Journal: bioRxiv

Article Title: KIF7 deletion in a mouse model of human ciliopathy alters cerebral cortex development

doi: 10.1101/2024.03.21.586159

Figure Lengend Snippet: Expression of Shh mRNA and SHH-Nter protein in the developing cortex. Expression of Shh transcripts detected by in situ hybridization and RNAscope in median (A) and caudal (B) E14.5 brain sections. In situ hybridization of forebrain sections with an antisens Shh probes shows strong expression of Shh mRNA in the ventral forebrain [MGE and MGE/LGE/septum boundary (A1), CGE (B1)], in the zona limitans (ZLI) of dorsal thalamus and the border of the 3 rd ventricle (B1). RNAscop of forebrain sections further confirmed the strong expression of Shh mRNA in the MGE/septum boundary that express Lhx-6 mRNA (a transcription factor expressed in post-mitotic interneurons in the ventral brain) (A2) and the CGE (B2). At higher magnification, Shh mRNA fluorescence (in green) labels a subpopulation of interneuron cell bodies (in red) in the MGE/septum boundary, in the subventricular zone of the MGE and in more dispersed cells in the MGE mantle (A2) and in the lateral and dorsal cortex (arrows, C1 and C2). Among Shh mRNA expressing cells in the cortex, some are principal glutamatergic cells (arrowhead, C1) and only few cIN express the transcript (C3). The graph represents the mean ± SEM in the IZ-CP layers and MZ layer. Expression of SHH-Nter protein in E14.5 median brain sections (D). Representative images are captured with epifluorecence (D1) and confocal microscop (D2). Confocal images are a merged stacks of 10 images and enlargment of pictures are shown. SHH expression is detected in blood vessels and in brigth dots all over the cortical neuropile. At the PSB and in the lateral part of the LGE, SHH-Nter is detectable in brigth elements aligned along the ventricular/subventricular axis. CX, cortex ; LGE, MGE and CGE, lateral, medial and caudal ganglionic eminence, V3, third ventricle; ZLI, zona intra-thalamica; Th, thalamus; Hyp, hypothalamus; PSB, palium/subpallium boundary; VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate; MZ, marginal zone. Scale bar: 500 µm (A, B, D1), 20 µm (C), 50 µm (D2).

Article Snippet: Primary antibodies were goat anti SHH-Nter (1:100, R&D system AF464), goat anti Netrin G1a (NG1a) (1:100, R&D system AF1166), rabbit anti TBR1 (1:1000, Abcam ab31940), rabbit anti TBR2 (1:1000, Abcam ab23345), rabbit anti PAX6 (1:100, clone poly19013, Covance PRB-278P), rabbit anti GSH2 (1:2000, Millipore ABN162), chicken anti MAP2 (1:500, Novus, NB30213), and rat CTIP2 (1:1000, Abcam ab18465).

Techniques: Expressing, In Situ Hybridization, RNAscope, Fluorescence

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Placental sonic hedgehog pathway regulates foetal growth via insulin-like growth factor axis in preeclampsia.

doi: 10.1210/jc.2019-00335

Figure Lengend Snippet: Figure 3. SHH pathway and trophoblast syncytialization. (A) Downregulation of the SHH pathway in primary CTBs. Cyclopamine decreased the expression of PTCH1 (left) and GLI2 (middle) at mRNA (n 5 6) and protein levels (n 5 3, right). (B) Cyclopamine-induced alteration of syncytium markers (n 5 6). (C) Fluorescent immunocytochemistry of CTBs treated with rSHH (200 ng/mL, 48 h, n 5 4) and cyclopamine (5 mM, 48 h, n 5 4). Data are presented as mean 6 SEM. Scale bars, 50mm. Representative images are shown in (C).

Article Snippet: Cyclopamine (5 mM, catalog no. sc-200929, Santa Cruz Biotechnology), an SMO inhibitor, and/or recombinant SHH (rSHH) protein (200 ng/mL, catalog no. 1314-SH, R&D Systems, Minneapolis, MN) were used to examine the potential roles of the SHH pathway in placental development.

Techniques: Expressing, Immunocytochemistry

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Placental sonic hedgehog pathway regulates foetal growth via insulin-like growth factor axis in preeclampsia.

doi: 10.1210/jc.2019-00335

Figure Lengend Snippet: Figure 5. Noncanonical SHH pathway and trophoblast syncytialization. (A) mRNA expressions of syncytium markers and b-hCG protein levels in primary CTBs treated with forskolin, an ADCY activator (20 mM, n 5 6). (B) Western blot analysis of CREB phosphorylation in CTBs treated with forskolin (20 mM, n 5 3), rSHH (200 ng/mL, n 5 3), and cyclopamine (5 mM, n 5 3). (C) Expressions of ADCY family in CTBs treated with cyclopamine (5 mM, 48 h, n 5 6). (D) Fluorescent immunocytochemistry of CTBs treated with forskolin (20 mM, 48 h, n 5 4). Data are presented as mean 6SEM. Scale bars, 50mm. Representative images are shown in (B) and (D). *P , 0.05, **P , 0.01, ***P , 0.001. NS, not significant.

Article Snippet: Cyclopamine (5 mM, catalog no. sc-200929, Santa Cruz Biotechnology), an SMO inhibitor, and/or recombinant SHH (rSHH) protein (200 ng/mL, catalog no. 1314-SH, R&D Systems, Minneapolis, MN) were used to examine the potential roles of the SHH pathway in placental development.

Techniques: Western Blot, Phospho-proteomics, Immunocytochemistry

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Placental sonic hedgehog pathway regulates foetal growth via insulin-like growth factor axis in preeclampsia.

doi: 10.1210/jc.2019-00335

Figure Lengend Snippet: Figure 6. Canonical and noncanonical SHH pathway in regulation of the placental IGF1R pathway. (A) Close correlation between the SHH pathway and the IGF axis in control and preeclampsia placentas (n 5 10 in each group). (B–D) Expressions of major components of the IGF axis in CTBs treated with (B) forskolin (20 mM, 24 h, n 5 5), (C) cyclopamine (5 mM, 24 h, n 5 6), and (D) GLI2 siRNAs (48 h, n 5 6). (E) Western blot analysis of IGF1R expression in CTBs (48 h, n 5 3) treated with forskolin (20 mM), cyclopamine (5 mM), GLI2 siRNAs, rSHH (200 ng/mL), and SQ22536 (100 mM). (F) Pathway analysis of microarray data demonstrating that the SHH pathway was closely associated with ADCY activation pathway, CREB phosphorylation pathway, and IGF1R pathway. (G) Schema of the SHH pathway that regulates the IGF1R pathway in the placenta. Data are presented as mean 6 SEM. Representative images are shown in (E). NS, not significant.

Article Snippet: Cyclopamine (5 mM, catalog no. sc-200929, Santa Cruz Biotechnology), an SMO inhibitor, and/or recombinant SHH (rSHH) protein (200 ng/mL, catalog no. 1314-SH, R&D Systems, Minneapolis, MN) were used to examine the potential roles of the SHH pathway in placental development.

Techniques: Control, Western Blot, Expressing, Microarray, Activation Assay, Phospho-proteomics