Hyaluronan in limb morphogenesis

Abstract

Hyaluronan (HA) is a large glycosaminoglycan that is not only a structural component of extracellular matrices, but also interacts with cell surface receptors to promote cell proliferation, migration, and intracellular signaling. HA is a major component of the extracellular matrix of the distal subapical mesenchymal cells of the developing limb bud that are undergoing proliferation, directed migration, and patterning in response to the apical ectodermal ridge (AER), and has the functional potential to be involved in these processes. Here we show that the HA synthase Has2 is abundantly expressed by the distal subridge mesodermal cells of the chick limb bud and also by the AER itself. Has2 expression and HA production are downregulated in the proximal central core of the limb bud during the formation of the precartilage condensations of the skeletal elements, suggesting that downregulation of HA may be necessary for the close juxtaposition of cells and the resulting cell-cell interactions that trigger cartilage differentiation during condensation. Overexpression of Has2 in the mesoderm of the chick limb bud in vivo results in the formation of shortened and severely malformed limbs that lack one or more skeletal elements. Skeletal elements that do form in limbs overexpressing Has2 are reduced in length, exhibit abnormal morphology, and are positioned inappropriately. We also demonstrate that sustained HA production in micromass cultures of limb mesenchymal cells inhibits formation of precartilage condensations and subsequent chondrogenesis, indicating that downregulation of HA is indeed necessary for formation of the precartilage condensations that trigger cartilage differentiation. Taken together these results suggest involvement of HA in various aspects of limb morphogenesis.

Fig. 1

Expression of Has2 during early chick limb bud development analyzed by in situ hybridization. (A–D) Frontal sections through stage 17/18 (A), stage 20 (B), stage 25 (C), and stage 27 (D) limb buds. In each panel the posterior border of the limb bud is on the right and the anterior border on the left. (E, F) Dark field (E) and bright field (F) images of a sagittal section through the AER of a stage 20 limb bud. At stages 17/18 (A), 20 (B), and 25 (C) Has2 is expressed in the distal posterior subridge mesoderm (red arrows). Little or no expression is detectable in the proximal central core (c in panel C) where precartilage condensations are forming. At stage 27 (D) Has2 is expressed in the distal mesoderm at the tips of the digits which is undergoing outgrowth, but is not expressed in the interdigital mesoderm or in the precartilage condensations of the digits. During early limb morphogenesis Has2 is also expressed in the AER (arrows in E, F).

Fig. 2

Adenoviral overexpression of Has2 in the mesoderm of the developing chick limb bud in vivo perturbs limb morphogenesis. (A–H) Alcian blue/Alizarin Red-stained skeletal elements of day 7 (A, B), day 9 (C, D), day 10 (E, F), and day 13 (G, H) contralateral control forelimbs (top of each panel) and Has2 infected forelimbs (bottom of each panel) resulting from microinjection of Has2 adenovirus at the onset of limb formation (stage 18). Digits 2, 3, and 4 in the day 9 control limb in panel C are indicated. Has2 infected limbs are shortened (92%) and several (42%) exhibit partial or complete absence of one or more skeletal elements. Skeletal elements that do form in the Has2 infected limbs are often misshapen and exhibit abnormal morphology (65%), and/or are positioned inappropriately (15%). In some cases, a rudimentary skeletal element is fused to and growing out of the shaft of the humerus (arrows in C, E, F), and in some cases (27%) the autopod contains only a digit 3, and digits 4 and 2 are absent.

Fig. 3

Alcian blue/Alizarin Red-stained skeletal elements of a day 13 contralateral control hindlimb (top) and Has2 RCAS retrovirus infected hindlimb (bottom) resulting from microinjection of the Has2 retroviral vector into the prospective leg forming region at stage 10. The femur in the Has2 infected limb is shortened and misshapen, and the zeugopod consists of a single abnormal skeletal element that articulates with two extremely small digits that bear no resemblance to normal digits.

Fig. 4

Overexpression of Has2 impairs formation of precartilage condensations and chondrogenesis by limb mesenchymal cells in vitro. (A–H) Phase contrast microscopy images of micromass cultures of stage 22–24 limb bud mesenchymal cells established at 5 × 10⁴ cells/10 μl (A–D) or 2.5 × 10⁴ cells/10 μl (E–H) infected with a control RCAS retrovirus (A,C; E,G) or a Has2 RCAS retrovirus (B,D; F,H). The RCAS control cultures form numerous precartilage condensations of closely packed rounded cells on day 2 (A) or day 3 (E), which subsequently enlarge and differentiate into cartilage nodules which deposit a refractile matrix (C,G). In contrast, only a few very small condensations form in cultures infected with the Has2 RCAS vector (B,F), and there a striking decease in the number of refractile cartilage nodules that differentiate (D,H).

Fig. 5

(A,B) Higher magnification phase contrast microscopy images of day 4 limb bud mesenchymal cell micromass cultures established at 2.5 × 10⁴ cells/10 μl and infected for 3 h at the onset of culture with a control RCAS (A) or Has2 RCAS (B) retroviral vector. Control cultures (A) form numerous precartilage condensations or nodules of closely packed rounded cells, whereas most of the cells of Has2 infected cultures (B) remain large, flattened, and spatially separated from one another.

Fig. 6

Retroviral overexpression of Has2 inhibits the accumulation of Alcian blue staining cartilage matrix by micromass cultures of limb bud mesenchymal cells established at 5 × 10⁴ cells/10 μl (A,B), 3.4 × 10⁴ cells/10 μl (C,D), or 2.5 × 10⁴ cells/10 μl (E,F). (A–E) Alcian blue-stained day 3 (A,B), day 4 (C,D), and day 6 (E,F) cultures infected with a control RCAS retrovirus (A,C,E) or with a Has2 RCAS retrovirus (B,D,F). There is a striking decrease in the extent of Alcian blue staining cartilage matrix in the Has2 infected cultures (B,D,F) compared to corresponding controls (A,C,E).

Fig. 7

(A, B) Distribution of hyaluronan (HA) in RCAS control (A) and Has2 RCAS (B) infected limb bud mesenchymal cell micromass cultures established at 2.5 × 10⁴ cells/10 μl as assayed by histochemical staining with a biotinylated HA binding protein (HABP). Relatively little HABP staining is detectable in day 5 RCAS control cultures (A), whereas widespread HABP staining is present throughout the Has2 infected cultures (B).