Growth differentiation factor 11 locally controls anterior-posterior patterning of the axial skeleton

Abstract

Growth and differentiation factor 11 (GDF11) is a transforming growth factor β family member that has been identified as the central player of anterior-posterior (A-P) axial skeletal patterning. Mice homozygous for Gdf11 deletion exhibit severe anterior homeotic transformations of the vertebrae and craniofacial defects. During early embryogenesis, Gdf11 is expressed predominantly in the primitive streak and tail bud regions, where new mesodermal cells arise. On the basis of this expression pattern of Gdf11 and the phenotype of Gdf11 mutant mice, it has been suggested that GDF11 acts to specify positional identity along the A-P axis either by local changes in levels of signaling as development proceeds or by acting as a morphogen. To further investigate the mechanism of action of GDF11 in the vertebral specification, we used a Cdx2-Cre transgene to generate mosaic mice in which Gdf11 expression is removed in posterior regions including the tail bud, but not in anterior regions. The skeletal analysis revealed that these mosaic mice display patterning defects limited to posterior regions where Gdf11 expression is deficient, whereas displaying normal skeletal phenotype in anterior regions where Gdf11 is normally expressed. Specifically, the mosaic mice exhibited seven true ribs, a pattern observed in wild-type (wt) mice (vs. 10 true ribs in Gdf11^-/- mice), in the anterior axis and nine lumbar vertebrae, a pattern observed in Gdf11 null mice (vs. six lumbar vertebrae in wt mice), in the posterior axis. Our findings suggest that GDF11, rather than globally acting as a morphogen secreted from the tail bud, locally regulates axial vertebral patterning.

Keywords: Cdx2-Cre; GDF11; skeletal patterning; tail bud.

Figure 1

Gdf11 expression is removed in posterior regions of Cdx2‐Cre; Gdf11 ^flox/flox mice. (a) Whole‐mount in situ hybridization of mouse embryos at E9.5. Gdf11 expression patterns of wt and Cdx2‐Cre; Gdf11 ^flox/flox embryos are shown. Dashed line with arrow heads indicates posterior regions that lack Gdf11 expression in a Cdx2‐Cre; Gdf11 ^flox/flox embryo. (b) Cells expressing Cdx2‐Cre are marked by GFP expression in Cdx2‐Cre; Gdf11 ^flox/+; Igs1 ^{CKI‐mitoGFP/+} embryo at E9.5. (c) Newborn wt, Gdf11 ^−/−, and Cdx2‐Cre; Gdf11 ^flox/flox pups. Both Gdf11 ^−/− and Cdx2‐Cre; Gdf11 ^flox/flox mice display extended torso and truncated tails. (d) Area expressing Cdx2‐Cre is labeled by GFP expression in newborn Cdx2‐Cre; Gdf11 ^flox/flox; Igs1 ^{CKI‐mitoGFP/+} mouse, and displayed laterally and ventrally. GFP, green fluorescent protein; wt, wild‐type [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

Cdx2‐Cre; Gdf11 ^flox/flox mice display abnormal skeletal patterning limited to posterior regions where Gdf11 expression is removed. (a–c) Alcian blue/Alizarin red staining of vertebral columns and vertebrosternal (true) ribs of newborn mice. True ribs attached to vertebral columns are shown in (c). Note that Cdx2‐Cre; Gdf11 ^flox/flox mice exhibit extended lumbar observed in Gdf11 ^−/− mice but display normal true ribs. (d) Cells expressing Cdx2‐Cre are marked by GFP expression in newborn Cdx2‐Cre; Gdf11 ^flox/flox; Igs1 ^{CKI‐mitoGFP/+} mouse. Red line points to the last (seventh) true rib. GFP, green fluorescent protein; wt, wild‐type [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

Craniofacial defects are observed in Gdf11 ^−/− mice, but not in Cdx2‐Cre; Gdf11 ^flox/flox mice. (a) Representative micro‐CT images of newborn mouse skulls shown ventrally. Red arrows point to palatine bones, and yellow arrows indicate pterygoid processes. Note that cleft palate is observed only in Gdf11 ^−/− mice. (b) Alcian blue/Alizarin red staining of newborn skulls. Boxed regions are shown at higher magnification. Yellow arrows indicate pterygoid processes. Cleft palate is observed in Gdf11 ^−/− mice, but not in Cdx2‐Cre; Gdf11 ^flox/flox mice. micro‐CT, micro–computed tomography; wt, wild‐type [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

Schematic representation of vertebral columns indicating that locally expressed GDF11, not GDF11 secreted from the tail bud, controls axial skeletal patterning. Cdx2‐Cre; Gdf11 ^flox/flox mice display an extended number of posterior vertebrae where GDF11 expression is removed, but normal patterning of anterior vertebrae. Cervical (orange)/thoracic (purple)/lumbar (sky blue) vertebrae, anterior tuberculi (small blue dots), sternums (red curves), and ribs (blue lines) are color‐coded as indicated. Gray‐dashed lines indicate normal vertebral positions: Six for the anterior tuberculum, 20 for the final thoracic vertebra, and 26 for the last lumbar vertebra. The green‐dashed line represents the upper limit of Cdx2‐Cre expression. GDF11, growth and differentiation factor 11; wt, wild‐type [Color figure can be viewed at wileyonlinelibrary.com]