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Development
2002 Dec 01;12924:5587-96. doi: 10.1242/dev.00154.
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Kremen proteins interact with Dickkopf1 to regulate anteroposterior CNS patterning.
Davidson G
,
Mao B
,
del Barco Barrantes I
,
Niehrs C
.
Abstract
A gradient of Wnt/beta-catenin signalling formed by posteriorising Wnts and anteriorising Wnt antagonists regulates anteroposterior (AP) patterning of the central nervous system (CNS) during Xenopus gastrulation. In this process, the secreted Wnt antagonist Dkk1 functions in the Spemann organiser and its anterior derivatives by blocking Wnt receptors of the lipoprotein receptor-related protein (LRP) 5 and 6 class. In addition to LRP6, Dkk1 interacts with another recently identified receptor class, the transmembrane proteins Kremen1 (Krm1) and Kremen2 (Krm2) to synergistically inhibit LRP6. We have investigated the role of Krm1 and Krm2 during early Xenopus embryogenesis. Consistent with a role in zygotic Wnt inhibition, overexpressed Krm anteriorises embryos and rescues embryos posteriorised by Wnt8. Antisense morpholino oligonucleotide (Mo) knockdown of Krm1 and Krm2 leads to deficiency of anterior neural development. In this process, Krm proteins functionally interact with Dkk1: (1) in axis duplication assays krm2 synergises with dkk1 in inhibiting Wnt/LRP6 signalling; (2) krm2 rescues microcephalic embryos induced by injection of inhibitory anti-Dkk1 antibodies; and (3) injection of krm1/2 antisense Mo enhances microcephaly induced by inhibitory anti-Dkk1 antibodies. The results indicate that Krm proteins function in a Wnt inhibition pathway regulating early AP patterning of the CNS.
Fig. 2. Expression analysis of Krm1 and Krm2 in the mouse. (A) Relative expression levels of mouse Krm1 and Krm2 in indicated tissues, as analysed by RT-PCR. Histone H4 was used as reference for sample normalisation. (B) Krm1 whole-mount in situ hybridisation of early (E8) and late (E8.5) headfold stage mouse embryos. (C) Whole-mount in situ hybridisation of E10.5 mouse embryos for Krm1 and Krm2 (top). Dissected anteriorlimb buds are shown in bottom panels, with arrows indicating staining in the apical ectodermal ridge (AER). br, branchial arches; f, forebrain; fl, forelimb; h, hindbrain; hl, hindlimb; m, midbrain; ms, mesanephros; np, nasal placode; ot, otic vesicle; ov, optic vesicle.
Fig. 3. Expression of Krm genes during Xenopus embryogenesis. (A) Developmental timecourse expression, as analysed by RT-PCR, at the indicated stages. Histone H4 was used for cDNA sample normalisation. (B-F) Spatial expression pattern of krm1 in Xenopus embryos, as analysed by whole-mount in situ hybridisation. (B) Control hybridisation of a stage 14 embryo using krm1 sense riboprobe. (C) Stage 14 embryo showing lateral neural plate expression, strongest in the anterior region. (D) Frontal view of late neurula, dorsal towards the top. (E) Sagittal midline cut of embryo shown in I, revealing expression in prechordal plate (pp). (F) Tailbudembryo showing krm1 expression in finmesenchyme, hatching gland (hg) and notochord (nc, see also inset of cross-section). (G-M) Spatial expression pattern of krm2. (G) Mid gastrula (stage 11) embryo showing expression in marginal zone but absence from dorsal region. Vegetal view, dorsal towards the top. (H) Early-mid neurula (stage 14) embryo showing lateral neural plate expression. Dorsal view, anterior towards the top. (I,J) krm2 expression in anteriormesoderm. Vibrotome section (50 μm) of horizontally cut stage15 embryos (I) and sagittally cut stage14 embryos (J). The inserts show the plane of the section, indicated by a horizontal line. Arrow in I indicates expression in anteriormesoderm. (K) Frontal view of late neurulaembryo (stage 18) showing anterior expression pattern. Dorsal towards the top. (L) Sagittal midline cut of embryo shown in K, revealing expression in prechordal plate (pp) tissue. Anterior is towards the left, dorsal is towards the top. (M) Lateral view of tailbud (stage 28) embryo showing expression in finmesenchyme, dorsal part of otic vesicle (ov), hatching gland (hg), branchial arches (br) and pronephric duct (pnd, see also inset in cross-section).
kremen1 (kringle containing transmembrane protein 1) expression in Xenopus lavis, NF stage 14 embryo, assayed by in situ hybridization, dorsal view, anteriorleft. Image by Davidson et al., 2002.
Xenopus kremen2 / kringle containing transmembrane protein 2 expression in stage 14 neurula (dorsal view- anterior is to the left). Image by Davidson et al., 2002.
