XB-ART-47667Int J Dev Biol January 1, 2013; 57 (9-10): 787-92.
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Kidins220 (Kinase D interacting substrate of 220 kDa)/ARMS (Ankyrin Repeat-rich Membrane Spanning) is a conserved scaffold protein that acts as a downstream substrate for protein kinase D and mediates multiple receptor signalling pathways. Despite the dissecting of the function of this protein in mammals, using both in vitro and in vivo studies, a detailed characterization of its gene expression during early phases of embryogenesis has not been described yet. Here, we have used Xenopus laevis as a vertebrate model system to analyze the gene expression and the protein localization of Kidins220/ARMS. We found its expression was dynamically regulated during development. Kidins220/ARMS mRNA was expressed from neurula to larval stage in different embryonic regions including the nervous system, eye, branchial arches, heart and somites. Similar to the transcript, the protein was present in multiple embryonic domains including the central nervous system, cranial nerves, motor nerves, intersomitic junctions, retinal ganglion cells, lens, otic vesicle, heart and branchial arches. In particular, in some regions such as the retina and somites, the protein displayed a differential localization pattern in stage 42 embryos when compared to the earlier examined stages. Taken together our results suggest that this multidomain protein is involved in distinct spatio-temporal differentiative events.
PubMed ID: 24307304
Article link: Int J Dev Biol
Species referenced: Xenopus laevis
Genes referenced: ank1 kidins220 myos tub tuba4b
Antibodies: GFAP Ab2 Kidins220 Ab1 Somite Ab1 Tuba4b Ab4
Article Images: [+] show captions
|Fig. 1. Spatio-temporal expression pattern of kidins220 mRNA in Xenopus laevis embryos. The developmental stages (st.) are indicated in each panel (A,B) Initial neural tube stage embryos (st. 19), (A) frontal view, dorsal to the top and (B) dorsal view, anterior to the bottom, respectively. (C) Early tail bud stage (st. 22) embryo showing kidins220 expression at the level of the head and in somites. In (A) and (C) the square brackets indicate the forming somites). (D) st. 27 embryo in which the labeled ophtalmic and maxillomandibular branches of the primordium of the trigeminal nerve are highlighted by white and light blue arrowheads, respectively. (E) Magnified view of st.33-34 embryo head, showing signal in forebrain (f), midbrain (m) hindbrain (h), eye vesicle (e), branchial arches (ba). Magnification of the somite region in stage 33-34 embryos, hybridized with Kidins220 (F) and mastr, respectively (G). The square brackets indicate individual somites. (H) Anterior portion of st. 35-36 embryo showing signal in the heart region (red arrowhead) and in otic vesicle (yellow arrowhead). (I) Magnification of somites and dorsal fin of stage 37-38 hybridized embryos. (C-I) Lateral views, anterior to the left, dorsal to the top. (J) Transverse microtome section of hybridized st. 37-38 embryo showing a specific signal in retinal ganglion cells (rgc) and in lens (l). Scale bars: (A-I) 250 um; (J) 50 um.|
|Fig. 2. Kidins220 localization pattern in somites and spinal neurons of DAB-stained embryos. Lateral views, anterior to the left, dorsal to the top (A-D). Sagittal section of a Kidins220-immunostained embryo (A) and Hoechst-counterstaining (B). The red square brackets indicate paraxial mesoderm. Whole-mount ab34790 anti-Kidins220-immunostaining (C) and Ab12101 anti muscle-immunostaining (D). Coronal sections of Kidins220 (Hoechst-counterstained) and Ab12101-stained embryos, respectively (E,F). The arrows in (E) show the Kidins220 signal in lateral regions of spinal cord. High magnification of a Kidins220-positive intersomitic junction (G). Transverse sections of Kidins220 and acetylated tubulin-immunostained embryos, respectively (H,I): the arrowheads show the exit point of a motor neuron that in (H) resulted broken because of sectioning. Sagittal section of a stage 42 embryo (J) and Hoechst-counterstaining (K). The arrowheads in (J) indicate motor neurons. The square brackets in (E,J,K) span somite length. Acet. tub.= acetylated tubulin; nt= neural tube; ij= intersomitic junction. Scale bars: (A,B,H,I) 50 um; (C,D) 250 um; (E,F) 25 um; (G,J,K) 10 um.|
|localFig. 3. Kidins220 expression in other different developmental regions of DAB-stained embryos. Coronal and transverse sections showed that Kidins220 was expressed in: hypothalamus and thalamus (A), trigeminal nerve (V) (B), the mandibular branch (arrow) of trigeminal nerve innervating the cement gland (C), otic vesicle (D), branchial arches (E), VII, IX, X cranial nerves (F), heart (G). Lateral view of the anterior portion of a whole-mount Kidins220-immunolabeled stage 42 embryo (H). Sagittal sections showing a view of head (I) and abdomen (J). (H-J) Anterior to the left, dorsal to the top. The red arrowheads in (H,J) indicate Kidins220-immunopositive cells around the intestine. The black arrowheads in (I) show the Kidins220 signal in the edges of a cranial muscle. The black arrowheads in (J) indicate a Kidins220-immunopositive cell localized in intestine wall. ba, branchial arches; cg, cement gland; e, eye; he, heart; hyp, hypothalamus; ov, otic vesicle; pit, pituitary; thal, thalamus; st, stomodeum. Scale bars: (A,B,D,G) 25 um; (C) 100 um; (E,F,I,J) 50 um; (H) 250 um.|
|Fig. 4. Whole mount immunohistochemistry experiments on stage 42 embryos. Lateral view of a posterior portion of DAB-stained embryo for Kidins220 (A). Parasagittal section performed laterally between the skin and the somites of an immunostained embryo (B) and Hoechst-counterstaining (C). Dashed lines in (A,B) highlight the pattern of the Kidins220 signal, organized in irregularly parallel rows to the side of intersomitic junctions. The square brackets in (B,C) span somite length. Lateral view of somite region of whole-mount double immunofluorescent staining for Kidins220 (red signal) and acetylated tubulin (green signal) (D-F). Magnified views of neurons in somite region (G-I), dorsal fin (J-L; M-O), abdominal region (P-R). The largest dashed box in (D) indicates the region magnified in (G-I); the smallest one, the region magnified in (J-L). White arrowheads in (G,I,J,L,M,O) highlight the spotted signal of kidins220 at some neural fiber. White arrowheads in (M,O) show specific cells in dorsal fin, positive for both Kidins220 and GFAP. The asterisks in (E,H,K) denote the acetylated tubulin-positive but Kidins220-negative ciliated cells. Scale bars: (A) 125 um; (B-F,J-O) 50 um; (G-I,P-R) 10 um.|
|Fig. 5. Sagittal cryosections of stage 42 embryos double-labeled for Kidins220 (red) and acetylated tubulin (green) by immunofluorescent staining. Kidins220-positive signal was present in dorsal and ventral regions of the spinal cord as well as in motor neurons (arrows) (A) as shown by the acetylated tubulin staining (B,C). Magnification of motor neurons double-stained by Kidins220 (D), acetylated tubulin (E) and merged (F). The dashed bar in (D) spans the somite length. Kidins220 protein in eye of st. 33-34 DAB-stained embryos (G) and st. 42 embryos double-immunolabeled for Kidins220 and acetylated tubulin (H-J). The open arrowhead in (H,J) indicates the Kidins220 signal in the optic nerve. The dashed circle indicates the position of lens in the eye. The white arrow in (H,J) denotes the presence of Kidins220 signal in a facial muscle near the eye. The white arrowheads in (H,J) indicate Kidins220 and acetylated tubulin-positive nerve fibers present in epidermis. ep, epidermis; l, lens; rgc, retinal ganglion cell; s, somite; sc, spinal cord; *, ciliated cell. Scale bars: (A-C) 50 um; (D-F,G-J) 25 um.|
|kidins220 (kinase D-interacting substrate, 220kDa) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 35 & 36, lateral view, anterior left, dorsal up.|