Hox genes specify vertebral types in the presomitic mesoderm

Abstract

We show here that expression of Hoxa10 in the presomitic mesoderm is sufficient to confer a Hox group 10 patterning program to the somite, producing vertebrae without ribs, an effect not achieved when Hoxa10 is expressed in the somites. In addition, Hox group 11-dependent vertebral sacralization requires Hoxa11 expression in the presomitic mesoderm, while their caudal differentiation requires that Hoxa11 is expressed in the somites. Therefore, Hox gene patterning activity is different in the somites and presomitic mesoderm, the latter being very prominent for Hox gene-mediated patterning of the axial skeleton. This is further supported by our finding that inactivation of Gbx2, a homeobox-containing gene expressed in the presomitic mesoderm but not in the somites, produced Hox-like phenotypes in the axial skeleton without affecting Hox gene expression.

Figure 1.

Patterning activity of Hoxa10 expressed with the Dll1 promoter. Skeletal staining of wild-type (A-C) and Dll1-Hoxa10 transgenic (D-F) newborns. (A,D) A global view of the animal, after removing the forelimbs for clarity. (D) In the transgenic, the ribs are missing from the area labeled with an asterisk. (B,E) An anterior view of the sternum with the associated cartilaginous part of the ribcage (mostly missing in the transgenic animal). (C,F) The sacral area. The arrow points to the lateral fusion between sacral vertebrae, missing in the transgenic embryo. (G,H) An in situ hybridization analysis of Hoxa10 expression in a Dll1-Hoxa10 transgenic embryo. (G) A dorsal view of the presomitic mesoderm. (H) A lateral view of an embryonic day 9.0 (E9.0) embryo.

Figure 2.

Patterning activity of Hoxa10 expressed in the somites. (A) Analysis of the expression activity of the sm promoter. The bacterial tetR gene was used as a reporter, and its expression was detected by in situ hybridization. The arrows indicate some somites, and the arrowhead indicates the presomitic mesoderm. (B) High-power view of the tailbud region of the embryo shown in A. The arrow indicates the last formed somite. (C) Ventral view of the thoracic area of a wild-type newborn. The sternum and the cartilaginous area of the ribcage were removed for clarity. (D) Lateral view of the thoracic area of an sm-Hoxa10 transgenic embryo, oriented rostral to the left and ventral at the bottom. The sternum (S), ribs (R), and vertebrae (V) are indicated. (E) Ventral view of the thoracic area of an sm-Hoxa10 transgenic embryo, oriented rostral at the top. The ossified area is strongly malformed. The sternum and the cartilaginous area of the ribcage were removed for clarity and are shown in F.

Figure 3.

Patterning activity of Hoxa11 expressed in the presomitic mesoderm and in the somites. (A) Ventral view of the lumbar and sacral area of a wild-type newborn. The indicates the position of vertebra 20, which is T13 in wild-type embryos; the indicates S1. (B,C) Ventral views of two Dll1-Hoxa11 transgenic embryos. In the embryo in B, S1 () is located in vertebra 24, and contains lateral protrusions in lumbar vertebrae (arrow). In the embryo in C, S1 is vertebra 26 and contains a lateral fusion between adjacent lumbar vertebrae (arrow in the blown-up region). (D) Thoracic area of a Dll1-Hoxa11 transgenic newborn showing fusions between the ossified area of adjacent ribs (arrows). (E) Sternum and the cartilaginous area of the ribcage of a Dll1-Hoxa11 transgenic newborn showing fusions between adjacent ribs (arrows). (F) Caudal region of a Dll1-Hoxa11 transgenic newborn showing fusions between adjacent vertebrae (arrows). (G,H) Cervical area of a wild-type (G) and a Dll1-Hoxa11 transgenic (H) newborn showing a lateral cartilaginous fusion between adjacent vertebrae (arrow in H). (I) Caudal area of a wild-type embryo. The arrow indicates the lateral process in a caudal vertebra. (J-L) The upper lumbar (J), thoracic (K), and sacral (L) areas of specific sm-Hoxa11 transgenic newborns. The arrow in J indicates an anteriorly projecting protuberance in a lumbar vertebra. The arrow in K indicates an anteriorly projecting protuberance at the base of a rib, and the arrowhead indicates an anteriorly projecting protuberance in a lumbar vertebra. The arrows in L show the unilateral anteriorization of the sacrum.

Figure 4.

Axial skeletal phenotype of Gbx2 mutant embryos. (A,B) Expression of Gbx2 in E10.5 embryos by in situ hybridization. (B)A close-up look at the tail tip. It is expressed in the presomitic mesoderm (arrow) but not in the somites (arrowheads point to some of them). (C,D) Ventral view of the lower thoracic, lumbar, and sacral areas of a wild-type (C) and a Gbx2 mutant (D) newborn. The sternum and the cartilaginous area of the ribcage were removed for clarity. The 21st vertebra, normally the first lumbar (L1), has an extra rib (arrow) in the Gbx2 mutants. (E,F) Cartilaginous area of the ribcage of a wild-type (E) and a Gbx2 mutant (F) newborn to show that the eighth rib (r8) is attached to the sternum in the Gbx2 mutants but not in the wild-type embryos.