Protogenin defines a transition stage during embryonic neurogenesis and prevents precocious neuronal differentiation

Abstract

Many Ig superfamily members are expressed in the developing nervous system, but the functions of these molecules during neurogenesis are not all clear. Here, we explore the expression and function of one of members of this superfamily, protogenin (PRTG), in the developing nervous system. Expression of PRTG protein is strong in the neural tube of mouse embryos between embryonic days 7.75 and 9.5 but disappears after embryonic day 10.5 when the neural progenitor marker nestin expresses prominently. Perturbation of PRTG activity in P19 embryonal carcinoma cells and in chick embryos, by either RNA interference or a dominant-negative PRTG mutant, increases neuronal differentiation. Using yeast two-hybrid screening and an in situ binding assay, we were able to identify ERdj3 (a stress-inducible endoplasmic reticulum DnaJ homolog) as a putative PRTG ligand. Addition of purified ERdj3 protein into the P19 differentiation assay reduced neurogenesis. This effect was blocked by addition of either a neutralizing antibody against PRTG or purified PRTG ectodomain protein, indicating that the effect of ERdj3 on neurogenesis is mediated through PRTG. Forced expression of ERdj3 in the chick neural tube also impairs neuronal differentiation. Together, these results suggest that expression of PRTG defines a stage between pluripotent epiblasts and committed neural progenitors, and its signaling plays a critical role in suppressing premature neuronal differentiation during early neural development.

Figure 1.

Expression of PRTG protein in the mouse embryo. A, Expression of rat prtg in the nervous system at different stages of development as analyzed by the Northern blot. Ad, Adult; Br, brain; Nt, neural tube. B, Specificity of PRTG antibodies. Cell lysates of HEK293T cells transfected with the control vector, prPRTGf (rat full-length PRTG), prPRTGΔc (rat PRTG without cytoplasmic domain), or prPRTGc (rat cytoplasmic tail), and rat E10.5 neural tube lysate (Embryo) were subjected to Western blot using α-PRTG1 and α-PRTG2 mAbs. Addition of peptide E (E; ectodomain) or peptide C (C; cytoplasmic tail) competes out the bands recognized by the monoclonal antibodies. C, Schematic structure of PRTG. Peptide E or C for generating antibodies is marked in blue. D–H, Transverse sections through the mid-body level of mouse at E7 (D), E7.75 (E), E8.25 (F), E9.5 (G), and E10.5 (H) were stained using α-PRTG2 mAb (red). The dorsal is up in all panels. D, At E7, PRTG labeling is weakly detected in the mesoderm (me), whereas Oct4 is restricted to the ectoderm (ec; green). E, At E7.75, PRTG is detected in neuroepithelium, mesoderm, endoderm, and posterior epiblasts (arrows). The yolk sac (ys) surrounding the embryo is nonspecifically stained by the secondary antibodies. F, G, PRTG protein is present in almost all cells between E8.25 and E9.5 (red) but not in the notochord (n), differentiating cardiac cells, and some mesenchymal cells. The endocardium is labeled with anti-endoglin antibody (green). ac, Atrial chamber; dm, dermomyotome; vc, ventricular chamber; sc, spinal cord. H, PRTG is not detectable at E10.5. I, Expression of PRTG (red) and SSEA1 (green) in a transverse section of E7 and E8.25 mouse embryos. J–N, Expression of PRTG (red) and nestin (J), Ascl1 (K), TuJ1 (L), MAP2 (M), and NeuN (N) (green) in adjacent transverse sections through the thoracic level of E9.5 (top) and E10.5 (bottom) mouse embryos.

Figure 2.

PRTG is expressed in early committed cells, and blockage of PRTG activity promotes neuronal differentiation in P19 cells. A, B, Differentiation of P19 cells was first induced by retinoic acid for 4 d (Day 1–4), and then the cells were cultured in serum-free medium for another 4 d (Day 5–8). A, Expression of prtg mRNA was analyzed by the Northern blot. MAP2, A neuronal marker; cyclophilin, RNA loading control. B, Expression of PRTG, Oct4, Sox1, and MAP2 in differentiating P19 cells was analyzed by Western blot. A nonspecific band with molecular weight 40 kDa recognized by α-PRTG2 was used as protein loading control (Ctrl). C, Quantification of protein amounts from the Western blots shown in B. Relative protein expression levels are normalized to the highest protein expression amount of each protein. D, Lysates of HEK293T cells transfected with shPRTG(m), control vector (UI4), or shS together with pmPRTGf were subjected to Western blot (WB) by anti-myc and anti-β-tubulin antibodies. E, Lysates of HEK293T cells transfected with pmPRTGf(myc), prPRTGf(HA), and prPRTGΔc(HA) were immunoprecipitated by anti-HA or anti-myc antiserum and then subjected to Western blot by anti-myc or anti-HA antiserum. F, Experimental procedure for P19 cell neuronal differentiation analysis. P19 cells were transfected with 0.4 μg of pAscl1 and 1.6 μg of the indicated plasmids, subjected to selection with G418 or puromycin, and then cultured in SF21 serum-free medium for 4 d. G, Differentiated neuronal cells were detected by TuJ1 staining. Scale bar, 50 μm. H, The percentage of cells labeled by TuJ1 in one whole well of the six-well plate was counted. Data are then normalized relative to the percentage of the control (pEF1A or UI4) and shown as the mean ± SEM (n = 3; **p < 0.01, by Student's t test).

Figure 3.

PRTG represses precocious neuronal differentiation in the developing chick neural tube. A–V, Control plasmids (UI4 or pCAG) or various chick PRTG constructs were electroporated into the right side (green) of chick neural tubes at HH stage 9–10. Two days after electroporation, the chick embryos were fixed and stained with DAPI (blue) and TuJ1 antibody (A–H, Q–V, red) or anti-HuC/D antibody (I–P, red). E–H are higher magnifications of the white boxes in C and D; M–P are higher magnifications of the white boxes in K and L. W, Regions of quantification are indicated. In each plane, the distance between ventricle and pial surface is measured. The medial part is defined as within three-quarters of that distance close to the ventricle. The lateral part is defined as within one-quarter of that distance close to the pial surface. X, Quantification of the relative fluorescence intensity of TuJ1 in the chick neural tube electroporated with the indicated plasmids. Quantification was performed as described in Materials and Methods. Data are shown as the mean ± SEM. The number of chick embryo analyzed in this assay is indicated in brackets. p value is shown by one-way ANOVA and post hoc least significant difference test. Y, The number of HuC/D⁺ cells in the medial region is quantified and shown as the mean ± SEM (**p < 0.01; ***p < 0.001, by Student's t test).

Figure 4.

ERdj3 is a putative ligand of PRTG. A, Experimental procedure for P19 cell differentiation assay used in the following analyses. B, Relative percentages of TuJ1⁺ neurons induced from Ascl1-transfected P19 cells in the presence of 10 μg/ml α-PRTG1 mAb, α-PRTG3 mAb, PRTGe–Fc, or Fc are shown (n = 3; ***p < 0.001, by Student's t test). C, Schematic representations of eight ERdj3 clones identified by yeast two-hybrid screening are shown, and their intensities of interaction with the PRTG ectodomain are displayed as β-galactosidase activity in an X-gal assay. D, Various prtg deletion mutants were examined for their ability to interact with C-terminal ERdj3. E, ERdj3 is a secreted protein. CM and total cell lysate (TCL) of HEK293T cells transfected with pERdj3 or the control vector were subjected to Western blotting using anti-myc antibody. β-tubulin, A cytosolic protein marker. F, ERdj3 binds PRTG. HEK293T cells were transfected with pPRTGf or control vector. Cells were then treated with ERdj3-containing CM in the presence or absence of PRTGe–Fc. PRTG expression was detected by rabbit anti-PRTG serum (W4; top row). Binding of ERdj3 on the cells was detected by anti-myc mAb (middle row). Cell nuclei were visualized with DAPI (bottom row). Binding of ERdj3 to PRTG-expressing cells was partially inhibited by 40 μg/ml PRTGe–Fc (the third column). Scale bar, 20 μm. G, Quantification of ERdj3 binding to PRTG-expressing cells. The results are normalized relative to fluorescence intensity of the cells transfected with pPRTGf in the presence of control CM. Results are shown as the mean ± SEM from a total of 50 cells (***p < 0.001, by Student's t test).

Figure 5.

ERdj3 inhibits neuronal differentiation through PRTG in P19 cell differentiation assay. A, P19 cells were transfected with indicated plasmids and pAscl1. Differentiated neurons were detected by TuJ1 staining 4 d after transfection. Scale bar, 100 μm. B, Quantitative results of A are shown as the mean ± SEM (n = 3; ***p < 0.001, by Student's t test). C, P19 cells were induced to differentiation and were cultured with purified 5 μg/ml ERdj3–Fc in the presence or absence of 10 μg/ml α-PRTG1 mAb, α-PRTG3 mAb, PRTGe–Fc, or Fc proteins. Quantitative results of TuJ1⁺ neurons are shown as the mean ± SEM (n = 3; *p < 0.05; **p < 0.01; ^#p = 0.053, by Student's t test).

Figure 6.

ERdj3 suppresses neuronal differentiation in the chick neural tube. Overexpression or knockdown of chick ERdj3 constructs were electroporated into the right side (green) of chick neural tubes at HH stage 9–10. Two days after electroporation, the chick embryos were fixed and stained with DAPI (blue) and TuJ1 (A, B, E, E′, red), anti-NeuN (C, D, F, F′, red), or anti-cCasp3 (G, H, red) antibodies. White boxed regions in A, C, and E are enlarged in B, D, and F, respectively. A, B, Overexpression of ERdj3 leads to fewer TuJ1⁺ cells (bracket in B) in the chick HH stage 17–20 embryo. C, D, Overexpression of ERdj3 reduces NeuN⁺ cells in the chick HH stage 17–20 embryo. There are more NeuN⁺ cells on the control side (arrowheads in D). Moreover, the NeuN⁺ cell on the electroporated side is an untransfected cell (NeuN⁺, GFP-negative cell; arrow in D). E–H, Knockdown of endogenous ERdj3 produces thinner chick neural tube at HH stage 17–20. E, F, More cCasp3-positive cells are detected in the shERdj3(g)-electroporated side (arrows in F). I, Quantitative results of the number of cCasp3⁺/GFP⁺ cells on the electroporated side shown in F. Data are shown as the mean ± SEM quantified from at least three embryos and five sections for each embryo (***p < 0.001, by Student's t test).

Figure 7.

Summary of PRTG expression and the potential function of ERdj3/PRTG signaling in the mouse embryo. Schematic representations of the protein amounts from the immunohistochemical staining in Figure 1 are shown in different colors. The intensity of the color indicates the protein levels. PRTG expression is between marker proteins for epiblasts and marker proteins for neural progenitor cells and thus depicts a distinctive transition stage during mouse neural development. By extrapolating experimental results obtained from P19 cells and the chick neural tube, it is likely that ERdj3/PRTG signaling plays a critical role in the suppression of premature neuronal differentiation in early mouse embryos. Whether ERdj3/PRTG signaling acts at the generation of neural progenitor cells and/or the differentiation of neural progenitor cells into neurons is currently unclear. ERdj3 may have an additional role associated with supporting survival of neuroectoderm.