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First published online 13 April 2005
doi: 10.1242/dev.01819

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Loss of the extraembryonic ectoderm in Elf5 mutants leads to defects in embryonic patterning

Martyn Donnison^*, Angela Beaton^*,, Helen W. Davey, Ric Broadhurst, Phil L'Huillier and Peter L. Pfeffer

AgResearch Crown Research Institute, Ruakura Campus, East Street, Hamilton 2001, New Zealand

View larger version (55K): [in a new window]   Fig. 1. Elf5 expression in the extraembryonic ectoderm (ExE). (A-E) Whole-mount in situ hybridisation analysis showing expression of Elf5 confined to the ExE and its derivative, the chorion, from E5.5 to E8.5. (D) Elf5 is restricted to the ectodermal layer of the chorion, as seen in a sagittal section. an, anterior neuroectoderm; ch, chorion; EPC, ectoplacental cone; epi, epiblast; m, mesoderm; ve, visceral endoderm; ys, yolk sac.

View larger version (23K): [in a new window]   Fig. 2. Loss of Elf5 function by homologous recombination. (A) Target disruption strategy of the Elf5 locus. The targeting construct is shown in the middle. The black box represents the long arm, the striped box the short homology arm of the targeting vector. The position of the external probe and the expected fragment sizes upon NcoI digestion in a Southern blot are indicated. (B) Southern blot of the NcoI-digested genomic DNA of wild-type (+/+) and heterozygous Elf5 mutant (+/–) mice derived from two separate ES cell lines, probed with the external probe depicted in panel A. (C) Embryos were genotyped by PCR, the 410 bp band corresponding to the targeted allele, the 580 bp to the wild-type locus.

View larger version (127K): [in a new window]   Fig. 3. Morphological defects seen in Elf5 homozygous mutant embryos. (A) Elf5-/- embryos could usually be morphologically recognised at E6.5 by their reduced size and the lack of the embryonic-extraembryonic constriction (arrow). (B,C) Haematoxylin and Eosin-stained sagittal cross sections revealed the presence of ectoderm, EPC and visceral endoderm (ve) in homozygous E7.5 mutants (C), but the absence of chorion (ch) and mesoderm (m), which are readily detectable in wild-type littermates (B). (D) At E8.5, approximately half of the Elf5 mutants did not express the pan mesodermal marker T (phenotypically more severe type I mutants), whereas the other half expressed T ectopically (type II mutants). (E) By E8.5, Elf5-/- embryo epithelium displayed an anterior neuroectodermal character, as shown by whole-mount in situ hybridisation with Otx2. D, distal; ee, embryonic ectoderm; P, proximal.

View larger version (125K): [in a new window]   Fig. 4. Elf5 deficient embryos lack the ExE. (A-N) Embryos are orientated with their proximal end at the top. Whole-mount in situ hybridisation of wild-type/heterozygous (on left side of all panels except M) and Elf5-/- embryos for the ExE markers Cdx2, Eomes, Fgfr2, Bmp4, Furin and Spc4, and the epiblast markers Oct4 and Otx2, as indicated. (A) At implantation stages, Cdx2 expression is seen in the polar trophectoderm (pTE) in both wild-type and Elf5-/- embryos. (B-J) None of the six ExE markers is expressed in the proximal region of mutant egg cylinders, indicating the absence of ExE tissue from as early as E5.5. (E) At E5.5, Fgfr2 is expressed more strongly in EPC than ExE tissue of wild-type embryos. In Elf5-/- counterparts, only the strong expression in the EPC capping the egg cylinder is seen. (H) Furin expression is detected in the EPC of E6.5 Elf5-/- mutants. (I,J) Spc4 is expressed in both EPC and ExE in E5.5 and E6.5 heterozygous embryos. In Elf5 homozygotes, it can only be detected at E5.5 in the EPC layer overlying the epiblast. (K,L) Oct4 is expressed up to the proximal margin of the embryo before gastrulation. (M,N) Expression of Otx2 extends into the proximal half of Elf5 deficient embryos, clearly seen in longitudinal sections (panel N).

View larger version (136K): [in a new window]   Fig. 8. Expression of markers for mesoderm and posterior epiblast in Elf5-/- embryos. (A) Nodal transcripts can no longer be detected in (type I) Elf5 null mutants by E6.5. (B) T/brachyury expression marking nascent mesoderm was never seen in E6.5 Elf5 deficient embryos. (C-E) The posterior epiblast/nascent mesoderm markers Cripto (C), Eomes (D) and Fgf8 (E) were either absent (type I mutants) or seen in the proximal region of the embryo, adjacent to the EPC (type II mutants). (F) No lateral mesoderm or extraembryonic mesoderm was formed in Elf5-/- embryos, as noted by the absence of Bmp4 transcripts (dorsal view, posterior at top of panel). (G) Cdx2, which in heterozygous E7.5 embryos marks the extraembryonic mesoderm and chorion, was not detected in Elf5 deficient embryos.

View larger version (77K): [in a new window]   Fig. 5. TS cells cannot be isolated from Elf5-/- embryos. (A,B) Cultures from Elf5+/– or wild-type E6.5 proximal ectoderm show distinct TS-like colonies (A), whereas Elf5-/- cultures yield no colonies. (B) Fifteen days after embryo dissociation only some giant cells remain. (C) Change in gene expression levels in wild-type TS colonies grown for 5 days in the absence versus presence of Fgf4/heparin, as measured by real-time RT-PCR.

View larger version (104K): [in a new window]   Fig. 6. Tetraploid aggregation chimeras composed of Elf5-/- epiblast are developmentally normal. (A) PCR genotyping of the tail tips of chimeric embryos reveals the presence of four embryos, indicated by arrows, composed solely of Elf5-/- cells. Embryo 5 had died and was not genotyped. (B,C) Chimeric embryos (embryo 13 shown) containing only wild-type (or wild-type and Elf5+/–) cells in embryonic and extraembryonic compartments (B) were morphologically indistinguishable from chimeric embryos (embryo 14 shown) containing only Elf5-/- diploid embryonic cells (C).

View larger version (56K): [in a new window]   Fig. 7. AVE formation in Elf5-/- mutant embryos. (A-C) Whole-mount in situ hybridisation analysis demonstrating that the AVE, as marked at E5.8 by Hex (A) and at E6.5 by Cer-l (B) expression, is formed and migrates to one side in Elf5-/- embryos. (C) By E7.5, Hesx1 transcripts are still apparent. (D) Nodal expression could consistently be detected in pregastrulation Elf5 null mutants.

View larger version (34K): [in a new window]   Fig. 9. The role of Elf5 in the TS cell lineage and the effects of its absence on embryonic development. (A) Model for the specification and maintenance of the TS lineage during early development. The effect of loss of function of the transcription factors Cdx2, Eomes and Elf5 is indicated (see text for discussion; TE, trophectoderm). (B,C) Model depicting the effect of the absence of ExE on embryonic development. (B) In wild-type pregastrula embryos, Furin and Spc4 protein produced by the ExE (green) diffuses (orange arrow) into the epiblast (blue), causing proteolytic activation of precursor Nodal (preNodal), which at these stages is produced from the intronic enhancer (InE) (Brennan et al., 2001; Norris et al., 2002). Activated Nodal protein positively acts (curved arrows) on its own transcription, as well as inducing the formation of the AVE (red). The lack of Elf5-dependent TS cell renewal in Elf5-/- mutants leads to the absence of ExE at E5.5. The EPC, now adjacent to the epiblast, produces Spc4, which is sufficient to activate preNodal in the epiblast, thereby leading to correct AVE formation in Elf5 null mutants. (C) By E6.0 to 6.5, when gastrulation commences, Nodal transcription has come under the control of the Nodal-independent PEE enhancer (Norris et al., 2002; Vincent et al., 2003). In Elf5 mutants, the absence of the ExE results in the loss of the factor(s) required for Nodal transcription from the PEE enhancer. Candidates for such factors are Bmps (dashed red arrow; see text). The loss of Nodal transcription in Elf5-/- embryos results in the absence of posterior gene expression and thus mesoderm (purple) is not formed.