Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme

Abstract

We identified hyaluronan synthase-2 (Has2) as a likely source of hyaluronan (HA) during embryonic development, and we used gene targeting to study its function in vivo. Has2(-/-) embryos lack HA, exhibit severe cardiac and vascular abnormalities, and die during midgestation (E9.5-10). Heart explants from Has2(-/-) embryos lack the characteristic transformation of cardiac endothelial cells into mesenchyme, an essential developmental event that depends on receptor-mediated intracellular signaling. This defect is reproduced by expression of a dominant-negative Ras in wild-type heart explants, and is reversed in Has2(-/-) explants by gene rescue, by administering exogenous HA, or by expressing activated Ras. Conversely, transformation in Has2(-/-) explants mediated by exogenous HA is inhibited by dominant-negative Ras. Collectively, our results demonstrate the importance of HA in mammalian embryogenesis and the pivotal role of Has2 during mammalian development. They also reveal a previously unrecognized pathway for cell migration and invasion that is HA-dependent and involves Ras activation.

Figure 1

Has2 and versican are expressed in similar domains in the E9.5 mouse. (a) Has2 expression. cm, cranial mesenchyme; fb, forebrain; mb, midbrain; fg, foregut diverticulum; 1, first branchial arch; ot, outflow tract; v, ventricle; a, atrium; st, region of developing septum transversum, including liver primordium and proepicardial organ; fm, foregut mesenchyme; am, periaortic mesenchyme; da, dorsal aorta. (b) Versican expression. (c and d) Higher-power views of the heart region boxed in a and b, respectively. Has2 and versican mRNAs are expressed in the endothelium (indicated by arrowheads) of the outflow tract (O) and myocardium (M) of the AV canal region. A, atrium; V, ventricle. (e and f) AV canal region and cardiac cushions (indicated by asterisks) of an E9.5 mouse embryo stained with hematoxylin and eosin (e) and for HA using a biotinylated HA-binding protein (f). E, endocardium. The boxed region contains endothelial cells that have transformed and are invading the underlying cushion tissue. These have abundant cell-surface HA (magnified in the inset). (g) Distribution of versican in the AV canal region, superimposing a Nomarski DIC image on a pseudocolored image of versican immunofluorescence. Note that the distribution of versican in g is similar to the distribution of HA in f. (h) Digital composite image of a Nomarski DIC image and a dark-field image of a ³⁵S-labeled in situ hybridization of Has2 mRNA in the AV canal and outflow tract region of an E10.5 heart. Mesenchymal cells within the AV canal and outflow tract cushions express abundant Has2 mRNA. The signal within the atrium results from light scattered by red blood cells, not from silver grains. Bars in a and b = 500 μm; bars in c–h = 100 μm.

Figure 2

Gene targeting of the Has2 locus. (a) Schematic of a portion of the Has2 locus with restriction sites, exons (filled boxes), the targeting vector, and resulting targeted locus. Homologous recombination replaces the end of intron 3 and the first 60 codons of exon 4 with PGK-Neo. The BamHI and EcoRI restriction fragments confirming the structure of PCR-positive ES clones are indicated. Arrowheads indicate the direction of transcription of PGK-Neo and the diphtheria toxin A chain. (b) PCR screening with PGK-Neo and flanking primer revealing predicted amplicons of 1.8 kb in two ES clones. (c) Southern blot analysis of BamHI genomic DNA digests from the parental ES line (control), the two targeted ES clones, a representative wild-type mouse, and a heterozygous mouse. Probe 2 (box 2 in a) detected the 12.5-kb and 5.4-kb restriction fragments corresponding to the wild-type and targeted alleles, respectively. DTA, diphtheria toxin A chain.

Figure 3

Abnormalities exhibited by Has2^–/– embryos. (a) Yolk sac of an Has2^+/– embryo. (b) Yolk sac from an Has2^–/– littermate. Cross-sections of the yolk sac stained with hematoxylin and eosin are shown in the inset. Note the presence of vitelline vessels (VV) containing nucleated red blood cells in the yolk sac of the Has2^+/– embryo. The endoderm and mesoderm are not fused in the Has2^–/– embryo, and the red blood cells are free within this space. (c and d) Representative wild-type and Has2^–/– embryos at E9.5. Note the diminished size, the bloodless heart, and distorted somites of the Has2^–/– embryo. (e and f) E9.5 wild-type and Has2^–/– embryos stained for the endothelial marker PECAM. Note the absence of an organized vascular network expressing PECAM in the Has2^–/– embryo. P, pericardium; E, endoderm; M, mesoderm; OpP, optic placode; OtP, otic placode; first and second pharyngeal pouches are numbered. Bars in c–f = 500 μm.

Figure 4

E9.5 Has2^–/– embryos lack alcian blue–staining glycosaminoglycans and HA in cardiac jelly. The cardiac jelly of wild-type embryos is rich in acidic glycosaminoglycans (blue stain in a) and HA (brown stain in c). In contrast, Has2^–/– embryos totally lack alcian blue–stained material (b) and HA (d). The heart of the Has2^–/– embryo has a characteristic constriction at the AV canal region (indicated by the arrows), but no endocardial cushions, which are indicated by the asterisks in a and c. Bars in a and b = 100 μm; bars in c and d = 250 μm.

Figure 5

FACE analysis of E9.5 embryo extracts for HA. Lane 1 contains disaccharide standards. Lanes 2–5 represent 5% of a single embryo (lanes 2 and 3 are wild-type; lanes 4 and 5 are Has2^–/–). The arrow indicates the ΔDiHA disaccharide derived from HA. Note the marked reduction of the ΔDiHA band in the individual Has2^–/– samples compared with wild-type controls. Additional analyses of material pooled from wild-type or Has2^–/– embryos and run at higher concentrations (equivalent to 40–60% of a single embryo) gave similar results (data not shown; see Table 1).

Figure 6

Ultrastructure of wild-type and Has2^–/– E9.5 mouse hearts. Scanning electron micrograph of the external structure of the heart from a wild-type (a–c) and an Has2^–/– embryo (d–f). Specimens were viewed from the left side (a and d), the front (b and e), and the right side (c and f). Note the apparent absence of the presumptive right ventricle and outflow tract in the Has2^–/– embryo compared with the wild type. Scanning electron microscopy of cross-sections of hearts from wild-type (g and h) and Has2^–/– embryos (i and j) reveal a lack of AV cushions and a compacted ventricle wall lacking trabeculations (arrowheads). There is a constriction at the site of the AV canal in the Has2^–/– embryo. LV, left ventricle; RV, right ventricle; AoP septum, aortic pulmonary septum. Asterisks indicate left posterior atrial wall. h and j are higher magnifications of g and i, respectively.

Figure 7

AV canal morphogenesis is deficient in Has2^–/– AV canal explants and is restored by exogenous HA or activated Ras. Top panel: AV canal explant morphogenesis in vitro. AV canal explants from E9.5 wild-type (a–c) or Has2^–/– embryos (d–h) were cultured on collagen gels. Explants from wild-type embryos exhibit abundant endothelial cell migration and invasion (image a is focused on the surface of the gel; b is focused below the surface). In contrast, there is no endothelial-cell migration in AV canal explants from E9.5 Has2^–/– embryos (d). Transfection with dominant-negative (DN) Ras cDNA significantly (P < 0.001) reduces endothelial migration and invasion in AV explants from wild-type embryos (c). Because migration and invasion begins during the 16-hour incubation before transfection, the degree of inhibition is probably underestimated (see Methods). AV canal explants from Has2^–/– embryos exhibit comparable morphogenesis after transfection with Has2 cDNA (e), in the presence of HA in the media (f), or in the collagen gel (g). Transformation in Has2^–/– embryos is also rescued by transfecting with constitutively active Ras (h). M, myocardium. Nomarski DIC optics. Bar in h = 200 μm. Bottom panel: Quantification of AV canal transformation in the presence or absence of either dominant-negative Ras (S17N) or constitutively active Ras (Q61L). The dominant-negative Ras significantly inhibited cell migration and invasion in viable wild-type explants, whereas constitutively active Ras restored cell migration and invasion in AV explants from Has2^–/– embryos to the same degree as wild-type explants. The scoring method is outlined in Methods. ^AP < 0.001.

Figure 8

Dominant-negative Ras inhibits HA-mediated endothelial-cell invasion in Has2^–/– AV canal explants. These images were obtained by laser scanning confocal microscopy after staining for α−smooth muscle actin. Exogenous HA (0.75 mg/mL of medium) was added to the culture medium in both Has2^–/– explants. (a) A collapsed Z series of 100 μm showing the characteristic transformation to mesenchyme and invasion of the collagen gel in the presence of exogenous HA. The dotted line indicates the previous location of the myocardium, which was removed. (b) Characteristic effect of transfection with dominant-negative Ras. In contrast to the rescued AV explant, an epithelial sheet has migrated over the surface of the collagen gel, but there are no invading mesenchymal cells. Similar results were obtained in three independent experiments.