Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION To account for the molecular nature of region-specific Dkk1 and noggin cooperate in organizers, a two-inhibitor model has been proposed, the mammalian head induction hallmark of which is differential inhibition of Wnt and BMP growth factor signals. Inhibition of BMP signaling Ivan del Barco Barrantes,1 Gary Davidson,1 leads to formation of the trunk organizer and simulta- Hermann-Josef Gro¨ne,2 Heiner Westphal,3 and neous inhibition of BMP and Wnt signaling promotes the 1,4 head organizer (Fig. 1A). This model is based on the find- Christof Niehrs ings that overexpression of BMP inhibitors alone leads to 1Divisions of Molecular Embryology, 2Department of trunk duplications, whereas coexpression of Wnt and Molecular and Cellular Pathology, Deutsches BMP inhibitors leads to ectopic head formation (Glinka Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 et al. 1997). Furthermore, genes encoding secreted Wnt Heidelberg, Germany; 3Laboratory of Mammalian Genes and antagonists such as cerberus, frzb, and dkk1 are pre- Development, National Institute of Child Health and Human dominantly expressed in anterior mesendoderm, where- Development, Bethesda, Maryland 20892, USA as all organizer derivatives express at least one of the Growth factor antagonists play important roles in medi- ating the inductive effects of the Spemann organizer in amphibian embryos and its equivalents in other verte- brates. Dual inhibition of Wnt and BMP signals has been proposed to confer head organizer activity. We tested the requirement of this coinhibition in Xenopus and mice. In Xenopus, simultaneous reduction of the BMP antago- nists chordin and noggin, and the Wnt antagonist dick- kopf1 (dkk1) leads to anterior truncations. In mice, com- pound mutants for dkk1 and noggin display severe head defects, with deletion of all head structures anterior to the mid-hindbrain boundary. These defects arise as a re- sult of a failure in anterior specification at the gastrula stage. The results provide genetic evidence for the dual inhibition model and indicate that dkk1 and noggin functionally cooperate in the head organizer. Received April 17, 2003; revised version accepted July 10, 2003. The primary body axis of vertebrate embryos arises and is patterned anteroposteriorly during gastrulation. Ante- Figure 1. Functional synergy of anti-Wnts and anti-BMPs in rior signaling centers that are required for induction of anterior development of Xenopus embryos. (A) Two-inhibitor head structures have been identified in gastrulae of all model: BMP and Wnt signaling pathways negatively regulate vertebrates. In amphibians, this induction is mediated by the Spemann organizer. Formation of the trunk organizer re- Spemann’s head organizer, a group of cells of anterior quires inhibition of BMP signals, whereas for formation of the mesendodermal fate that is localized in the gastrula up- head organizer both Wnt and BMP signals need to be inhibited. per dorsal blastopore lip (Harland and Gerhart 1997; De Differential expression in mesendoderm and chordamesoderm Robertis et al. 2000). In mouse gastrulae, anterior signal- of secreted anti-Wnts (Dkk1, Frzb1, Crescent/Frzb2, and Cer- ing centers, as defined by heterotopic transplantation, berus) as well as anti-BMPs (Noggin, Chordin, Xnr3, and Fol- are the anterior visceral endoderm (AVE; Thomas and listatin) mediates regional-specific induction. (B) Specificity of Beddington 1996; Tam and Steiner 1999) and the anterior noggin and chordin antisense Morpholino oligonucleotides mesendoderm (AME), whose progenitors are the early- (Mo): Embryos were injected at the two-cell stage with mRNA and midgastrula organizers (Camus et al. 2000; Kinder et encoding either noggin-AP (5 ng) or chordin-AP (2 ng) and again al. 2001). Subsequently, during early neural patterning, at the four- to eight-cell stage with either noggin or chordin Mo the anterior neural ridge induces and promotes forebrain, (25 ng). Protein lysates were prepared at stage 13 and heated at and inducing signals include FGF8 and Tlc (Rubenstein 65°C for 30 min to inactivate endogenous alkaline phosphatase. et al. 1998; Houart et al. 2002; for reviews, see Bedding- The remaining AP activity was measured. (C) Xenopus embryos ton and Robertson 1998; Davidson and Tam 2000; Perea- were injected at the four-cell stage in two dorsal blastomeres Gomez et al. 2001). with a total of 50 ng control Mo or 25 ng each of noggin and chordin Mo as indicated. Zero percent (n = 36) of control Mo and 10% (n = 34) of nog/chd Mo injected embryos were eyeless. Embryos injected into the blastocoel of early gastrulae with 0.1 [Keywords: Dkk1; Noggin; Wnt; BMP; Spemann organizer; gastrula] µg of anti-Dkk1 antibody (anti-Dkk1) showed 0% (n = 32) eye- 4 Corresponding author. less embryos. Coinjection of nog/chd Mo together with anti- E-MAIL [email protected]; FAX 49-6221-42-4692. Article published online ahead of print. Article and publication date are Dkk1 antibody leads to enhanced head defects (75% eyeless at http://www.genesdev.org/cgi/doi/10.1101/gad.269103. embryos; n = 36). GENES & DEVELOPMENT 17:2239–2244 © 2003 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/03 $5.00; www.genesdev.org 2239 Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press del Barco Barrantes et al. BMP antagonists chordin, noggin, follistatin, Xnr3,or bryos injected with a combination of two different Mos cerberus (for review, see Niehrs 1999; Yamaguchi 2001). against chordin, which inhibit both pseudoalleles Although Nodal antagonists can also promote head (Oelgeschläger et al. 2003). However, embryos coinjected structures (Piccolo et al. 1999), this may be an indirect with both noggin and chordin Mos show shorter trunks consequence of blocking the formation of trunk meso- and minor microcephaly (Fig. 1C), consistent with re- derm and its associated caudalizing signals, such as sults in the mouse, where they have redundant functions Wnt8. Rather, cerberus-like/lefty double mutants reveal (Bachiller et al. 2000; Anderson et al. 2002). If limiting a requirement of anti-Nodals to restrict primitive streak doses of a specific inhibitory anti-Dkk1 antibody (Glinka formation to the posterior end of mouse embryos (Perea- et al. 1998; Kazanskaya et al. 2000) are injected in addi- Gomez et al. 2002). tion to nog/chd Mo, the anterior phenotype is enhanced, Consistent with the two-inhibitor model, inhibition of leading to headless and eyeless embryos (Fig. 1C, anti- Wnt signals is required for anterior specification. Ze- Dkk1 + nog/chd Mo). brafish mutants for the negative intracellular regulators of Wnt signaling tcf3/headless and axin/masterblind Dkk1 and Noggin synergize in mouse head induction display anterior defects (Heisenberg et al. 2001; Kim et al. 2001; van de Water et al. 2001). Interference with the We next tested the requirement for dual BMP/Wnt inhi- Wnt antagonist dkk1 in Xenopus and mouse leads to bition genetically in mice. Dkk1 starts to be expressed in head truncations (Glinka et al. 1998; Kazanskaya et al. the AVE at embryonic day 6 (E6; data not shown), and in 2000; Mukhopadhyay et al. 2001). Furthermore, Six3 the anterior mesendoderm (AME) at E7 (Glinka et al. mutant mice lack forebrain structures, and this appears 1998). Homozygous dkk1 mutant animals display lack of to be due to derepression of Wnt1 (Lagutin et al. 2003). head and brain structures anterior to the mid-hindbrain These negative regulators of the Wnt signaling pathway boundary, as well as limb defects (Mukhopadhyay et al. may help establish a gradient of Wnt activity patterning 2001). Noggin is expressed in the node and AME at E7.5 the antero–posterior axis (Kiecker and Niehrs 2001; (McMahon et al. 1998; Bachiller et al. 2000), where its Houart et al. 2002). expression partially overlaps with that of dkk1 (Fig. A requirement for BMP inhibition for normal anterior 4A,B, below). In mice lacking noggin, normal gastrula- specification is supported by mice double mutant for the tion and anterior central nervous system patterning take BMP antagonists noggin and chordin, which show a place, although at later stages abnormalities in posterior range of anterior defects (Bachiller et al. 2000; Anderson spinal cord, somites, and limbs are observed (McMahon et al. 2002). Likewise, zebrafish with reduced BMP ac- et al. 1998). Importantly, dkk1 and noggin heterozygous tivity have expanded anterior neural fates (Nguyen et al. mice are phenotypically normal and viable, and hence 1998; Barth et al. 1999; Fekany-Lee et al. 2000). intercrosses were set up between them. Although these data strongly support the role of BMP and Wnt antagonists in promoting rostral cell fates, an impor- tant prediction in the above model for head induction that remains untested is that coinhibition of BMP and Wnt signaling is required for anterior speci- fication. To test if BMP and Wnt an- tagonists act synergistically, we have created compound mutant mice for the Wnt antagonist dkk1 (Glinka et al. 1998; Mukhopadhyay et al. 2001) and the BMP antagonist noggin (Smith and Harland 1992; McMahon et al. 1998). Analysis of these mice reveals a syner- gistic effect of both types of antagonists in the specification of anterior cell fates. Results and Discussion Dkk1 and anti-BMPs synergize in Xenopus We initially tested if anti-Wnts and anti-BMPs act synergistically for head formation in Xenopus. Injection of Figure 2. Dkk1+/− Nog+/− mice have head defects. Frontal (A,B,C,D) and lateral Morpholino-antisense oligonucleotides (AЈ,BЈ,CЈ,DЈ) views of wild-type (A,AЈ), mild (B,BЈ), strong (C,CЈ), and severe (D,DЈ) (Mo) acting specifically against noggin newborn mutant animals. Lateral view of skeletal preparations from wild-type (AЉ), (nog) or chordin (chd; Fig. 1B) alone mild (BЉ), and severe case (DЉ) newborn heads reveal gradual loss of maxillar (mx), does not result in head defects (data not mandibular (mn), and other bones anterior to the parietal bone (p).