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First published online 16 November 2005
doi: 10.1242/dev.02157

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Non-cell-autonomous role for Cripto in axial midline formation during vertebrate embryogenesis

Jianhua Chu¹, Jixiang Ding^1,*, Katherine Jeays-Ward², Sandy M. Price¹, Marysia Placzek² and Michael M. Shen^1,

¹ Center for Advanced Biotechnology and Medicine and Departments of Pediatrics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
² Centre for Developmental and Biomedical Genetics, Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, UK

View larger version (56K): [in a new window]   Fig. 1. Cripto expression and gene targeting. (A-C) Expression of Cripto in nascent mesoderm, node and axial mesendoderm of a wild-type late-streak stage embryo; frontal section in B is counterstained with Methyl Green. (C) High-power view of boxed region in B shows expression in midline mesendodermal cells, but not in epiblast or visceral endoderm cells being displaced during gastrulation. (D,E) Widespread ß-galactosidase staining in a heterozygous CriptolacZ/+ embryo at the neural plate (late allantoic bud) stage; transverse section in E shows staining in the nascent mesoderm and definitive endoderm, with most intense staining in the axial mesendoderm; no expression is detected in the primitive streak or neuroectoderm. (F-H) Generation of hypomorphic and null alleles of Cripto. (F) The targeting strategy for the Cripto3loxP allele inserts a floxed PGK-neo cassette into the intron between Cripto exons 5 and 6. Following germline transmission, the PGK-neo cassette was deleted by crossing the Cripto3loxP/+ mice with EIIa-Cre transgenic mice; complete excision results in the null allele Criptodel. (G) Southern blot detection of the targeted allele in ES genomic DNA digested with XbaI. An 8 kb fragment is detected by probe A for the wild-type allele, and a 5.5 kb fragment (arrow) is detected for the Cripto3loxP allele. (H) Southern blot detection of the targeted allele using probe B in ES genomic DNA digested with HindIII; 11 kb (wild-type) and 6.8 kb (targeted) fragments are detected. Positions of molecular standards at 10 and 5 kb are indicated. Scale bars: 100 µm in A-D; 50 µm in E. ame, axial mesendoderm; B, BamHI; de, definitive endoderm; E, EcoRI; epi, epiblast; H, HindIII; mes, nascent mesoderm; nd, node; ne, neuroectoderm; ps, primitive streak; ve, visceral endoderm; X, XhoI; Xb, XbaI.

View larger version (83K): [in a new window]   Fig. 2. Holoprosencephaly phenotypes in Cripto3loxP/null embryos. (A-C) Lateral (A) and ventral (B,C) views of wild-type and Cripto3loxP/null embryos at 8.25 dpc, showing anterior truncation (arrow in A) and fusion of somite pairs across the midline (C). (D-G) Lateral (D,E) and coronal (F,G) views of wild-type and Cripto3loxP/null embryos at 9.0 dpc, showing forebrain reduction (arrows in E,G) and fusion of optic vesicles. (H) Lateral view of wild-type and Cripto3loxP/null embryos at 10.5 dpc, showing greatly reduced forebrain and midbrain (arrow) in the mutant. (I) Lateral views of 11.5 dpc wild-type and Cripto3loxP/null embryos, with reduced telencephalon (arrow) in the mutant. (J,K) Coronal sections of 11.5 dpc wild-type (J) and Cripto3loxP/null mutant (K) littermates, stained with hematoxylin and eosin. While the wild-type embryo has distinct telencephalic and diencephalic vesicles, the Cripto hypomorph displays a single prosencephalic vesicle (arrow); the hindbrain appears unaffected. (L-N) Transverse sections through wild-type and Cripto3loxP/null mutant embryos at the late neural plate/early head-fold stages, stained with Nuclear Fast Red. The wild-type embryo (L) has a midline prechordal plate that is absent in the Cripto hypomorph (M,N, arrow). Scale bars: 500 µm in A,D,E,H,I; 200 µm in B,C,F,G,J,K; 50 µm in L-N. di, diencephalic vesicles; hb, hindbrain; nt, neural tube; op, optic vesicles; pp, prechordal plate; so, somite pairs; te, telencephalic vesicles.

View larger version (104K): [in a new window]   Fig. 3. Marker analysis of Cripto3loxP/null embryos. (A) Expression of Shh in wild-type and two Cripto3loxP/null littermates at 8.25 dpc. Expression in the rostral diencephalic ventral midline, ventral midline and hindgut is completely (arrow) or partially (arrowheads) absent in the less severely affected Cripto3loxP/null embryo (middle) and is abolished in the more severely affected embryo (right). (B) Expression of Hex in the anterior definitive endoderm at 7.5 dpc is lost in a Cripto3loxP/null littermate. (C) En1 expression around the midbrain-hindbrain boundary in both wild-type and Cripto hypomorph embryos. (D) Six3 marks the ventral forebrain but is absent in a Cripto3loxP/null littermate. (E,F) Fgf8 is expressed in the telencephalic commissural plate and the midbrain/hindbrain boundary (E), but is absent from the anterior end of a Cripto3loxP/null embryo (F). Scale bars: 500 µm. cp, commissural plate; hg, hindgut; mhb, midbrain-hindbrain boundary; rdvm, rostral diencephalic ventral midline; vfb, ventral forebrain; vm, ventral midline.

View larger version (133K): [in a new window]   Fig. 4. Axial mesendoderm/definitive endoderm defects in Cripto3loxP/null embryos. Wild-type and Cripto3loxP/null embryos at the late head-fold stage (A-L) or early neural plate stage (M-P) are shown as whole mounts and corresponding transverse sections; B,D,F,H,J,L are counterstained with Methyl Green. (A-D) Expression of Shh in the prechordal plate is absent in the Cripto3loxP/null embryo; instead, a small number of expressing cells (arrows) can be found displaced from the axial midline. (E-H) Expression of Foxa2 in the notochordal plate and prospective floor plate is absent in the Cripto3loxP/null embryo. (I-L) Gsc expression in the prechordal plate, anterior diencephalic ventral midline neuroectoderm and foregut endoderm is completely abolished in the Cripto3loxP/null mutant. (M-P) Expression of Cer1 marks the anterior definitive endoderm, but is mostly absent except for scattered staining cells (arrow) in the Cripto hypomorph. Scale bars: 100 µm. ade, anterior definitive endoderm; advm, anterior diencephalic ventral midline; fg, foregut endoderm; pfp, prospective floor plate; pp, prechordal plate.

View larger version (40K): [in a new window]   Fig. 5. Strategy for analysis of Cripto in aggregation chimeras. (A) Morula aggregation is performed between wild-type unmarked embryos and Rosa26-marked embryos from a cross between Cripto heterozygous parents [adapted from Rivera-Perez et al. (Rivera-Perez et al., 1999)]. One-quarter of the chimeras will contain marked Cripto-null cells (Rosa26/+; CriptolacZ/del +/+), while the remaining three-quarters will be phenotypically wild type and serve as internal controls. (B) Extra-embryonic regions from ß-galactosidase-stained chimeric embryos genotyped by PCR; analysis of a representative litter is shown.

View larger version (69K): [in a new window]   Fig. 6. Contribution of Cripto null mutant cells to prechordal mesoderm, notochord and foregut endoderm in chimeric embryos. (A-L) Analysis of Rosa26/+; CriptolacZ/del +/+ chimeras generated by morula aggregation; whole-mount embryos were stained for ß-galactosidase activity, and cryosections were counterstained with Nuclear Fast Red. (A-E) Medium-grade Rosa26/+; CriptolacZ/del +/+ chimera at the late head-fold stage. Transverse sections (B,D) show random distribution of marked Cripto mutant cells in the headfolds and allantois; high-power views (C,E) show contribution of marked Cripto mutant cells to the prechordal plate and notochordal plate. (F-J) Medium-high-grade Rosa26/+; CriptolacZ/del +/+ chimera at 9.5 dpc. Marked Cripto mutant cells contribute with high efficiency to all tissues examined (G,I); high-power views (H,J) show contribution of marked Cripto mutant cells to the notochord, floor plate and foregut endoderm. (K,L) High-grade Rosa26/+; CriptolacZ/del +/+ chimera with abnormal morphology; note the presence of mesoderm (arrow in L). (M-O) Analysis of medium-grade chimera generated by blastocyst injection of marked CriptolacZ/del ES cells. Mutant cells contribute efficiently to the prechordal plate, anterior diencephalic ventral midline and foregut endoderm. Scale bars: 100 µm. advm, anterior diencephalic ventral midline; al, allantois; fg, foregut endoderm; fp, floor plate; hf, headfolds; nc, notochord; np, notochordal plate; pp, prechordal plate.

View larger version (67K): [in a new window]   Fig. 7. Addition of exogenous Cripto alters the differentiation of chick node/head process mesoderm explants. (A,E) Schematic drawings showing regions dissected at HH stage 4--4 (A) and HH stage 5 (E). (B-D,J-L) Explants of HH stage 4 Hensen's node cultured in vitro for 20 hours in the presence of 293T cell conditioned medium, either untransfected (B-D) or transfected with Cripto (J-L). Gsc-expressing prechordal mesoderm and Hex-expressing anterior endoderm differentiate with or without Cripto (B,C,J,K). 3B9-expressing notochord cells differentiate in control cultures (D) but not in explants exposed to Cripto (L). (F-I,M-P) Explants of HH stage 5 posterior head process mesoderm cultured in vitro for 20 hours (F-H,N-P) or 4 hours (I,M). Control cultures express 3B9 (H) and Fgf10 (M) but not Gsc or Hex (F,G,M). Explants exposed to Cripto protein express Gsc and Hex (I,N,O), but downregulate 3B9 and Fgf10 (M,P). Explant cultures (H,P) were counterstained with DAPI. (Q,U) Schematic drawings showing bead implant at HH stage 4 (Q) and level of sections analyzed at HH stage 9 (U). (R-T,V-X) Serial adjacent sections of HH stage 9 embryos, in which control beads (R-T) or Cripto-soaked beads (V-X) were placed adjacent to Hensen's node at HH stage 4. Axial mesoderm cells of control embryos display notochord properties, expressing 3B9 and Shh, but not Gsc (R-T). Axial mesoderm cells of Cripto-exposed embryos display prechordal mesoderm properties, expressing Shh and Gsc, but not 3B9 (V-X). (Y-BB) Explants of HH stage 5 head process mesoderm cultured in vitro for 20 hours. Control cultures and cultures exposed to SB-431542 express 3B9 (Y,AA). Cripto prevents expression of 3B9 (Z). The Cripto-mediated loss of 3B9 is abolished by treatment with SB-431542 (BB).

View larger version (9K): [in a new window]   Fig. 8. (A) Cell-autonomy and non-cell-autonomy of genes in the Nodal signaling pathway [adapted from Schier and Shen (Schier and Shen, 2000)]. (B,C) Schematic model for Cripto function in mediating Nodal activity as a cis-acting GPI-linked co-receptor (B), as well as a trans- acting soluble factor (C) [adapted from Yan et al. (Yan et al., 2002)].