XB-ART-47669Int J Dev Biol January 1, 2013; 57 (9-10): 779-86.
Xnr3 affects brain patterning via cell migration in the neural-epidermal tissue boundary during early Xenopus embryogenesis.
Neural induction and anteroposterior neural patterning occur simultaneously during Xenopus gastrulation by the inhibition of BMP and Wnt signaling, respectively. However, other processes might be necessary for determining the neural-epidermal boundary. Xenopus nodal-related-3 (Xnr3) is expressed in dorsal blastula and plays a role in neural formation. In this study, we analyzed how Xnr3 affects neural patterning to identify novel mechanisms of neural-epidermal-boundary determination. In situ hybridization revealed that ventro-animal injection with Xnr3 shifted the lateral krox20 expression domain anteriorly and reduced Otx2 expression. The mature region of Xnr3 is necessary for these effects to occur, and the pro-region accelerated them. Phalloidin labeling revealed that cells around the neural-epidermal boundary lost their slender shape following Xnr3 injection. Moreover, we analyzed the cell migration of ectodermal cells and found specific Xnr3-induced effects at the neural-epidermal boundary. These findings together suggested that Xnr3 affects anterior ectoderm migration around the neural-epidermal boundary to induce a specific neural pattern abnormality. Change of the shape of surrounding ectodermal cells and the specific migratory pattern might therefore reflect the novel mechanism of neural-epidermal boundary.
PubMed ID: 24307296
Article link: Int J Dev Biol
Genes referenced: dap egr2 en2 foxg1 muc2 nodal nodal3.1 nodal3.2 odc1 otx2 rax snai2 sox2
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|Fig. 1. Xnr3 injection induced an abnormal neural pattern in Xenopus embryos. (A,B) Superficial phenotype of 2-day tadpole after Xnr3 injection. (A) An uninjected embryo. (B) An embryo injected with 100 pg of Xnr3 mRNA into the ventral animal pole (VAP) region at the 4-cell stage. (C-N) Whole-mount in situ hybridization (WISH) of stage-18 embryos. Normal embryos (C,E,G,I,K,M) and embryos injected with 40 pg of Xnr3 mRNA into the VAP region at the 4-cell stage (D,F,H,J,L,N) showing the spatial expression of xOtx2 (C, D), Xrx1 (E, F), CG1 (G, H), krox20, En2, and xBF1 (I, J), slug (K, L), and Sox2 (M, N). In (I,J), krox20/En2 expression extended laterally (black arrow) and curved anteriorly (white arrow). In (K,L), 200 pg of lacZ was also injected. Arrows in (L) show the anterior elongation of the slug expression domain. (O) Quantitative levels of brain marker genes measured by RT-PCR. Experiments were carried out with the anterior region dissected from stage-18 embryos. 0 pg (lane 1), 40 pg (lane 2), or 100 pg (lane 3) of Xnr3 mRNA were injected into the VAP of 4-cell embryos, and the expressions of xOtx2 (lane 1), Xrx1 (column 2), xCG1 (column 3), krox20 (column 4), En2 (column 5), slug (column 6), Sox2 (column 7) and ODC (column 8) were analyzed. (P-U) Expression patterns of neural markers in late neurula after treatment of the embryos with RA. Expression of slug (P-R), krox20, and xOtx2 (S-U). The embryos were treated with 5 x 10-7 M RA (Q,T) or 10-6 M RA (R,U).|
|Fig. 2. Temporal changes in the specific neural pattern induced by Xnr3 injection. 100 pg of Xnr3 mRNA was injected into both ventro-animal hemispheres of 4-cell embryos (B,D,F,H,J,L,N,P,R). The expression of krox20 (A-F), Xrx1 (G-L), or xOtx2 (M-R) were observed at Stage 14 (A,B,G,H,M,N), Stage 16 (C,D,I,J,O,P) or Stage 18 (E,F,K,L,Q,R). The arrows in (E,F) indicate the neural-epidermal boundary|
|Fig. 3. The mature region of Xnr3 contributed to abnormal neural patterning. (B-M) Spatial expression of neural marker genes was observed by WISH. 750 pg of pXtnr3 (C,G,K), 100 pg of mXtnr3 (D,H,L), or 40 pg of Xnr3 mRNA (E,I,M) was injected into the VAP region of 4-cell embryos. pXtnr3 and mXtnr3 indicate the pro-region and the mature-region, respectively, of Xtnr3. Embryos were examined with probes for krox20 and En2 (B-E), Xslug (F-I), xOtx2 (L-M). pXtnr3 did not affect the expression patterns, whereas mXtnr3 injection induced patterns similar to those observed after injection of Xnr3. Arrow indicates the change in expression pattern. (N) Synergistic enhancement of the activity of mXtnr3 by pXtnr3 expression. Spatial expression of En2/krox20 at stage 18 was assessed by WISH, and the results are expressed in a bar graph. The severity of the effect neural is categorized into three classes: Class 1, Class 2 and Class 3 are represented by white, orange and red, respectively.|
|Fig. 4. Xnr3 injection specifically affected the shape of cells at the epidermal-neural boundary. (A) The experimental procedure is illustrated in a schematic diagram. Alexa 568 and Xnr3 mRNA were injected into the DAP and the VAP region, respectively. At stage 18, the epidermal-neural boundary regions of the anterior end of neural plate and lateral neural plate were dissected and stained with phalloidin-Alexa488. (B-G) Double-fluorescent labeling of the anterior (B-D) or lateral (E-G) region of a normal embryo. The Alexa568 dextran staining pattern (B,E), the phalloidin staining pattern (C,F), and a merged image (D,G) are shown. The Alexa568-positive region approximately shows the presumptive neural plate. (H-M) Doublestained patterns in the anterior region (H-J) and lateral region (K-M) of an Xnr3-injected embryo. The Alexa568 dextran staining pattern (H, K), the phalloidin staining pattern (I, L), and a merged image (J, M) are shown.|
|Fig. 5. Xnr3 specifically caused defects in neuralepidermal boundary cell migration. (A) Schematic overview of the experiment. Alexa568-dextran (Red) and 100 pg of Xnr3 mRNA were injected into the DAP and VAP, respectively, of 4-cell embryos. After injecting Alexa488 (Green) into one cell in the epidermal-neural boundary region at the 128-cell stage, the injected embryos were examined at three points of stage: NP, neural plate; CG, cement gland. (B-G) Observation of migratory cells labeled with Alexa488 dextran during neurulation. Double-labeled embryos were injected with 0 pg (B-D) or 100 pg of Xnr3 mRNA, and then observed at stage 9 (B-G), stage 14 (B’-G’), or stage 18 (B”-G”). These embryos were all anteriorly viewed. B-B” and E-E” are embryos in that Alexa488 was ventrally injected. Similarly, C-C” and F-F”, or D-D” and G-G” are injected into dorso-anterior (around animal pole) or into dorso-posterior (near marginal zone), respectively. The white line indicates the midline, and the asterisk marks the anterior end of the Alexa568-positive region. The Alexa488-positive domain is indicated by a dotted white line. The pink dots and the blue dots indicate the anterior end and the posterior end, respectively, of the Alexa488-positive area. (H,I) Summary of the results in regard to the migration of ventral cells (H) and dorsal (I) cells. In these diagrams, the result of normal embryo (left half) and Xnr3 injected embryo (right half) is shown. The red line and the short yellow line mark the midline of the embryo and anterior end of neural groove, respectively. The arrow indicates the movement of the migratory cells from Stage 14 to Stage 18. Black and Red arrows indicate mild changes and severe changes, respectively, in comparison with normal embryos. (J) Assessment of gastrulation defect by Xnr3 injection. In the left picture, L1 indicates the distance between the anterior end of the neural groove and the anterior end of the Alexa568 region, and L2 indicates the diameter of the embryo. L1/ L2 is shown in a bar graph. The error bars represent the standard error (SE). The numbers below the titles are the numbers of embryos examined. (K) The angle between the yolk plug and the anterior end of the neural plate (angle 1, black bars), and the angle between the yolk plug and the anterior end of the dorsal mesoderm (angle 2, red bars) are observed with half sections of the embryos. The results are shown in a bar graph. The error bars represent the standard error (SE). The numbers below the titles are the numbers of embryos examined.|