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Insufficient VEGFA activity in yolk sac endoderm compromises haematopoietic and endothelial differentiation

Annette Damert^1,*,, Lucile Miquerol^2,,, Marina Gertsenstein², Werner Risau^1, and Andras Nagy^2,¶

¹ Max Planck Institute for Physiological and Clinical Research, Bad Nauheim, Germany
² Mount Sinai Hospital, Samuel Lunenfeld Research Institute, Toronto, Canada
^* Present address: Paul-Ehrlich-Institute, Langen, Germany
Present address: Laboratoire de génétique et physiologie du développement, IBDM- campus de Luminy, Marseille, France
These authors contributed equally to this work
In memoriam: Professor Werner Risau (18 December 1953-13 December 1998)

View larger version (18K): [in a new window]   Fig. 1. Knock-in of IRES-lacZ into the VEGFA locus. (A) Vegf IRES-lacZ knock-in alleles with insertion of the reporter cassette at different positions of the Vegf 3'UTR. LoxP sites remaining after excision of the selectable marker are indicated by arrowheads. Sequences are not drawn to scale. (B) Schematic representation of the wild-type, targeted (Vegflo+Neo) and Neo-excised (Vegflo) allele. Exons are depicted as boxes. Probes used in Southern blot analysis and expected fragment lengths are indicated. (C) Southern blot analysis with Probes 1 and 2 on EcoRV digested genomic DNA showed proper homologous recombination both at the 5' and 3' homology arms of the target vector, respectively. The Southern blot analysis with Probe 1 on BglII digested genomic DNA showed successful removal of the loxP flanked neo selectable marker.

View larger version (99K): [in a new window]   Fig. 2. Phenotypical consequences of homozygous IRES-lacZ knock-in into the Vegf locus. +/+, wild type; –/–, Vegflo/lo. (E-H) -PECAM specific immunohistochemistry; (I-T) Haematoxylin/Eosin-stained sections. (A,B,E,F) In the 8.5 dpc yolk sac, defects in vascularisation (B,F) lead to formation of endothelial cell-lined ‘lacunae’ (arrows). (I,J) Few ‘blood islands’ (arrowheads) devoid of primitive erythrocytes are observed in Vegflo/lo embryos (J) in comparison with wild-type littermates (I). (C,D) Growth retardation in Vegflo/lo (D) becomes obvious shortly before death at 8.5-9.0 dpc (compare with wild type in C). (G,H,K,L) Lumen formation of the dorsal aortae is impaired (H,L) in the aorta-gonad-mesonephros region at 8.5 dpc when compared with wild type (G,K). Dorsal aortae are indicated with asterisks (G,H) and arrowheads (K,L). (M-P) Intra-embryonic defects with delayed heart development and lack of blood cells in the sinus venosus (sv) (N,P) compared with wild type (M,O). Endocardium (enc) and cardinal veins (white arrowheads) are formed properly. Dorsal aortae are indicated with black arrowgeads. (Q-T) Phenotypic alterations in the yolk sac are obvious as early as 8.0 dpc. Embryos show a reduction in the number of blood islands and those that are present are devoid of primitive erythrocytes.

View larger version (75K): [in a new window]   Fig. 3. Expression of endothelial (VEGFR1, VEGFR2 and VEZF) and haematopoietic (EKLF) markers is disturbed in Vegflo/lo embryos. Whole-mount in situ hybridisation for the marker gene expression was performed on 8.0 and 9.0 dpc wild-type (+/+) and Vegflo/lo (–/–) embryos. Note the presence of intersomitic vessels sprouting from the dorsal aorta in 9.0 dpc Vegflo/lo (–/–) embryos that hybridised to the VEGFR2 probe.

View larger version (157K): [in a new window]   Fig. 4. Expression of VEGFR2 in wild-type (A,C) and Vegflo/lo (B,D) embryos at 9.0 dpc. Sections are derived from whole-mount in situ hybridisation with a VEGFR2 probe. Transverse sections show VEGFR2 expression in the endocardium (enc) and the cardinal vein (cv) in Vegflo/lo embryos (B) and in the endothelial cells lining one of the rare blood islands (bl; D).

View larger version (31K): [in a new window]   Fig. 5. Aberrant splicing of the Vegf-lacZ fusion RNA results in loss of exon 8-coding sequences and the complete IRES. (A) Schematic representation of mouse VEGFA isoforms (VEGF 188, VEGF 164 and VEGF 120) (, VEGFR1 binding site; , VEGFR2 binding site) and the predicted Vegf-lacZ fusion RNA followed by the actual RNA generated by aberrant splicing (primers used in RT-PCR analysis are indicated as arrows). (B) Sequence comparison of the VEGFA exon7/8 junction (top) and the exon 7/lacZ splice junction of the Vegflo RNA (bottom). The out-of-frame lacZ start codon and the stop codon, which terminates the translation of the fusion protein, are underlined. (C) RT-PCR analysis of the Vegflo allele (lo, lane 3). Oligo dT-primed cDNA was amplified using the primers indicated in A, amplification from the VegflacZKI-allele is shown for comparison (KI, lane 2). (D) Quantitative analysis of VEGFA-immunoreactive material in 8.5 dpc mouse embryos of different genotypes. Amounts of VEGFA immunoreactive material are given in pg/ml.

View larger version (106K): [in a new window]   Fig. 6. Rescue of blood island formation by wild-type visceral endoderm. (A) Schematic representation of the aggregation setup: tetraploid Vegf wild-type (wt) GFP+ embryos were aggregated with diploid Vegflo/lo embryos (F2), resulting in chimaeric visceral endoderm and Vegflo/lo mesoderm. (B) Whole-mount photograph of a 9.0 dpc embryo and its chimaeric yolk sac (ys), which was taken under GFP visualising light conditions. The black arrowhead points to trace contribution of tetraploid cells to the gut epithelium. White arrowheads indicate the remains of the yolk sac attached to the embryo. (C) One of the GFP-positive patches in the yolk sac. (D) Photograph of the same area as in C using light conditions that allow the visualisation of GFP-negative cells as well. (E) The same area as in C,D after lacZ staining. (F) Appropriate (green arrow) and impaired (blue arrow) blood island formation in the chimaeric yolk sac. Higher magnifications of the respective areas are shown in G,H. (I) ß-galactosidase staining reveals blood islands only in areas where the visceral endoderm is Vegf wild type (lacZ-negative cells, green arrows), whereas blood islands cannot be formed in opposition to Vegflo/lo (lacZ-positive cells, blue arrows) endoderm. (L) Higher magnification of the boxed area in I. The short range rescue of blood island formation by VEGFA secreted from wild-type visceral endoderm is marked by an asterisk. (J,K) Extensive wild-type tetraploid contribution to the yolk sac visceral endoderm is not sufficient to rescue intra-embryonic vascular defects. Lumina of the dorsal aortae (black arrows) are barely detectable (J). An increase in the number of blood cells (in the heart in K and sinus venosus in J (red arrows) is observed with increasing wild-type tetraploid contribution. h, heart; sv, sinus venosus.

View larger version (91K): [in a new window]   Fig. 7. Vegflo/lo visceral endoderm cannot support blood island formation in wild-type mesoderm. (A) Schematic representation of the aggregation setup. Tetraploid Vegflo/lo embryos were aggregated with Vegf wild-type (wt) GFP+ ES cells resulting in completely Vegflo/lo-derived visceral endoderm and wild-type mesoderm. (B-D) Whole-mount photographs of a 9.5 dpc chimaeric embryo and yolk sac derived from tetraploid Vegflo/lo/Vegf wild-type GFP+ ES cell aggregation. Embryo proper (B) and vitelline vessels (arrowhead in D) are wild-type GFP+ whereas Vegflo/lo-lacZ positive cells constitute the yolk sac visceral endoderm (C). (E) Uniform and intense GFP fluorescence in a 9.5 dpc chimaeric yolk sac derived from tetraploid Vegflo/+/Vegf wild-type GFP+ ES cell aggregation indicates proper expansion of mesoderm-derived cell lineages. (G,H) ß-galactosidase staining of yolk sac derived from tetraploid Vegflo/lo/wild-type GFP+ ES cell (G) and Vegflo/+/wild-type GFP+ ES cell (H) aggregations. Only few blood islands devoid of primitive erythrocytes are seen in association with Vegflo/lo visceral endoderm (G), whereas blood island formation proceeds normally when supported by Vegflo/+ visceral endoderm (H). (F,I,J) Normal vascular but not haematopoietic development in the embryo proper. Dorsal aortae (arrowheads) show appropriate lumen formation. The number of blood cells in the heart atrium (at) and sinus venosus (sv) is reduced compared with completely wild-type embryos.