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Wilson JM
,
Martinez-De Luna RI
,
Smith R
,
King MW
,
Mescher AL
,
Neff AW
,
Belecky-Adams TL
.
???displayArticle.abstract??? Ddx39, a DEAD-box RNA helicase, is a part of the homeostatic machinery that regulates the switch between cellular proliferation and differentiation. Ddx39 was shown to be differentially regulated in Xenopus laevis using a differential screen of mRNAs from regenerating limbs (King et al., 2003). Here, the expression patterns of Ddx39 in developing limb and nervous system are reported. Ddx39 was detected by RT-PCR in the Xenopus embryo, the earliest stage examined. Localization of the message by whole-mount in situ hybridization at stage 17 showed it to be localized primarily to the developing nervous system. Ddx39 was present in the ventricular region of the developing neural tube up to and including stage 48, and was also localized to the headmesenchyme, pharyngeal arches, and paraxial mesoderm. Strong label was also present in the developing limb buds at stages 48-55. Analysis of expression patterns in cryosections of the developing eye at stage 38 and 47 showed Ddx39 in the ciliary marginal zone (CMZ) adjacent to the neural retina and within the lensepithelium. Ddx39 was also present in the anterioreye during fibroblast growth factor 2 (FGF2)-mediated retinal regeneration. BrDU incorporation analyses and double-label studies with proliferating cell nuclear antigen showed that Ddx39 message was restricted to a subpopulation of proliferating cells in the developing and regenerating optic cup.
Fig. 4. Ddx39 expression was found in the developing limbs in st 48â 55. Expression was observed in the developing limb buds of stage 48, 50, 52, 53, and 55 tadpoles (Aâ E).
Fig. 5. Ddx39 is expressed in the ciliary marginal zone of the retina and ventricular zone of the neural tube. In situ hybridization was performed on coronal sections through stage 38 (E and F) and 48 (Câ F) optic cup and stage 48 neural tube (G and H). Lower magnification for each field is shown on the right of the figure. Boxed areas on the right side of the figure indicate the region shown at higher magnification on the left side of the figure. Ddx39 message was apparent in the lensepithelium and the CMZ of the optic cup at both stages examined. Embryos incubated with sense probe showed no apparent label (E and F). Magnification Bar in (A) =50â î¼m and applies to photomicrographs shown in (A), (C), (E), and (G). Magnification Bar in (B) =50â î¼m and applies to photomicrographs in (B), (D), (F), and (H).
Fig. 3. Ddx39 expression was detected in the developing neural plate, neural tube, branchial arches, and otic vesicls. WISH was performed on embryos from st 17 to 28. Photomicrographs of wholemounts and sections through the wholemounts for st 17 (A and B), 22 (C), 26 (D and E), and 28 (F and G) are shown. Ddx39 expression was restricted primarily to the developing nervous system by stage 17 (A). This pattern was confirmed by section analysis (B) which showed labeled neural plate (NP) and no expression in the ectoderm (Ec) and endoderm (En). The headmesoderm (Ms) appeared to be very lightly labeled. The neural tube, including the optic cups (OC), the branchial arches and otic vesicle were labeled at st 22 (C) and 26 (D). WISH using sense control probe showed only weak background levels of labeling (E). Stage 28 tadpoles showed similar labeling patterns to those obtained at earlier stages including expression in the facial mesenchyme and pharyngeal arches and higher levels of expression in the otic vesicle, optic cup, and neural tube proper (F and G). WISH and section analysis at st 38 showed Ddx39 to be expressed in the neural tube (NT), otic vesicles (OV) and headmesenchyme (Ms), and was absent in notochord (Nc), endoderm (En), and heart (HT, H,I). Abbreviations are as follows: NP; neural plate, Ec; ectoderm, En, endoderm, Ms; mesoderm, OV; otic vesicle, BA; branchial arches, Ph; pharynx. Magnification Bar in (B) =50 μM and is applicable to photomicrographs in (B), (G) and (I).
Fig. 6. Ddx39 expression is found at the anterior margin of regenerating retina. FGF2-soaked heparin-acrylic beads were inserted into retinectomized stage 53 tadpoles and the tadpoles were allowed to regenerate retina for 0 (A and B), 2 (C and D), 4, (E and F), 7 (G and H) and 12 days (I and J) after surgery and cryosections through the regenerating retina underwent in situ hybridization for Ddx39. (A and B) Immediately following retinectomy, a large gap in the anterior portion of the eye remains where the retina, CMZ, and lens were removed. No remaining retinal or CMZ cells were apparent. (C and D) Two days post-retinectomy, there were still no retinal or CMZ cells apparent. By this time, the anterior portion of the eye had closed. (E and F) By 4 days post-retinectomy, cells were apparent in the anterior half of retinectomized eyes in which FGF2 beads had been placed. Only the cells at the anterior-most edge of the progenitor population were expressing Ddx39 (arrows). (G and H) By 7 days post-retinectomy a good portion of eye was filled with retinal and CMZ progenitors. However, only cells at the anterior edge of the progenitor population expressed Ddx39 (arrows). (I and J) Twelve days post-retinectomy much of the retina appeared to be regenerated and layers were apparent. As was noted at pervious times, however, the Ddx39 expression was still apparent only at the anterior margins of regenerating retina. (K and L) The layered appearance of the regenerated tissue at Day 12 can be appreciated in sections stained with hematoxylin and eosin. L; lens, B; bead. Magnification Bar in (A) and (B) =100 lm. Magnification Bar in (A) is applicable to photomicrographs in (A), (C), (E), (G), (I), (K). Magnification Bar in (B) is applicable to photomicrographs in (B), (D), (F), (H), (J), (L).
Fig. 7. Ddx39 is expressed in a subpopulation of proliferating cells in the ciliary marginal zone (CMZ). Sections from tadpoles injected with BrDU were double-labeled for Ddx39 by in situ hybridization (A) and BrDU immunohistochemistry (C). DAPI staining is shown to highlight the nuclei of the CMZ and layers of the developing retina (B and F). An overlay of the Ddx39 and BrDU photomicrographs is shown in (D). Sections through stage 48 tadpoles (E) were double-labeled with Ddx39 in situ hybridization (E) and proliferating cell nuclear antigen (PCNA) (G). An overlay of photomicrographs shown in (E) and (G) is shown in (H). Sections through sections of 7 day post-retinectomized eyes were double-labeled for Ddx39 mRNA (I) and PCNA (K) and stained for DAPI (J). DAPI label of the section shows the regenerated CMZ as well as adjacent disorganized regenerating retina (J). An overlay of photomicrographs in (I) and (K) show that Ddx39 was found in the proliferating cells of the regenerating retina (L). Magnification Bar in (A) =50 lm and applies to photomicrographs in (A).
