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The ADAM family of transmembrane metalloproteinases has important functions in fertilization, development and disease, and is widely distributed throughout the Metazoa. In this study, we identified a novel ADAM protein in Xenopus tropicalis (X. tropicalis) with closest overall sequence similarity to the Xenopus ADAM10 protein. Based on comparisons of available sequence information, putative orthologs of this ADAM (which we designate ADAM41) are identified in several other vertebrate species including non-placental mammals, but absent from placental mammals and aves. ADAM41 mRNA is maternally deposited in X. tropicalis with subsequent zygotic expression detected in the neural plate at neurula stages. Antisense morpholino knockdown of ADAM41 results in a delay in early neuronal marker expression, which can be rescued by a non-targeted ADAM41 transcript. Thus, ADAM41 is likely required for maintaining proper timing of neurogenesis in X. tropicalis.
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22811267
???displayArticle.pmcLink???PMC3695402 ???displayArticle.link???Int J Dev Biol ???displayArticle.grants???[+]
Fig. 3. Whole-mount in situ hybridization for adam41. Sense (S) and antisense (AS) transcripts of adam41 were used as probes for whole-mount in situ hybridization to detect the expressions of adam41 messenger at different developmental stages indicated. (A,G) Animal pole view; (B,C,H,I) dorsal view; (D,J) anterior-dorsal view; (E,F,K,L) side view. Arrowheads point to the blastopore (BP).
Fig. 4. Inhibition of ADAM41 translation by a specific antisense morpholino. (A) A morpholino (MO) was designed to target underlined sequence covering the translational start site (indicated in red) in adam41 mRNA. (B) ADAM41 morpholinos blocked the translation of myc-tagged exogenous ADAM41 mRNA. The indicated morpholinos were co-injected with exogenous myc-tagged adam41 mRNA into X. tropicalis embryos at one-cell stage, and Western blot was carried out using an anti-myc antibody. Each lane contains 10 embryo equivalent of total protein.
Fig. 5. In situ hybridization of neural markers in normal X. tropicalis embryos. (A) Neural induction in Xenopus involves multiple events. Early neural induction signals activate proneural genes that specify neural fate of ectodermal cells. Ngnr-1 is a strongly expressed proneural gene in X. tropicalis. Proneural genes coordinate with neurogenic genes, such as notch/delta, to define neuronal pattern. (B-E) Dorsal view of X. tropicalis embryos demonstrating primary neuron patterning as seen by whole-mount in situ hybridization for the neuron marker N-tubulin. Three stripes of neurons, medial neurons (m), intermediate neurons (i), and lateral neurons (l), form in parallel to each other (arrows in B and C) on both sides of dorsal midline axis. Arrowhead in (B) points to the trigeminal ganglion (TG) in the anterior neural plate of the embryo. (F-J) Whole-mount in situ hybridization for the proneural gene ngnr-1 in X. tropicalis embryos at different stages. (F) Ngnr-1 message is first expressed dorsally as three stripes on each side of the midline at st.11.5 (arrows denote dorsal ngnr-1 expression, and star indicates blastopore surrounding circular yolk plug). At this stage ngnr-1 is also expressed in the anterior region (G, anterior view of embryo with dorsal side at the top). The dorsal stripes elongated and migrated toward the midline
Fig. 6. Knockdown of ADAM41 caused a developmental delay at neurula stages. Embryos were injected in one cell at 2-cell stage with the indicated morpholinos. Injected side is marked by yellow dot in each panel, and dotted yellow lines indicate midline position in (A-E). (A-E) Injection of ADAM41 morpholino resulted in a delay of ngnr-1 expression pattern in the trigeminal ganglia at late gastrula stages. The side injected with ADAM41 morpholino had reduced ngnr-1 expression while the uninjected side had normal ngnr-1 expression pattern at st.12 (A,B are different views of the same embryo). The ADAM41 morpholino injected side started to express ngnr-1 at st.12.5 (C) (anterior view). By stage 13, expression of ngnr-1 in the trigeminal ganglion at the uninjected side had significantly decreased (D,E are different views of the same embryo). In contrast, ngnr-1 was more pronounced in the ADAM41 morpholino injected side compared with uninjected side at st. 13; see arrowheads in (E). (F-M) Effects of ADAM41 knockdown on the expression of ngnr-1 and N-tubulin at st. 13. Control morpholino did not alter the early patterns of primary neurons (G,I) or anterior neural placodes (F,H). ADAM41 knockdown resulted in an enlarged expression domain of ngnr-1 as the embryos developed (I, arrow). Anterior ngnr-1 expression decreased and was restricted to neural placodes including the trigeminal ganglion (TG) in st.12/13 embryos (arrowheads in H and J; embryo in H is tilted to the left in order to give a better view of anterior right side TG staining). The yellow dashed line defines the midline of each embryo.
Fig. 6. Knockdown of ADAM41 caused a developmental delay at neurula stages. Embryos were injected in one cell at 2-cell stage with the indicated morpholinos. Injected side is marked by yellow dot in each panel, and dotted yellow lines indicate midline position in (A-E). (A-E) Injection of ADAM41 morpholino resulted in a delay of ngnr-1 expression pattern in the trigeminal ganglia at late gastrula stages. The side injected with ADAM41 morpholino had reduced ngnr-1 expression while the uninjected side had normal ngnr-1 expression pattern at st.12 (A,B are different views of the same embryo). The ADAM41 morpholino injected side started to express ngnr-1 at st.12.5 (C) (anterior view). By stage 13, expression of ngnr-1 in the trigeminal ganglion at the uninjected side had significantly decreased (D,E are different views of the same embryo). In contrast, ngnr-1 was more pronounced in the ADAM41 morpholino injected side compared with uninjected side at st. 13; see arrowheads in (E). (F-M) Effects of ADAM41 knockdown on the expression of ngnr-1 and N-tubulin at st. 13. Control morpholino did not alter the early patterns of primary neurons (G,I) or anterior neural placodes (F,H). ADAM41 knockdown resulted in an enlarged expression domain of ngnr-1 as the embryos developed (I, arrow). Anterior ngnr-1 expression decreased and was restricted to neural placodes including the trigeminal ganglion (TG) in st.12/13 embryos (arrowheads in H and J; embryo in H is tilted to the left in order to give a better view of anterior right side TG staining). The yellow dashed line defines the midline of each embryo.
B
A(J) and decreased N-tubulin staining in trigeminal ganglion (L), typical of earlier stage control or uninjected embryos[SW1]. The distance between the lateral neuronal stripe and midline increased compared to the uninjected side (compare arrows in M with those in I).
Fig. 7. Developmental delay phenotype caused by knockdown of ADAM41 was rescued by an exogenous ADAM41 transcript. (A-D) Co-injection of the ADAM41 rescue transcript into one cell of 2-cell stage embryos restored ngnr-1 and N-tubulin expression in trigeminal ganglia in ADAM41 morphants (B,D). Arrowheads point to the trigeminal ganglia, and the injected side is denoted with a yellow dot in each panel. (E) Summary of the effect of ADAM41 rescue transcript on the expanded ngnr-1 expression caused by ADAM41 morpholino. More than 80% of ADAM41 morphants displayed expanded ngnr-1 expression in the trigeminal ganglion. Co-injection of 200pg of ADAM41 rescue transcript reduced the number of abnormal embryos to 60%, and less than 20% embryos showed expanded ngnr-1 expression in the trigeminal ganglion when 400pg rescue transcript was used.
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