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First published online 13 May 2004
doi: 10.1242/dev.01137

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The Wnt co-receptors Lrp5 and Lrp6 are essential for gastrulation in mice

Olivia G. Kelly^*, Kathy I. Pinson^*, and William C. Skarnes^,

Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720-3200, USA

View larger version (59K): [in a new window]   Fig. 1. Lrp5/6 mutant alleles and expression of Lrp5 and Lrp6 in early embryos. (A, top) Genomic structure and restriction map of the region surrounding exon 1 (black box) of the Lrp5 gene. Primers used to amplify the wild-type allele, Lrp5-U1 and Lrp5-D1, are indicated by arrows. (A, bottom) Targeting vector used to replace part of exon 1 of Lrp5 with the IRES-ß-galactosidase (ß-gal) reporter and MC1-neomycin phosphotransferase (neo) selection cassette by homologous recombination. Primers used to amplify the mutant allele, neo and Lrp5-D1 are indicated by arrows (see Materials and methods for further details). IRES, internal ribosome entry site; TK, thymidine kinase. (B, top) Genomic structure of part of the Lrp6 gene. Primers used to amplify the wild-type allele, Lrp6-U1 and Lrp6-D1 are indicated by arrows. Exons are indicated by black boxes and the arrowhead shows the position of the gene-trap insertion in intron 5. (B, bottom) Genomic structure of the mutant Lrp6 allele. The gene trap vector contains the splice acceptor (SA) of the mouse engrailed 2 gene (En2), the transmembrane domain of rat CD4 and the ß-galactosidase-neo (ß-geo) reporter. Primers used to amplify the mutant allele, Lrp6-U1 and en2 are indicated by arrows. (C) PCR genotyping of Lrp5 and Lrp6 mutations. Ethidium bromide-stained 1.5% agarose gel of DNA products produced by PCR amplification of genomic DNA using the genotyping primers shown in A and B. Numbers on the left indicate the size of the molecular weight markers in base pairs. (D) Northern blot analysis of 20 µg of total RNA isolated from adult kidneys of Lrp5+/+ and Lrp5-/- mice probed with the 3'UTR of Lrp5 (top) and reprobed with actin cDNA (bottom) as a loading control. (E-I) Expression of Lrp6-ß-galactosidase reporter fusion protein. Anterior is towards the left in all panels. Control embryo (E) and Lrp6+/- embryos (F-I) were stained with X-gal. Lrp6 is widely expressed at 6.5 dpc (F) and 7.5 dpc (G-I). Line in G indicates the plane of section in H. Transverse (H) and sagittal (I) sections of 7.5 dpc Lrp6+/- embryos showing ß-galactosidase expression in the embryonic ectoderm (ee), mesoderm (me) and definitive endoderm (de) emerging from the primitive streak (ps). (J-N) Whole-mount RNA in situ analysis of Lrp5 expression in wild-type embryos. Anterior is towards the left in all panels. Embryos were stained with sense control (J) and antisense (K-N) riboprobes. At 6.5 dpc (K), Lrp5 is expressed throughout the ectoderm. At 7.5 dpc (L-N) Lrp5 expression is highest in the visceral endoderm overlying the extra-embryonic, but not embryonic region. Line in L indicates the plane of transverse section shown in M. Transverse (M) and sagittal (N) sections of stained embryos showing Lrp5 expression in the visceral endoderm (ve), embryonic ectoderm (ee), but not in nascent mesoderm (me) or definitive endoderm (de) emerging from the primitive streak (ps).

View larger version (66K): [in a new window]   Fig. 2. Functional redundancy between Lrp5 and Lrp6 in the limb. (A-C) Dorsal views of skeletal preparations of 18.5 dpc forelimbs stained with Alizarin Red showing regions of ossification in Lrp5+/- (A), Lrp5-/- (B) and Lrp5+/-;Lrp6+/- (C) littermates. Arrows show ossification centers in the phalanx that are lost in Lrp5-/- and Lrp5+/-;Lrp6+/- forelimbs. (D,E) Ventral views of adult forelimbs showing loss of a post-axial digit (arrow) in Lrp5+/-;Lrp6+/- mice (E) compared with a wild-type littermate (D). (F,G) Side views of newborn pups showing normal limbs (F) compared with defects in a Lrp5-/-;Lrp6+/- mutant littermate (G). (H-M) Skeletal preparations of newborn limbs stained with Alizarin Red and Alcian Blue to indicate bone and cartilage formation, respectively. Lrp5-/-;Lrp6+/- forelimbs (I,J) and hindlimbs (L,M) show severe defects compared with a wild-type forelimb (H) and hindlimb (K). The mutant forelimb in I is from the newborn pictured in G.

View larger version (61K): [in a new window]   Fig. 3. Mesodermal migration and patterning defects in Lrp5+/-;Lrp6-/- embryos. Whole-mount RNA in situ analysis of marker genes in normal and Lrp5+/-;Lrp6-/- mutant embryos between 7.5 dpc and 8.0 dpc. Anterior is towards the left in all panels. (A-D) Analysis of T expression. Control (A) and mutant (B) embryos showing expression of T in the primitive streak. Lines indicate level of transverse sections shown in C and D. Transverse section of a control embryo (C) showing T expression in the primitive streak (ps) and its rapid downregulation in nascent mesoderm migrating away from the streak (arrow). In the mutant embryo (D), T expression is seen within ectodermal cells and a few mesenchymal cells, but is downregulated in most of the mesenchymal cells that have accumulated in the abnormal primitive streak (arrowhead). Fewer mesodermal cells are seen migrating away from the streak (arrow). (E-H) Analysis of Lhx1 expression. Control (E) and mutant (F) embryos showing Lhx1 expression in mesendoderm. Line in F corresponds to level of transverse section in H; arrow in F indicates the allantois. Transverse section (G) through a slightly younger 7.5 dpc control embryo (inset) than pictured in E, showing Lhx1 expression in the primitive streak (ps) and in nascent mesoderm that has migrated laterally (arrow). Transverse section (H) of the mutant embryo shown in F showing Lhx1 expression in mesenchymal cells that have accumulated at the abnormal primitive streak (arrowhead). (I,J) Analysis of Ebaf expression. A control embryo (I) shows expression in the primitive streak and in wings of nascent mesoderm, whereas expression in the mutant embryo (J) is confined to cells accumulating at the posterior primitive streak. (K,L) Analysis of Tbx6 expression. A control embryo (K) shows expression in the primitive streak that is lost the mutant embryo (L). (M,N) Analysis of sprouty 2 (Spry2) expression. Expression is detected in primitive streak of both control (M) and mutant (N) embryos. All embryos were photographed at the same magnification, as were the sections.

View larger version (90K): [in a new window]   Fig. 4. Expanded anterior primitive streak derivatives and increased Nodal expression in Lrp 5+/-;Lrp6-/- embryos. Whole-mount RNA in situ analysis of marker gene expression in normal and Lrp5+/-;Lrp6-/- mutant embryos. Anterior is towards the left in all panels except B, which is an anterolateral view. Embryos in A-D and K-P are between 7.5 dpc and 8.0 dpc stages, whereas embryos in E-J are at 8.5 dpc. (A,B) Analysis of Foxa2 expression. A control embryo (A) shows expression in the axial mesendoderm, which is greatly expanded in the mutant embryo (B). (C,D) Analysis of Hex expression. A control embryo (C) shows expression in the ADE, which is broader in the mutant embryo (D). (E-H) Analysis of Shh expression. Control embryos (E,G) show expression in the axial mesoderm that is greatly (F) or mildly (H) reduced in mutant embryos. (I,J) Analysis of T expression. Control (I) and mutant (J) embryos show expression in the axial mesoderm. (K-P) Analysis of Nodal expression. Control embryos show expression in the primitive streak (K) that is downregulated by the late streak stage (M) and restricted to the node by the headfold stage (O). Mutant embryos (L,N,P) fail to properly downregulate expression in the primitive streak as development proceeds. Photos taken at the same magnification are grouped as follows: A-D; E,F; G-J; K-P.

View larger version (85K): [in a new window]   Fig. 5. Expanded anterior neurectoderm in Lrp5+/-;Lrp6-/- embryos. Whole-mount RNA in situ analysis of marker gene expression in normal and Lrp5+/-;Lrp6-/- mutant embryos. Anterior is towards the left in all panels except D, which shows an anterior view. Embryos in A,B and C-H are at 8.0 dpc and 8.5 dpc, respectively. (A-D) Analysis of Six3 expression. Control embryos (A,C) show expression in anterior neurectoderm that is greatly expanded in mutant embryos (B,D). Inset in D shows a dorsal view of the mutant embryo, dissected out of the yolk sac. (E,F) Analysis of Gbx2 expression. A control embryo (E) shows expression in the anterior hindbrain, which is also evident in the mutant embryo (F), particularly in a dorsal view (inset, arrow) of the anterior region of the embryo. (G,H) Analysis of Pou5f1 expression. A control embryo (G) has no expression, whereas the mutant embryo (H) shows expression in the abnormal posterior region. Photos taken at the same magnification are grouped as follows: A,B; C-H.

View larger version (100K): [in a new window]   Fig. 6. Double homozygous mutant embryos fail to form a primitive streak and its derivatives. Histological and wholemount in situ analysis of marker gene expression in normal and Lrp5-/-;Lrp6-/- mutant embryos at 7.0-7.5 dpc. Where possible, anterior is towards the left. For some mutant embryos, the orientation is ambiguous. (A,B) Sagittal histological sections stained with Hematoxylin and Eosin. In the normal embryo (A), mesoderm (me) is apparent between the embryonic ectoderm (ee) and either the visceral endoderm (ve) or definitive endoderm (de), and the amnion (am) has formed. In the mutant embryo (B), no mesoderm or definitive endoderm is detected. (C,D) Analysis of Bmp4 expression. A control embryo (C) shows expression in the extra-embryonic mesoderm, whereas the mutant embryo (D) shows expression in only a faint proximal ring (arrow). (E,F) Analysis of Fgf8 expression. A control embryo (E) shows expression at the primitive streak that is absent in the mutant embryo (F). (G,H) Analysis of T expression. A control embryo (G) shows expression at primitive streak that is lost in the mutant embryo (H). (I,J) Analysis of Tbx6 expression. A control embryo (I) shows expression at the primitive streak that is missing in the mutant embryo (J). (K,L) Analysis of Wnt3 expression. A control embryo (K) shows expression in the primitive streak and nascent mesoderm that is lost in the mutant embryo (L). (M,N) Analysis of Lhx1 expression. A control embryo (M) shows expression in the primitive streak, nascent mesoderm, and the AVE. The mutant embryo (N) shows expression in a small distal patch (arrow) that most probably corresponds to the AVE. (O,P) Analysis of Hex expression. A control embryo (O) shows expression in the ADE. The mutant embryo (P) shows reduced expression that most probably corresponds to the AVE. (Q,R) Analysis of Cer1 expression. A control embryo (Q) shows expression in the AVE and definitive endoderm. The mutant embryo (R) shows reduced expression that most probably corresponds to the AVE. (S,T) Analysis of Hesx1 expression. A control embryo (S) shows expression in the anterior axial mesendoderm and overlying anterior neurectoderm. The mutant embryo (T) shows greatly expanded expression that likely corresponds to anterior neurectoderm. Photos taken at the same magnification are grouped as follows: A,B; C,D,G,H,K-T; E,F,I,J.