XB-ART-7778Int J Dev Biol 2001 Dec 01;458:845-52.
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Xerl, a novel CNS-specific secretory protein, establishes the boundary between neural plate and neural crest.
A novel gene, Xerl, has been found as a CNS-specific gene encoding a secretory protein. In order to clarify a function of Xerl, we first examined Xerl-expressing areas during early development. Comparison with XlSox-2-positive neural plate and ADAM13-positive neural crest showed that Xerl expression was limited within the neural plate area. Microinjection of Xerl mRNA into 2- or 4-cell stage embryos indicated that Xerl overexpression caused the regional expansion of XlSox-2- and NCAM-positive neural plate, which was concomitant with the outer shift of ADAM13-positive region. The Xerl injection resulted in incomplete neural closure because of the local overproduction of the neuroepithelium. In contrast, loss of function analysis of Xerl indicated that Xerl inhibition caused the ectopic differentiation of neural crest cells. In the conjugation experiment using chordin-injected animal caps, Xerl promoted chordin-induced XlSox-2 expression, whereas Xerl inhibition caused ADAM13expression even in the injection with a high dose of chordin. Animal cap assays also showed that Xerl expression was induced by chordin. In the functional analysis using truncated forms of Xerl, Xerl deltaL (lacking LNS domain) worked as a dominant negative form that induced the overproduction of neural crest cells. These results suggest that Xerl is involved in the boundary formation of the neural plate through exclusion of neural crest cell differentiation.
PubMed ID: 11804027
Article link: Int J Dev Biol
Species referenced: Xenopus
Genes referenced: adam33 chrd.1 crb2 krt12.4 ncam1 npb snai2 sox2
Article Images: [+] show captions
|Fig. 1. Comparison of the gene expression patterns of Xerl, XlSox-2 and ADAM13. (A,C,E) Embryos before neural fold closure (st.15). The left is a dorsal view and the right is an anterior view for each picture. (B,D,F) Embryos after neural fold closure (st.20). The left is a dorsal and the right is also an anterior view. (A) Xerl expression at the neural plate stage. Xerl expression was seen in the anterior neural plate and the posterior neural groove. (B) Xerl expression after neural fold closure. The opened arrowhead indicates the narrow region of gene expression between diencephalon and midbrain. (C) XlSox-2 expression at the neural plate stage. XlSox-2 expression was wider than Xerl expression. (D) XlSox-2 expression after neural fold closure. The opened arrowhead indicates the diencephalon/midbrain boundary. (E) ADAM13 expression at the neural plate stage. ADAM13 expression was complementary to Xerl and XlSox-2. (F) ADAM13 expression after neural fold closure. The arrowhead indicates the diencephalon/midbrain boundary.|
|Fig. 2. Effect of Xerl overexpression on anterior neural folding. (A) Selection of GFP- positive embryos. GFP and Xerl mRNA were co-injected into one dorsal blastomere of 4- cell embryos, and the GFP-positive injected side was recognized under fluorescence microscope at stage 14/15. (B) GFP mRNA-injected control (st.20, anterior view). The closure of the anterior neural plate was completed. (C) Xerl mRNA-injected embryo (st.20, anterior view). The neural fold in the injected side shifted to the distal-posterior from midline as shown by incomplete neural closure (arrow). (D) The same embryo of (C) (st.20, dorsal view). The neural fold in the injected side was smaller than the counter side (arrow). (E) Cross-section of the anterior neural plate of the Xerl-injected embryo (st.15). There was no significant change in the injected side (left). (F) The future mid-hindbrain level section of the anterior neural plate of the Xerl-injected embryo (st.17). Arrowheads indicate the neural plate boundary. Lateral shift of the neural plate boundary occurred in the injected side. (G) Cross- section of the future midbrain of the Xerl-injected embryo (st.20). Abnormal cell mass was observed in the injected side (broken- lined circle and arrowheads). inj, injected side; npb, neural plate boundary; nc, notochord.|
|Fig. 3. Gene expression in the Xerl-injected embryo. Synthesized mRNAs of GFP or Xerl were injected into one dorsal blastomere of 4-cell stage embryos, and neural marker gene expression was examined by whole mount in situ hybridization at the stage 15 (A- E) or 20 (F and G). (A) XlSox-2 expression showing the neural plate area (dorsal view). Arrowhead indicates expansion of the XlSox-2-positive area in the Xerl-injected side. (B) XK81A1 expression showing the epidermal area (dorsal view). Arrowheads indicate the posterior end of the head neural plate. (C) NCAM expression showing the neural plate area (anterior view). Arrowhead indicates expansion of NCAM expression. (D) ADAM13 expression showing future cephalic neural crest cells (anterior view). Arrowheads indicate the posterior ends of neural crest cells. (E) Xslug expression showing neural crest cells (dorsal view). Arrowhead indicates the outer and posterior shift of neural crest cells in the Xerl injected side. (F) ADAM13 expression showing cephalic neural crest cells. ADAM13 expression was not seen at the anterior disclosure. (G) XlSox-2 expression (st. 20, anterior view). XlSox-2 expression is seen in the Xerl-injected area (arrow). (H) Cross section at the midbrain level. The dorsal midline is indicated with the broken line. The opened arrowhead shows XlSox-2 expression in the abnormal tissues in the Xerl-injected side. Numbers in each picture indicate the frequency of the corresponding phenotype.|
|Fig. 4. Neurogenesis of antisense Xerl-injected embryo. (A) Control embryo (st.15). GFP mRNA was injected into the right side. (B) Antisense Xerl-injected embryo (st.15). The neural fold in the injected side was larger than that of the control side. (C) GFP fluorescence showing the injected side of B. (D) Cross section on the anterior neural plate of (B). The injection was performed to the right. Arrowheads indicate the neural plate/neural crest boundary. The opened arrowhead at the injected side indicates the overproduction of the neural fold cells. (E) XlSox-2 expression (st.12). Lateral and marginal expression of XlSox-2 in the injected side (right) was fainter than that in the control side. (F) (Left) ADAM13 expression in the antisense Xerl-injected embryo (st.15). Arrowheads indicate enhanced expression of ADAM13 in the injected side. Inner shift of the ADAM13-expressing region (arrows) shows the reduction of the neural plate region. (Right) Xslug expression in the antisense Xerl injected embryo (st.15). Arrowheads indicate an increase and inner shift of expression. (G) Chordin expression (st.12). Normal chordin expression in the axial mesoderm was observed in control and in the antisense Xerl- injected embryos. Numbers on the picture indicate the frequency of the corresponding phenotype. inj, injected side; cont, control side; npb, neural plate boundary.|
|Fig. 5. Conjugated animal cap assay for testing chordin- Xerl interaction. (A) Schematic diagram illustrating the conjugated animal cap assay. Conjugates were made by combining animal cap from chordin-injected embryos (wild) and animal cap from experimental embryos injected with various test samples (albino) at stage 8.5. Gene expression was examined by whole mount hybridization at stage 17. (B-L) The upper-right corner indicates the injected sample or combination of conjugates. The lower-left corner indicates the probe used for gene expression analysis. The lower-right corner indicates the count of conjugates ex- pressing the same phenotype. (B) Xerl expression in chordin/control conjugates. Xerl expression was seen at the center of the conjugate. (C) XlSox-2 expression in chordin/control conjugates. XlSox-2 expression was seen near the border. (D) XlSox-2 expression in chordin/Xerl conjugates. XlSox-2 expression was seen in the whole conjugate. (E) XlSox-2 expression in chordin/antisense Xerl conjugates. XlSox-2 expression was not seen in the antisense Xerl-injected side. (F) XlSox-2 expression in Xerl- injected animal caps. Sufficient XlSox-2 expression was not detected, although a weak signal was seen in some specimens. (G) Xerl expression in XlSox-2-injected animal caps. No expression was observed. (H) ADAM13 expres- sion in chordin/control conjugates. No expression was observed. (I) ADAM13 expression in chordin/Xerl conjugates. No expression was observed. (J) ADAM13 expression in chordin/antisense Xerl conjugates. The expression was clearly recognized in the antisense Xerl-injected side. (K) ADAM13 expression in chordin-injected animal caps. No expression was observed. (L) ADAM13 expression in animal caps co-injected with chordin and antisense Xerl. Ectopic ADAM13 expression was induced.|
|Fig. 6. Truncated Xerl overexpression. (A) Scheme illustrating truncated con- structs. (B) Anterior view of the Xerl ∆E- injected embryo (st. 20). Opened arrowhead indicates extra epithelial tissues within the anterior disclosure. The inset is a fluorescent view of the same embryo. (C) Dorsal view of the Xerl ∆L-injected embryo (st.17). Opened arrowhead indicates the inner shift of the end of the anterior neural fold. White arrowhead indicates the overgrowth of neural fold. The inset is a fluorescent view of the same embryo. (D) ADAM13 expression in the Xerl ∆E-injected embryo (st.15). Arrowhead indicates the distal-posterior shift of the neural crest. The number of embryos showing the same phenotype is indicated at the right corner. (E) ADAM13 expression in the Xerl ∆L-injected embryo (st.15). The left panel is the anterior view. The right panel is the dorsal view of the same embryo. Arrowhead indicates the overgrowth of neural crest.|