least in part, be responsible for the cell autonomous abnor- between the primitive streak (including the node) of the egg malities in cell adhesion and/or movement that we observe in cylinder and the tail bud of much more advanced embryos. Fur- chimeras. If this is the case, we would predict that chimeras thermore, the distribution of mutant cells and the constant size made between wild-type and α5−/α5− cells would exhibit the of the caudal domain of T gene expression in wild-type same anomalous mutant cell behaviour as seen with T/T cells. embryos (Herrmann, 1991; Kispert, 1994) give little indication that streak regression occurs in the mouse. In contrast, the Abnormal notochord development streak and node appear to be incorporated into the tailbud, In zebrafish, it has been shown that homozygous no tail (ntl) becoming fully internalised once posterior neuropore closure embryos, which lack the zebrafish homologue of the T gene, has occurred (Fig. 5). The accumulation of T/T cells prevents form axial mesoderm (except in the very caudal extremity of this closure in chimeras and thus descendants of the streak and the embryo) but that this fails to differentiate into mature node are left exposed on the dorsal amniotic surface of the tail. notochord (Halpern et al., 1993). Transplantation of ntl cells In intact T/T mutants, neural tube closure occurs later than in into wild-type embryos does not rescue this defect (they fail to wild-type littermates (Wilson and Beddington, unpublished colonise notchordal tissue) and in reciprocal transplants donor observations), and delayed posterior neuropore closure in het- wild-type cells can still form notochord in spite of a mutant erozygotes is documented by Gruneberg (1958), where trans- environment. Consequently, the failure of ntl mutant cells to verse sections of the caudal regions of intact T/+ and T/t differentiate into notochord appears to be a cell autonomous mutants show that the posterior neuropore remains open up to defect. However, the axial mesoderm that does form in ntl a more rostral level than in wild-type littermates. mutants can induce floorplate from the ventral neural tube indi- In Xenopus, cell lineage experiments indicate that cells in cating that the specification of floorplate seems to precede the late gastrula dorsal blastopore lip are the progenitors of the definitive notochord differentiation. Similar results have been tailbud hinge region at later stages (Gont et al., 1993). In the found in T/T mouse embryos where floorplate differentiates in mouse, transplantation experiments have shown that tailbud regions of the trunk where normal notochord is missing mesoderm and 8.5 dpc primitive streak cells have equivalent (Rashbass et al., 1994). histogenetic potential and that orthotopically grafted 8.5 dpc Sections of chimeras show that occasionally T/T cells can be primitive streak cells give rise to descendants in the tailbud 24 incorporated into stretches of wild-type notochord. However, it hours later as well as to trunk paraxial mesoderm and other is more common for short (<100 µm) lengths of an additional tissues (Tam and Tan, 1992). DiI labelling of both the node ‘notochord’ composed entirely of mutant cells to form on one and primitive streak at 8.5 dpc also shows that both contain or both sides of a central wild-type notochord in the tail (Fig. cells that contribute to the tailbud at 9.5 dpc, but the node only 3F-H). Frequently, fusions are seen between this mutant gives rise to notochord while the primitive streak descendants ‘notochord’ tissue and the mutant ventral neural tube (Fig. are responsible for generating paraxial mesoderm (Wilson and 3G,H). Like the experiments in zebrafish, these observations Beddington, unpublished data). Thus, the transition from gas- imply a cell autonomous defect in T/T mutant cells, which com- trulation via the primitive streak to axis elongation by supply promises their differentiation into mature notochord, making of new tissue from the tailbud appears to involve a constant intercalation with wild-type notochord cells an exception. population of cells, which retain their topographical relation- 886 V. Wilson and others ship with respect to prospective fates and become internalised embryogenesis. In Postimplantation Development in the Mouse. CIBA during posterior neuropore closure. Consequently, the gradual Found. Symp. 165, 78-86 accumulation of T/T cells in the streak will eventually result in Herrmann, B. G., Labeit, S., Poustka, A., King, T. R. and Lehrach, H. (1990). 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