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First published online 9 April 2008
doi: 10.1242/dev.018853

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Dkk1 and Wnt3 interact to control head morphogenesis in the mouse

¹ Embryology Unit, Children's Medical Research Institute, University of Sydney, Locked Bag 23, Wentworthville, New South Wales, NSW 2145, Australia.
² Faculty of Medicine, University of Sydney, Locked Bag 23, Wentworthville, New South Wales, NSW 2145, Australia.
³ Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, National Institute of Health, Bethesda, MD 20892, USA.
⁴ The Walter and Eliza Hall Institute of Medical Research, 1G, Royal Parade, Parkville, Victoria 3050, Australia.

View larger version (65K): [in this window] [in a new window]   Fig. 1. Enhanced expression of the WNT reporter in the Dkk1-/- embryo. (A-J) Expression pattern of the BATGal reporter in (A-E) wild-type and (F-J) Dkk1-/- embryos (A,F, E7.0; B,G, E7.5; C,H, E7.75; D,I, E8.5; E,J, E9.5). In the mutant embryos, the lacZ expression domain expands to the anterior germ layer tissues at E7.0-7.75, encompasses the whole head folds at E8.5 and reaches the rostral-most region of the truncated head. Scale bar: 50 µm.

View larger version (84K): [in this window] [in a new window]   Fig. 2. Impact of loss of Dkk1 on mesoderm and endoderm differentiation and anterior morphogenesis. (A) Dkk1-/- embryo shows an expanded domain (black asterisk) of BATGal (2/2 embryos) and Axin2 expression (3/3 embryos) in the head folds and anterior tissues, and loses head structures anterior to the upper hindbrain level (red arrow) with an abrupt truncation of Shh-expressing axial mesoderm and floor plate. Scale bar: 100 µm. (B,C) Marker analysis of the wild-type and Dkk1-/- embryos at E7.0 (B: Foxa2, Chrd and Lhx1; C: Cer1 and Sox17), E7.5 (B: Mixl1, T and Tbx6), E8.5 (C: Foxa2) and E9.0 (C: Sox17). Scale bar: 50 µm. Markers of anterior visceral endoderm are expressed in the mutant embryos (black asterisk). The domain (indicated by lines) of Foxa2 and Chrd expression in the node area is broader, and T expression extends more anteriorly in the head process mesoderm. The endoderm is formed (Cer1), but fewer Sox17- expressing cells are present in the mutant embryo (black arrow). Sox17 expression is absent in the foregut of the E9.0 mutant embryo. At E8.5, the head truncation is clearly visible (Foxa2: whole-mount embryo, red arrow), and the histology (transverse section of the head) shows reduced Foxa2 expression in the foregut (red asterisk) and the neural plate (green bar).

View larger version (81K): [in this window] [in a new window]   Fig. 3. Expression of WNT signalling pathway components in the mutant embryo. (A,B) Double in situ hybridization showing Dkk1 (orange) and Wnt3 (purple) expression domains. (A) At E6.5, the Dkk1 expression domain is juxtaposed to the Wnt3 domain. (B) At E7.25, the two domains become wider apart. (C-G') Wild-type (C-G) and Dkk1-/- (C'-G') E7.0 embryos: the domain of Wnt3 (C,C') remain unchanged, but that of Wnt2b (D,D'; six out of six mutant embryos) and Wnt8a (E,E'; one out of three mutant embryos) are slightly expanded in Dkk1-null embryos. The expression domain of Sfrp1 (F,F') is reduced while that of Srfp5 (G,G') is unchanged. (H) The domain of Dkk1 expression becomes more restricted with the decrease in Wnt3 gene dose. (I) RT-PCR analysis of Dkk1 expression in NIH3T3 cells expressing lacZ, Wnt1,Wnt3a, Wnt4, Wnt5a, Wnt7a and Wnt11. -RT, no reverse transcription control; Hprt, loading control. (J) Reduced domain of Dkk1 expression in the Dkk1+/-;Wnt3+/- embryo, compared with Dkk1+/+;Wnt3+/+, Dkk1+/+;Wnt3+/- and Dkk1+/-;Wnt3+/+ embryos (top row, lateral view; bottom row, frontal view). In situ hybridization for H and J was performed under the same conditions so that the staining result can be compared among embryos of each set. Scale bar: 50 µm.

View larger version (56K): [in this window] [in a new window]   Fig. 4. Anterior defects of the compound Dkk1+/-;Wnt3+/- mutant embryo. (A) The E9.5 Dkk1+/-;Wnt3+/- embryos display the four categories of phenotypes (Class I-IV) with different degrees of head and trunk defects, compared with single heterozygous (Dkk1+/-;Wnt3+/+ and Dkk1+/+;Wnt3+/-) embryo with normal head morphology and single homozygous mutants showing head truncation (Dkk1-/-;Wnt3+/+) or arrested development at gastrulation (Dkk1+/+;Wnt3-/-, broken white line marks the embryonic/extra-embryonic border of the embryo). (B) BATgal reporter is expressed ectopically in cells in the anterior region (red asterisks) of the E7.75 compound Dkk1+/-;Wnt3+/- mutant embryo. Analysis of marker expression reveals the loss of forebrain tissue at E8.5 (Fgf8, Hesx1 and Six3). In the Dkk1+/-;Wnt3+/- embryos, Fgf8 expression is reduced in the commissural plate (arrow) but not altered in the isthmus (black asterisk). Tissues anterior to the Hesx1-expressing domain are reduced (red asterisk). Six3 expression is reduced in the ventral forebrain (red asterisk). At E9.5, the expression domain of Nkx2.1, which marks the ventral forebrain is reduced but that of Pax6 for dorsal forebrain tissues is maintained. Broken line indicates the length of the forebrain, which is slightly shorter in the mutant embryo).

View larger version (50K): [in this window] [in a new window]   Fig. 5. Partial rescue of the head and forebrain defects in the Dkk1-/- embryo with reduced Wnt3 gene dose. (A) Comparison of the morphology of the Dkk1+/+;Wnt3+/+, Dkk1-/-;Wnt3+/- and Dkk1-/-;Wnt3+/+ embryos showing the almost complete to partial restoration of head structures in the Dkk1-/-;Wnt3+/- embryo at E9.5 and E10.5. (B) Marker analysis of wild-type (Dkk1+/+;Wnt3+/+), `rescued' (Dkk1-/-;Wnt3+/-) and Dkk-null (Dkk1-/-;Wnt3+/+) embryos (E8.5: TopGal, Six3 and Fgf8; E9.5: En1 and Dlx5). The presence of TOPGal-free and Six3-expressing domains (arrows) show the restoration of forebrain tissue in the `rescued' embryos. Expression of Fgf8 is present in the commissural plate (arrow) of the rescued embryo but is weaker than the wild type, whereas the isthmus is restored to a normal position (asterisk) that is shifted rostrally in the Dkk1-/-;Wnt3+/+ embryo. En1 is restored to nearly wild-type pattern in the midbrain of the rescued embryo (asterisk). Dlx5 expression (arrows) reveals the improved development of the branchial arch in the rescued embryo.

View larger version (58K): [in this window] [in a new window]   Fig. 6. The scheme of genetic interaction and the requirement of Dkk1 and Wnt3 activity in anterior morphogenesis. (A) The modulation of WNT3 signalling by the antagonistic action of DKK1 maintains a correct level of patterning activity. (B) Halving the dose of both genes in the Dkk1+/-;Wnt3+/- embryo would theoretically produce a `balanced' level of patterning activity but at a reduced level (thinner arrow and inhibitory connector). (C) Because of the requirement of WNT3 signalling to regulate the expression of Dkk1, the reduced level of signalling associated with the Wnt3+/- genotype and the loss of one copy of transcription target Dkk1 gene in the Dkk1+/-;Wnt3+/- embryo have resulted in a reduction of DKK1 activity to below the heterozygous (Dkk1+/-) level, which may be inadequate for antagonizing WNT3 signalling (dotted inhibitory connector). This may lead to an unbalanced (and reduced) WNT3 signalling activity (broken arrow) that disrupts head morphogenesis in the Dkk1+/-;Wnt3+/- embryos. (D-H) The schematic profile of agonist (WNT3) and antagonist (DKK1) activity (red, WNT3 signal; green, DKK1 activity; white, no activity; shaded, reduced activity) in embryos of different Dkk1 and Wnt3 genotypes, correlated with the pattern of WNT reporter (TOPGal) expression in the E7.75 and E9.5 embryos, and the phenotypic consequences. (D) A balanced Dkk1 and Wnt3 activity in the wild-type embryo enables normal pattering and morphogenesis of anterior (head) structures. (E,F) The unbalanced agonist and antagonist activity leads to an elevated WNT signalling (revealed by the expanded TOPGal expression domain) and abnormal phenotypic outcome in embryos of (E) Dkk1-/-;Wnt3+/+ (Dkk1 null) and (F) Dkk1+/-;Wnt3+/- (compound heterozygous) embryos. (G) In Dkk1-/-;Wnt3+/- (rescued) embryos, the TOPGal expression domain is very similar to that of (D) the wild-type embryo. (H) Wnt3-null embryos do not develop beyond gastrulation and show no TOPGal expression when examined at E7.75 or E9.0, which may indicate a complete shutdown of WNT signalling function owing to arrested development.

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