Cdx1 and Cdx2 have overlapping functions in anteroposterior patterning and posterior axis elongation
Eric van den Akker1,*,
Sylvie Forlani1,
,
Kallayanee Chawengsaksophak2,
Wim de Graaff1,
Felix Beck2,3,
Barbara I. Meyer4 and
Jacqueline Deschamps1,
1 Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
2 Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Australia
3 Department of Biochemistry, University of Leicester, Leicester LE1 7RH, UK
4 Department of Molecular Cell Biology, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany
* Present address: Institute of Hematology, Erasmus University, Dr Molewaterplein 50, 3015 GR, Rotterdam, The Netherlands
Present address: Unité de Biologie Moléculaire du Développement, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France
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Fig. 1. (A-F)Ventral views of the upper cervical area in a wild type (A) and in different genotypic combinations between Cdx1 and Cdx2 mutant alleles (B-F). Rostral is towards the top. The position of the basioccipital (bo), exoccipital (eo) and the first three cervical vertebrae (C1-C3) is indicated. In Cdx1–/– (D) and Cdx1–/–/Cdx2+/– (F) mutants, fusion of C1 to the exoccipital and basioccipital bones is clearly visible (anterior transformation of C1') and the third vertebra has an axis-like morphology (C3'), indicating C2 to C1 and C3 to C2 transformations, respectively.
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Fig. 2. Side views of the cervical and upper thoracic region of the same wild-type (A) and Cdx mutant newborns (B-F) as in Fig. 1. Rostral is towards the left, dorsal towards the top. The first rib emanates from the eighth vertebra and the long spinous process (asterisk) is located on the ninth vertebra in both the wild-type and Cdx1+/– mutant. Like the wild type, the Cdx1+/– newborn displayed in C has seven cervical vertebrae (indicated by numbers C1-7) and a normal atlas and axis. The Cdx2+/– animal (B) shown here displays a posterior shift of the anterior tuberculum (at) from the sixth to the seventh vertebra (C7 to C6 transformation) and the long spinous process is visible on the tenth instead of the ninth vertebra (T3 to T2 transformation). The Cdx1–/– animal in D lacks dorsal and lateral parts of the first vertebra and the remaining ventral part is fused to the exoccipital bone. Clearly visible are the atlas (with ventral arch) and axis-like morphologies of the second and third cervical vertebrae. As in the Cdx2+/– mutant in B, the position of the anterior tuberculum and the long spinous process is shifted posteriorly by one vertebra. The ninth vertebra (T2) is the first vertebra bearing ribs (T1 to C7 transformation – T1'). In the Cdx1+/–/Cdx2+/– mutant shown in (E) the atlas looks normal. The second vertebra has a partial atlas-like and the third vertebra an axis-like morphology (respectively indicating C2 to C1 and C3 to C2 transformations). At lower levels, similar transformations are visible as in the Cdx1–/– mutant (D). More severe transformations are visible in the Cdx1–/–/Cdx2+/– mutant (F) compared with the Cdx1–/– mutant. The first vertebra in F also lacks dorsal and lateral structures but is more completely fused to the exoccipital bone than the first vertebra in D, and not only the third but also the fourth vertebra has an axis-like morphology (C4 to C2 transformation). The anterior tuberculum is present on the eighth vertebra (T1'), the ninth vertebra has lost its rib (T2'), and the long spinous process is present on the eleventh vertebra, all indicating anterior transformation of vertebrae by two segments in the Cdx1–/–/Cdx2+/– mutant.
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Fig. 3. Ventral views of the ribcage of the same animals as in Fig. 1 and Fig. 2. Rostral is towards the top. Wild types (A) have seven ribs emanating from the 8th to 14th vertebrae (T1 to T7), attached to the sternum. The Cdx2+/– (B) and Cdx1+/– (C) animals shown here have an extra rib attached to the sternum at one side (T8 to T7 transformation). The arrow in B indicates a slight second rib defect (rib partly anteriorised) in the Cdx2+/– mutant. The Cdx1–/– (D) and Cdx1+/–/Cdx2+/– (E) mutants displayed here show bilateral attachment of ribs, emanating from the 9th to 15th vertebrae, to the sternum and bilateral presence of (partial) ribs on the 21st vertebra (L1 to T13 transformation: L1’). In the Cdx1–/–/Cdx2+/– mutant shown in F, ribs are emanating from the 10th to 16th vertebrae (the latter indicating T9 to T7 transformation) and a complete pair of ribs is visible on L1. The arrow points at two fused ribs. v, vertebra.
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Fig. 4. Dorsal view of the lumbosacral region of the same animals as in Figs 1-3. Rostral is towards the top. The wild type (A), Cdx2+/– (B) and Cdx1+/– (C) mutants shown have the last pair of ribs attached to T13 (see also Fig. 3) and 6 lumbar vertebrae. In the Cdx1–/– mutant (D), one complete and one incomplete rib is visible on L1 (L1'). More caudally no abnormalities are present. In the Cdx1+/–/Cdx2+/– (E) and Cdx1–/–/Cdx2+/– (F) mutants, in addition to the partial and complete ribs present on L1, partial (arrow in E) and complete (F) shifts in the position of the sacrum (S1 to L6 transformation: S1') are visible.
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Fig. 5. Loss of Cdx functional alleles causes a shift in the anterior expression boundary of Hoxb8 in the paraxial mesoderm. Sagittal sections of day 11.5-12 whole-mount Hoxb8lacZ+/– embryos stained with X-gal are viewed under dark field conditions. A and B are slightly younger than C-F, but our analysis has shown that the rostral Hoxb8 and Hoxb8lacZ expression boundaries were identical at both stages. In the wild type (A), Hoxb8lacZ expression is visible in pre-vertebra (pv) 7. In the Cdx2+/– mutant (B), expression is almost undetectable in pv7 and expression in pv8 appears to be reduced. In the Cdx1+/– (C) embryo, the first Hoxb8lacZ expressing prevertebra is pv7 (arrow), as in wild type (A). In Cdx1–/– (D) mutant embryos, weak expression is visible in pv7. This expression has disappeared in Cdx1+/–/Cdx2+/– mutant embryos (E), the first expressing vertebra being pv8 (arrow) In Cdx1–/–/Cdx2+/– mutants, either weak or no (F) expression was visible in pv8. Note the lower expression of Hoxb8lacZ in the prevertebrae in E,F (compared with C,D at the same stage). This lower expression correlates with the compound Cdx mutant genotype. Vertebral abnormalities [close approximation of pv1 and the basioccipital (bo)] are already apparent at this stage in the Cdx1–/– and Cdx1–/–/Cdx2+/– mutants.
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Fig. 6. Cdx mutations lead to a slight posterior shift of the rostral expression boundaries of Hoxd4 and Hoxb9. Whole-mount 9.5-day embryos (23-29 somites) were hybridised with a Hoxb4 (A-D) or a Hoxb9 probe (E-H). (A,E) Wild types; (B,F) Cdx1-null mutants; (C,G) Cdx2 heterozygotes; and (D,H) compound Cdx1null/Cdx2 heterozygotes. The expression boundaries in the somitic mesoderm are indicated, the arrows pointing at the most rostral strongly expressing somite. For Hoxb9 (E-H), a slight posterior shift in the neural tube was noticed as well, the expression boundary being located at the level between somites 6 and 7 (wild type and Cdx2 heterozygotes), between somites7 and 8 (Cdx1 null), and in the middle of somite 8 (double mutants).
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Fig. 7. The anterior expression boundary of Hoxb8 in both the neurectoderm and paraxial mesoderm is located more posteriorly in Cdx1–/–/Cdx2+/– compared with Cdx1+/– mutants during an early phase of Hoxb8 expression. (A) Day 9 (17 somites) Cdx1–/–/Cdx2+/–/Hoxb8lacZ+/– and Cdx1+/–/Hoxb8lacZ+/– mutant embryos were stained overnight with X-gal. The neurectodermal boundary of Hoxb8lacZ expression in the Cdx1+/– embryo (right) is located at a level halfway up the fifth somite, while in the Cdx1–/–/Cdx2+/– embryo (left), the boundary is located halfway the sixth somite. (B) The level of Hoxb8lacZ expression in the mesoderm is lower in the Cdx1–/–/Cdx2+/– (left) than in the Cdx1+/– (right) embryo, and the rostral boundary of expression in the paraxial mesoderm is located more posteriorly (somite 12 compared with 11, respectively). The embryo on the right in A is curved in such a way that only its rostral part is visible.
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Fig. 8. Tail truncations in Cdx1/Cdx2 compound mutants. (A-D) Ventral and (E-H) lateral view of the same newborn individuals of genotypes indicated below, stained for bone and cartilage. (A,E) Mice heterozygous for the Cdx1 mutation have the same tail length as wild types, and have 30-32 caudal, of a total of 60-62 vertebrae. (B,F) Mice heterozygous for the Cdx2 mutation have 26-28 caudal, out of a total of 56-58 vertebrae. (C,G) Mice that are double heterozygotes Cdx1+/–/Cdx2+/– were found to have 15-20 caudal from a total of 45-50 vertebrae. (D,H) Cdx1–/–/Cdx2+/– mutant mice were found to have only 6-11 caudal, out of a total of 36-41 vertebrae. The anatomical boundaries between each type of vertebrae are shifted 1 or 2 pv more posteriorly in these mice (due to anterior transformations at all axial levels). In addition, they have a severely truncated tail. In B,F, parts of the hindlimbs were removed for photographic purposes.
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Fig. 9. Whole-mount in situ hybridisation of a Cdx1+/– (left) and a Cdx1–/–/Cdx2+/– (right) 8.5-day embryos with a brachyury (T) probe. The double mutant had 15 somites, and the heterozygote Cdx1 13 somites. Clearly visible is the abnormally bent and posteriorly truncated tail bud in the double mutant. The posterior neuropore is also more widely open in the double mutant than in the Cdx1+/– embryo. The T expression domain in both embryos, and in other mutants and controls examined extends anteriorly up to a position located one somite more posterior than the last formed somite. The T expression level is also unaltered in double mutants compared with Cdx1+/–, but the expression domain is posteriorly truncated. T expression encompasses the abnormally curved and shorter tail bud in the mutant, labelling the territory affected by the Cdx compound mutation at this stage.
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Fig. 10. Fusion between spinal ganglia in Cdx compound mutants. Sections of 14.5 day Cdx double mutants reveal fusions between spinal ganglia at cervical and upper thoracic levels (arrows in E,F). A section of a wild-type embryo is shown as a control (D). A section of a 12.5 day double mutant embryo carrying a Hoxb8lacZ knock in allele also reveal fusions between spinal ganglia at cervical levels (C, arrows). It is compared with a wild-type (A) and with a Hoxb8 heterozygote knock-in (B) embryo section.
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Fig. 11. Other skeletal abnormalities observed in Cdx1+/–/Cdx2+/– and Cdx1–/–/Cdx2+/– mutants. Split digit 1 in one of the hindlimbs of a Cdx1+/–/Cdx2+/– (A) and a Cdx1–/–/Cdx2+/– mutant (B). (C,D) Fusion of ribs and vertebrae (arrows) in a Cdx1–/–/Cdx2+/– mutant.
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© The Company of Biologists Ltd 2002
