Downregulation of ADAM10 inhibits cell proliferation in the ventral spinal cord.

<p>After electroporation at E4, transverse sections were used for detection at E6. The transfected cells are marked by green fluorescence, and the control morpholinos (Fluo-m) and the untrasfected side (left side) serve as controls. The immune reactive cells are stained by red. Cell nuclei are labeled by DAPI (blue). (A–X) Immunohistochemistry was performed in adjacent sections with antibodies against BrdU (A–D), Pax6 (E–L), NKx6.1 (M–P), Pax7 (Q–T), NKx2.2 (U–X), respectively. Arrows in (A–D) indicate decrease of BrdU-labeled cells; Arrows in (I–O) indicate the immune negative region within the transfected side. (Y) Quantitative data of the cell number in different domains normalized by the number of the control side, which is set to be 1. All data are presented as mean ± SEM from at least 3 independent experiments (**<i>p</i><0.01, and ***<i>p</i><0.001 compared to the control). Scale bar, 200 µm in (I) for (A–W).</p

ADAM10 Negatively Regulates Neuronal Differentiation during Spinal Cord Development

<div><p>Members of the ADAM (a disintegrin and metalloprotease) family are involved in embryogenesis and tissue formation via their proteolytic function, cell-cell and cell-matrix interactions. ADAM10 is expressed temporally and spatially in the developing chicken spinal cord, but its function remains elusive. In the present study, we address this question by electroporating ADAM10 specific morpholino antisense oligonucleotides (ADAM10-mo) or dominant-negative ADAM10 (dn-ADAM10) plasmid into the developing chicken spinal cord as well as by in vitro cell culture investigation. Our results show that downregulation of ADAM10 drives precocious differentiation of neural progenitor cells and radial glial cells, resulting in an increase of neurons in the developing spinal cord, even in the prospective ventricular zone. Remarkably, overexpression of the dn-ADAM10 plasmid mutated in the metalloprotease domain (dn-ADAM10-me) mimics the phenotype as found by the ADAM10-mo transfection. Furthermore, in vitro experiments on cultured cells demonstrate that downregulation of ADAM10 decreases the amount of the cleaved intracellular part of Notch1 receptor and its target, and increases the number of βIII-tubulin-positive cells during neural progenitor cell differentiation. Taken together, our data suggest that ADAM10 negatively regulates neuronal differentiation, possibly via its proteolytic effect on the Notch signaling during development of the spinal cord.</p></div

Electroporated ADAM10 morpholinos induce donwregulation of endogenous ADAM10 protein during development of the spinal cord.

<p>After electroporation at E4, the embryos were collected and transverse sections were cut at E6. The transfected cells are marked by green fluorescence. The control morpholinos and the untransfected side of the spinal cord serve as controls. The immune reactive cells are stained red. Cell nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI) as blue. (A–H) The expression of ADAM10 protein in the spinal cord transfected with control morpholinos (Fluo-m; A–D) or ADAM10 morpholinos (AM10-m; E–H). Arrows indicate transfected regions in the ventricular zone, and arrowheads in the mental layer. (I–L) Apoptotic cells (red, arrowheads) measured by TUNEL assay. (M, N) Representative Western blots (M) and semi-quantitative Western blot analysis (N) of ADAM10 protein, including a pre-mature (pADAM10) and a mature (mADAM10) form, in the transfected (tran) and untransfected side (untran) of ADAM10-morpholinos (AM10-m) or control morpholinos (Fluo-m) transfected embryos. GAPDH is used as a loading control. The amount of ADAM10 protein is normalized by the number of the control side, which is set to be 1. All data are presented as mean ± SEM from at least 3 independent samples (**<i>p</i><0.01 compared to control). Scale bar, 200 µm in (A) for (A–L).</p

Downregulation of ADAM10 drives differentiation of radial glial cells in the ventral spinal cord.

<p>After electroporation at E4, transverse sections were used for detection at E6. The transfected cells are marked by green fluorescence and the control morpholinos (Fluo-m) and the untransfected side (left side) serve as controls. The immune reactive cells are stained by red. Cell nuclei are labeled by DAPI (blue). Immunohistochemistry was performed in adjacent sections with antibodies against Nestin (A–H) and vimentin (3CB2; I–M), respectively. Arrows in A–D indicate transfected regions (green) and in E–K the inhibition of nestin (E–H) and vimentin (I–K) in the transfected region. Noted that no change of viementin expression in the transfected region above the floor plate is found (arrows in L, M). Scale bar, 200 µm in (A) for (A–K).</p

Overexpression of a dominant-negative ADAM10 mutated in the metalloprotease domain (dn-ADAM10-me) promotes neuronal differentiation in the developing spinal cord.

<p>After electroporation at E4, transverse sections at E6 were used for immunostaining. The dn-ADAM10-me (AM10-me) transfected cells are marked by green fluorescence and pCAGGS-GFP (pCA-GFP) transfection and untransfected side (left side) serve as controls. The immune reactive cells are stained by red color. Cell nuclei are labeled with DAPI (blue). (A–C) Representative Western blots (A) and semi-quantitative Western blot analyses (B, C) of ADAM10 protein, including a pre-mature (pADAM10) and a mature (mADAM10) form, and the cleaved Notch1 (cle Notch1) in the transfected (tran) and untransfected side (untran) of pCAGGS-ADAM10 (pCA-AM10) or dn-ADAM10-me (AM10-me) transfected embryos. GAPDH is used as a loading control. The amount of ADAM10 and cle Notch1 protein is normalized by the number of the control side, which is set to be 1. All data are presented as mean ± SEM from at least 3 independent samples (*<i>p</i><0.05, **<i>p</i><0.01 compared to control). (D–G) Apoptotic cells (red, arrowheads) measured by TUNEL assay. (H–A’) Immunostaining using antibodies against NeuN (H–K), MNR2 (L–S), and NKx6.1 (T–A’), respectively. Arrows in (H–K) and (P–S) indicate ectopic immune reaction in the prospective ventricular zone of the transfected region (green); in (L–O) and (T–W) no change in the transfected region (green); in (X–A’) decrease of endogenous NKx6.1 expression. Scale bar, 200 µm in (D) for (D–A’).</p

Inhibition of ADAM10 affects Notch targeted Hes5 and Gli genes in the ventral spinal cord (A–H) and in vitro cultured cell system (I–M).

<p>(A–H) After electroporation at E4, transverse section at E6 was used for in situ hybridization. The ADAM10-mo transfected cells are green and the left side serves as a control. In situ hybridization using the antisense probes for Hes5 (B), Hes1 (C), Gli1 (F), Gli2 (G), and Gli3 (H) are labeled purple and cell nuclei are blue. Arrows indicate the downregulation of mRNA expression (purple color) in the transfected region (green). (I) Western blot analysis reveals that premature ADAM10 (pADAM10), the cleaved Notch1 (NICD), and Hes5 proteins are decreased in the ADAM10 siRNA transfected human neural progenitor cells at differentiation day 3, when compared to the control group (n = 2). GAPDH was used as a loading control. (J–L) Three days after differentiation, cells were stained against βIII-tubulin using antibody Tuj1 (green) in control siRNA (J) and ADAM10 siRNA transfected cells (K). Cell nuclei are labeled with DAPI (blue). Quantitative analyses reveal that the number of Tuj1-positive cells (L) significantly increase in the ADAM10 siRNA transfected cells compared to the control group (*<i>p</i><0.05). Abbreviations: cle Notch1, cleaved Notch1. Scale bars, 200 µm in (A) for (A–H); 100 µm in (J) for (J, K).</p

Downregulation of ADAM10 expression promotes neuronal differentiation in the developing spinal cord.

<p>Two days after electroporation, the embryos were collected and transverse sections cut at E4 or E6. The transfected cells are marked by green fluorescence. The control morpholinos and the untransfected side of the spinal cord serve as controls. The immune reactive cells are stained red. Cell nuclei are labeled blue with 4′,6-diamidino-2-phenylindole (DAPI). (A–P) NeuN immunostaining (red) in sections transfected with control morpholinos (Fluo-m; A–D, I–L) and ADAM10 morpholinos (AM10-m; E–H, M–P) at E2 (A–H) or E4 (I–P), respectively. Arrows indicate the transfected region in the ventricular zone of the basal plate, arrowheads in (G) of the alar plate and arrowheads in (M–O) above the floor plate. Scale bar, 200 µm in (A) for (A–P).</p