tgfβr2  (Bioss)

Journal: EMBO Reports

Article Title: ADAMTS2 promotes radial migration by activating TGF-β signaling in the developing neocortex

doi: 10.1038/s44319-024-00174-x

Figure Lengend Snippet: ( A ) The plasmid construct used for luminescent imaging of TGF-β signaling. TGF-β-responsive elements were tandemly inserted into the upstream of Emerald Luc conjugated with the PEST sequence. ( B – D ) 2xTRE-Eluc-Pest plasmids were electroporated at E14.5 along with GFP-expression plasmids. The luminescence imaging was performed using cultured slices prepared at E16.5. Compared with the control ( B ), the luminescence signals were diminished when 50 μM RepSox was added ( C ), or Adamts2 si-RNA was co-electroporated ( D ). ( E ) Enlarged images revealed that a control migrating neuron transiently showed strong luminescence emission during the multipolar-to-bipolar transition from 12 to 18 h. Arrows indicate migrating neurons with positive luminescent signals. ( F ) Quantification of the luminescence signals ( N = 3 slices from three independent experiments). The data were taken 4.5 h after the start of imaging. ( G ) Overexpression of TGF-β2 rescues the migration impairment phenotype of the Adamts2 KO heterozygous mouse cortex. Plasmids expressing TGF-β2 or TGF-βRII under the NeuroD1 promoter were used to determine if they could rescue the phenotype of impaired radial migration in the Adamts2 KO heterozygous mouse embryonic cortex. The phenotype was rescued by overexpression of TGF-β2 but not by overexpression of TGF-βRII. In utero electroporation was performed at E14.5, and the brains were fixed at E17.5. Compared with heterozygous brains in which GFP was electroporated (HT-GFP), the proportion of neurons distributed in BIN 1 and 2 significantly increased in the TGF-β2 rescued brains (HT-TGFb2) ( N = 6 sections for each group. Three sections from two brains each were analyzed). A graph quantifying the migration status is shown at the bottom. ( H ) Hypothetical model for the functions of ADAMTS2. The migrating multipolar neurons transiently secrete ADAMTS2 around the bottom part of the SP layer, which cleaves TGF-β-related ECM proteins such as LTBP1 and versican. After these initial cleavages, active TGF-β- is released from the ECM, which initiates the activation process of TGF-β signaling, leading to the multipolar-to-bipolar transition and switching of the migration mode. Data information: The statistical significance for each pair was measured by unpaired, two-tailed t-tests (* p < 0.05; ** p < 0.01; *** p < 0.001). Scale bars: ( E ) 10 μm; ( G ) 50 μm.

Article Snippet: The primary antibodies used for immunostaining were mouse anti-neurocan (Oohira et al, ), rabbit anti-cleaved versican (ab19345, Abcam), rabbit anti-Fibrillin-2 (bs12166R, Bioss), rabbit anti-LTBP1 (ab78294, Abcam), chicken anti-GFP (ab13970, Abcam), rabbit anti-MAP2 (AB5622, Merck Millipore), mouse anti-TIMP2 (ab1828, Abcam), rabbit anti-pSmad3 (ab52903, Abcam), and rabbit anti-TGF-βRII (bs0117R, Bioss), mouse anti-CS monoclonal antibody, CS-56 (C8305, SIGMA), rabbit anti-Ki67 (Novocastra), and rat anti-Tbr2 (SIGMA), Nestin (SC-33677, Santa Cruz).

Techniques: Plasmid Preparation, Construct, Imaging, Sequencing, Expressing, Cell Culture, Control, Over Expression, Migration, In Utero, Electroporation, Activation Assay, Two Tailed Test

Journal: EMBO Reports

Article Title: ADAMTS2 promotes radial migration by activating TGF-β signaling in the developing neocortex

doi: 10.1038/s44319-024-00174-x

Figure Lengend Snippet: ( A – H ) Cortical sections from Lpar1-EGFP mice, in which SP neurons expressed EGFP, were immunostained with antibodies against TGF-β signaling-related proteins. Timp2 and p-Smad were expressed at the upper part and the lower part of the SP layer, respectively. ( I , I’ ) TGF-βRII immunoreactivities were localized at the SP layer and the upper part of the intermediate zone. ( J ) The expression of CTGF, a direct downstream target of TGF-β signaling, was down-regulated in the cerebral cortex of Adamts2 KO mice. The expression levels of CTGF were measured by Q-PCR using mRNAs isolated from the cerebral cortex ( N = 3 sections for each group: three different embryos (E18) were used for collecting cerebral cortex). All sections were E15.5. Data information: The statistical significance for each pair was measured by unpaired, two-tailed t-tests (* p < 0.05; ** p < 0.01; *** p < 0.001). Scale bars, 100 μm.

Article Snippet: The primary antibodies used for immunostaining were mouse anti-neurocan (Oohira et al, ), rabbit anti-cleaved versican (ab19345, Abcam), rabbit anti-Fibrillin-2 (bs12166R, Bioss), rabbit anti-LTBP1 (ab78294, Abcam), chicken anti-GFP (ab13970, Abcam), rabbit anti-MAP2 (AB5622, Merck Millipore), mouse anti-TIMP2 (ab1828, Abcam), rabbit anti-pSmad3 (ab52903, Abcam), and rabbit anti-TGF-βRII (bs0117R, Bioss), mouse anti-CS monoclonal antibody, CS-56 (C8305, SIGMA), rabbit anti-Ki67 (Novocastra), and rat anti-Tbr2 (SIGMA), Nestin (SC-33677, Santa Cruz).

Techniques: Expressing, Isolation, Two Tailed Test

Journal: EMBO Reports

Article Title: ADAMTS2 promotes radial migration by activating TGF-β signaling in the developing neocortex

doi: 10.1038/s44319-024-00174-x

Figure Lengend Snippet: ( A ) Overexpression of TGF-βRII in the migrating neurons impaired radial neuronal migration ( N = 8 sections for each group; two fetuses from two mother mice were collected, and we used two sections from each brain for quantification. Control and overexpression were counted in pairs on the same litter). ( B ) Inhibitors of TGF-β receptor (50 μM RepSox and 1 μM LDN212854) disturbed radial neuronal migration in the cultured slices ( N = 3 slices from three independent experiments). A graph quantifying the migration status of each experiment ( A , B ) is shown on the right side of each. ( C ) Selected images from the time-lapse recordings of F-actin dynamics shown in Movie (left). CAG-Lifeact and RFP plasmids were electroporated at E14.5. The brain slices were prepared at E16.5 and cultured in the presence or absence of 50 μM RepSox. The multipolar-to-bipolar transition was impaired in the presence of the inhibitor. Arrows indicate neurites. In control, cells became bipolar and had leading process, whereas those in the TGFβ inhibitor-treated group were observed to have multipolar neurites. Morphological analyses indicated that the multipolar-to-bipolar transition was impaired in the inhibitor-treated slices ( N = 4 imaging areas from two experiments) (right). Data information: The statistical significance for each pair was measured by unpaired, two-tailed t-tests (* p < 0.05; ** p < 0.01; *** p < 0.001). Scale bars: ( A , B ) 50 μm; ( C ) 10 μm.

Article Snippet: The primary antibodies used for immunostaining were mouse anti-neurocan (Oohira et al, ), rabbit anti-cleaved versican (ab19345, Abcam), rabbit anti-Fibrillin-2 (bs12166R, Bioss), rabbit anti-LTBP1 (ab78294, Abcam), chicken anti-GFP (ab13970, Abcam), rabbit anti-MAP2 (AB5622, Merck Millipore), mouse anti-TIMP2 (ab1828, Abcam), rabbit anti-pSmad3 (ab52903, Abcam), and rabbit anti-TGF-βRII (bs0117R, Bioss), mouse anti-CS monoclonal antibody, CS-56 (C8305, SIGMA), rabbit anti-Ki67 (Novocastra), and rat anti-Tbr2 (SIGMA), Nestin (SC-33677, Santa Cruz).

Techniques: Over Expression, Migration, Control, Cell Culture, Imaging, Two Tailed Test