|
FIG. 3. Expression of Xnrs in Stage 9.5 embryos. Equivalent amounts of full-length digoxigenin-labeled RNAs were used as probes to enable comparison of levels of expression. All panels are vegetal views of embryos, with the dorsal side on the upper part of the image. Xnr2, Xnr5, and Xnr6 are strongly expressed in endoderm at this stage. Xnr1 was very weakly expressed in endoderm and no Xnr4 expression was detected at this stage. Specific staining is pur- ple; the brown region located around embryos is naturally occurring pigment. Reproduced with permission of the Publisher, John Wiley & Sons.
|
|
FIG. 4. Comparison of the expression pattern of Xnr5 mRNA species and Xnr2 mRNA. Xnr5 mRNA was detected under the dorsal lip from Stage 8, before MBT, to Stage 10. Xnr2 was first detected at Stage 9 and was expressed over a wider area than Xnr5s. The zone of Xnr2 expression region moved from dorsal to ventral with increasing stage. Wild-type embryos were fixed at Stages 8, 8.5, 9, 10, 10.5, or 11 and examined by modified whole-mount in situ hybridization using probes for Xnr5 and Xnr2. All embryos are viewed from the vegetal pole with dorsal facing upward. Reproduced with permission of the Publisher, John Wiley & Sons.
|
|
FIG. 5. Comparison of the expression pattern of duplicated Xnr5 and Xnr2 in deep endoderm of sectioned embryos. (a) Xnr5 mRNA was strongly expressed in the dorsal superficial layer of endoderm, and in the deep layers of both dorsal and ventral endoderm. Xnr2 expression was only detected in the superficial layer of endoderm. (b) Higher magnification of Xnr5 expression at Stage 9. (c) Higher magnification of su- perficial Xnr2 expression at Stage 9.5. All embryos were hemi-sectioned prior to hybridization through the animalegetal axis and dorsalentral axis, and oriented with the dorsal side to the right in the panel. Wild-type embryos were fixed at Stages 8, 8.5, 9, 9.5, 10, and 10.5 and examined by modified whole-mount in situ hybridization using probes for Xnr5 and Xnr2. Reproduced with permission of the Publisher, John Wiley & Sons.
|
|
FIG. 6. Expression pattern of genes downstream to Xnr5 in the deep mesendodermal region of embryos. All embryos were hemisectioned prior to hybridization through the animal-vegetal axis; dorsal side to right. Wild-type embryos were fixed at Stages 9 (a), 9.5 (f), 10 (k), 10.5 (p), and 11 (u) and examined by whole-mount in situ hybridization using probes specific for Xbra, Xwnt8, gsc, cerberus, and Xlefty/ Xatv. In the dorsal side, Xbra was detected in the most animal side of the mesoderm (f), gsc was localized more vegetally, including the endoderm region (h) and Xlefty/Xatv was detected between these two regions in the mesoderm (j). The expression of cerberus was localized more vegetally than gsc (i). In the ventral side, Xbra was localized to the most animal side of the marginal zone, similar to the dorsal side (k), Xwnt8 was detected more vegetally (l), and Xlefty/Xatv was expressed between Xbra and Xwnt8 (t,y). Note that all of these mesendodermal markers were detected in the inner mesendodermal layers and not in the superficial layer. (z-1) Double staining showed relationship of expressing domains. FITC-labeled Xbra and gsc probes were detected in emerald green. DIG-labeled gsc and Xlefty/Xatv probes were detected in brown. Right panels are higher magnification of left panels. All albino embryos were oriented with dorsal side facing the reader. (z-2) Xnr5-induced downstream genes in animal cap explants. Xnr5 dose-dependently induced downstream genes. Xbra was induced by a low amount of Xnr5 mRNA (0.05 pg), and Xlefty/Xatv was weakly induced by moderate doses. gsc, Xnr2, chordin, and cerberus were only induced by a higher amount of Xnr5 mRNA (1 pg). Sibling embryos (WE, Stage 10.5) served as positive control. Reverse- transcriptase-negative samples (WE-) showed absence of genomic DNA contamination. Reproduced with permission of the Publisher, John Wiley & Sons.
|
|
FIG. 7. Xnr5 induces a negative feedback loop. (a) Xlefty/Xatv in- hibited Xnr-mediated induction of downstream genes. cmDer also inhibited this Xnr5 activity but only at RNA amounts 10- to 100-fold higher than those for Xlefty/Xatv. (b-v) Xnr5 and Xnr6 expression were enhanced or prolonged after the peak of the expression by overexpression of Xlefty/Xatv and CerS, whereas the expression of Xnr2, Xbra, gsc, cerberus, and Xwnt8 was inhibited. (b,e,h,k,n, q,t) control uninjected embryos. (c,f,i,l,o,r,u) Xlefty/Xatv-overexp- ressing embryos. (d,g,j,m,p,s,v) CerS-overexpressing embryos. (bd) Xnr5 expression at Stage 9.75, (e) Xnr6 expression at Stage 9.75, (h) Xnr2 expression at Stage 9.75, (k) Xbra expression Stage at 10.5, (n) gsc expression at Stage 10.5, (q) cerberus exp- ression at Stage 10.5, (t,u) Xwnt8 expression at Stage 10.5. Reproduced with permission of the Publisher, John Wiley & Sons.
|