Expression of Ptdsr during embryogenesis and in adult tissues
The observed perinatal lethality indicates that Ptdsr plays an important role during development. Analysis by RT-PCR (data not shown) showed that Ptdsr is expressed early in development, because we were able to detect Ptdsr transcripts in ES cells and embryos at all developmental stages. To analyze in more detail the temporal and spatial expression patterns of Ptdsr, and to correlate expression patterns with observed pathological malformations, we made use of a Ptdsr-β-geo gene-trap reporter mouse line generated from a Ptdsr gene-trap ES cell clone. This line has an insertion of β-galactosidase in the 3' region of the gene (Figure 2a).
We first examined Ptdsr expression by X-Gal staining in heterozygous embryos staged from E9.5 to E12.5. These developmental stages were chosen so as to investigate Ptdsr expression in affected organs prior to the onset of pathological malformations in Ptdsr -/- embryos. At E9.5 we found Ptdsr expression in the developing neural tube, somites, heart, gut and branchial arches (Figure 2b). At E10.5, Ptdsr expression remained high in the developing nervous system, with most intense staining in the forebrain, hindbrain and neural tube. At this stage of embryogenesis, high levels of Ptdsr expression could also be detected in the developing limb buds and eyes (Figure 2b). Ptdsr expression was altered at E12.5, with most intensive β-galactosidase staining in the eyes, developing condensations of the limb buds, neural tube and brain (Figure 2b). Transverse sections of X-Gal-stained embryos at E12.5 showed an asymmetric expression pattern in the neural tube with intense staining of the central mantle layer but no expression in the dorsal part of the neural tube (for example, the roof plate; Figure 2c). Expression in dorsal root ganglia lateral to the neural tube and in the somites was observed; Ptdsr was expressed throughout the somite structure (myotome, dermatome and sclerotome; Figure 2d). Expression boundaries between somites were evident, with no expression in the segmental interzones, which correspond to the prospective intervertebral discs (Figure 2d). Transverse sections of the developing eye at E12.5 revealed strong Ptdsr expression in the inner layer of the neural cup, which will later develop into the neural retina. Furthermore, Ptdsr expression was detected in the primary lens fiber cells of the developing lens (Figure 2e). We carefully investigated whether Ptdsr is expressed from E10.5 to E12.5 in the developing kidney and lungs, but no expression could be detected indicating that Ptdsr expression is required only at later stages in the development of these organs (see below).
Hybridization of a multiple-tissue northern blot revealed a single transcript of about 1.8 kb in almost every tissue analyzed in adult mice (Figure 2f). The most prominent expression was observed in testis, thymus, kidney, liver and skin, with moderate to low expression in lung, small intestine, spleen, stomach and skeletal muscle. Thus, Ptdsr is ubiquitously expressed throughout embryogenesis and in adult tissues, although at different levels.