Additional sex combs-like 1 belongs to the enhancer of trithorax and polycomb group and genetically interacts with Cbx2 in mice

Abstract

The Additional sex combs (Asx) gene of Drosophila behaves genetically as an enhancer of trithorax and polycomb (ETP) in displaying bidirectional homeotic phenotypes, suggesting that is required for maintenance of both activation and silencing of Hox genes. There are three murine homologs of Asx called Additional sex combs-like1, 2, and 3. Asxl1 is required for normal adult hematopoiesis; however, its embryonic function is unknown. We used a targeted mouse mutant line Asxl1(tm1Bc) to determine if Asxl1 is required to silence and activate Hox genes in mice during axial patterning. The mutant embryos exhibit simultaneous anterior and posterior transformations of the axial skeleton, consistent with a role for Asxl1 in activation and silencing of Hox genes. Transformations of the axial skeleton are enhanced in compound mutant embryos for the polycomb group gene M33/Cbx2. Hoxa4, Hoxa7, and Hoxc8 are derepressed in Asxl1(tm1Bc) mutants in the antero-posterior axis, but Hoxc8 expression is reduced in the brain of mutants, consistent with Asxl1 being required both for activation and repression of Hox genes. We discuss the genetic and molecular definition of ETPs, and suggest that the function of Asxl1 depends on its cellular context.

Fig. 1

Asxl1^tm1Bc mutant adult mice exhibit reduced body and testis weight, hypothymia, and splenomegaly. (A) Body weight is significantly reduced in Asxl1^tm1Bc mutant (mut) mice (n=12 wt, 5 +/mut, 12 mut/mut). (B) Asxl1^{tm1Bc/ tm1Bc} mice are hypothymic (n=11). (C) Asxl1^{tm1Bc/ tm1Bc} mice exhibit splenomegaly (n=12). (D) Asxl1^{tm1Bc/ tm1Bc} mice have reduced testis weight (n=5 +/+, 6 mut/mut; p=0.08). *, p<0.05; average values are shown with error bars indicating one SD. Black bars indicate samples from wild type; grey bars heterozygotes; white bars Asxl1^tm1Bc/tm1Bc mutants.

Fig. 2

Alterations of the axial skeleton of newborn Asxl1^tm1Bc mutant and Asxl1 ^tm1Bc; Cbx2 ^tm1Ykf compound mutant mice. Bone and cartilage were stained with alizarin red and alcian blue respectively. Lateral views of the cervical regions and scapulae of cleared skeletons (A–C). (A) Wildtype mouse with cervical 1 (C1), cervical 2 (C2), anterior arch (aa), supraoccipital (SO) and exoccipital (E) bones indicated. Notice the association of the aa with C1 via cartilage, the intact C1, and the separation between the SO and EO. (B) Asxl1^tm1Bc/tm1Bc mouse. The C2 vertebra is transformed towards the C1 vertebra shown by an association of the now larger aa with C2 (black arrow) and C1. The C1 vertebra is anteriorly transformed toward the SO and EO bones of the skull indicated by a split within the C1 vertebra (grey arrow). (C) Asxl1^{+/ tm1Bc}; Cbx2^{+/ tm1Ykf} compound mutant. The C2 vertebra is anteriorly transformed towards the C1 vertebra, while the aa is reduced in size (black arrow). More severe anterior transformations are indicated by fusion of the C1 vertebra to the EO (long grey arrow). The SO is reduced (short grey arrow); the scapula (star) exhibits defective ossification. Lateral views of the thoracic regions of cleared skeletons, where the first 3 thoracic (T) vertebrae are numbered (D–E). (D) Wild-type mouse. (E) Asxl1^tm1Bc/tm1Bc mouse with a C7 to T1 posterior transformation is indicated by an ectopic rib on the C7 vertebra, shown with a black arrow. Dorsal views of the thoracic and upper lumbar (L) regions of cleared skeletons (F–G). (F) Wildtype mouse, showing two ribs on T13, and absence of ribs on L1. (G) Asxl1^tm1Bc/tm1Bc mouse exhibiting size reduction of one T13 rib, noted with a black arrow. The cartilage associated with the T13 rib is still present. Ventral views of the thoracic region of cleared skeletons (H–I). (H) Wildtype mouse showing 7 normal vertebrosternal ribs numbered in yellow. (I) Asxl1^tm1Bc/tm1Bc; Cbx2^{+/ tm1Ykf} mutant mouse with defects in the sternum, notably reduced ossification of the 5^th sternebra (black arrow), and the presence of only 6 vertebrosternal ribs attached to the sternum on the left side.

Fig. 3

Asxl1^tm1Bc/tm1Bc embryos exhibit altered Hox expression. In situ hybridization of Hoxa4, Hoxa7, and Hoxc8 riboprobes to wildtype and Asxl1^tm1Bc/tm1Bc mutant E10.5 embryos. Anterior boundaries of ectoderm (blue) and mesoderm (red) expression are shown by coloured arrowheads.