Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Cilk1 Is Essential for Mesenchymal Cilia Maintenance and Epithelial-mesenchymal Crosstalk in Intestinal Villus Morphogenesis

doi: 10.1016/j.jcmgh.2025.101672

Figure Lengend Snippet: Cilk1 is essential for the maintenance of mesenchymal cilia during intestinal development. ( A ) Immunofluorescence analysis showing cilia distribution in both epithelial and mesenchymal cells during different stages of intestinal development. ( B ) Quantification of the percentage of ciliated cells in epithelial and mesenchymal compartments between Cilk1 WT and Cilk1 KO intestines (n = 3 per group). Scale bars: epithelial cilia, 5 μm (E12.5, E16.5 and E18.5) and 10 μm (E14.5); mesenchymal cilia, 5 μm (E12.5), 10 μm (E14.5), and 2 μm (E16.5 and E18.5). ( C ) Confocal microscope image of cilia in the intestine using the Arl13b-mCherry;Centrin2-EGFP transgenic mouse embryo, confirming the dynamic ciliogenesis in epithelial and mesenchymal cells. Scale bars: epithelial cilia, 5 μm (E12.5), 10 μm (E14.5), and 5 μm (E16.5); mesenchymal cilia, 10 μm (all stages). ( D ) Expression levels of ciliogenesis-related genes including transcription factors such as Foxj and Rfx families, quantified by qRT-PCR in epithelial and mesenchymal tissues from E16.5 intestines (n = 3 per group). Error bars represent SEM. Statistical significance is indicated by asterisks, with the following P values: ∗ P < .05, ∗∗ P < .01, ∗∗∗∗ P < .0001 (Student’s t -test).

Article Snippet: Arl13b-mCherry; Centrin2-EGFP double transgenic mice express mCherry in cilia and EGFP in centrosomes was used for staining primary cilia with centrosome (Tg(CAG-Arl13b/mCherry)1Kvand Tg(CAG-EGFP/CETN2)3-4Jgg/KvandJ, Jackson Laboratory, stock #027967).

Techniques: Immunofluorescence, Microscopy, Transgenic Assay, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Methods to assess neuronal primary cilia electrochemical signaling

doi: 10.1101/2025.04.01.646689

Figure Lengend Snippet: A primary cilia toolbox. Genetically encoded fluorescent reporters for identification, Ca 2+ concentration and GPCR signaling within primary cilia. Previously published expression constructs or transgenic animal models are listed with corresponding citations.

Article Snippet: Cilia-targeted identification reporters (ARL13B-EGFP; ARL13B-mCherry); Ca 2+ sensors (ARL13B-GFP-RGECO1; 5-HT6R-RGECO1) were custom synthesized (VectorBuilder) into the lentiviral cassettes (pLV[Exp]-puro-CMV) to generate lentivirus (10^8 titer) solutions encoding for each reporter or sensor.

Techniques: Concentration Assay, Expressing, Construct, Transgenic Assay

Journal: bioRxiv

Article Title: Methods to assess neuronal primary cilia electrochemical signaling

doi: 10.1101/2025.04.01.646689

Figure Lengend Snippet: On DIV 2-3, hippocampal WT cultured cells were transduced overnight with the lentivector ARL13B-GFP-RGECO1 (MOI= 1-2) and 5 days after transduction, ratiometric experiments were performed under a confocal microscope. Ciliated neurons were then filmed (1 frame/sec) before and after the application of ionomycin alone (1 μM) ( A ) or in a combination with Ca 2+ (10 μM) ( B ). ROIs were placed on both base and tip of the cilia. To verify the role of stored calcium in the influx to the primary cilia, thapsigargin (Thapsi, 2 μM, blue, n= 5) or DMSO (control, red, n= 5) treatments were performed for 3 min followed by the application of ionomycin together with Ca 2+ 10 μM. Interestingly, we observed a small increase in calcium influx in the cilia seconds after thapsigargin application. After 3 min, the sensor response to ionomycin together with Ca 2+ 10 μM in the cilia was smaller when compared to control group (DMSO) ( C ). When cells were permeabilized with α-hemolysin (α-HL, 10 μg/mL) for 3 min, the sensor also detected calcium changes in the primary cilia upon external application of 500 nM calcium (n= 9). Ionomycin 1 μM was used as control (n= 6) (D). Fluorescence intensity was calculated by the ratio of RGEGO1 (red)/GFP (green) and normalized by the fluorescence average of the initial 30 sec of filming.

Article Snippet: Cilia-targeted identification reporters (ARL13B-EGFP; ARL13B-mCherry); Ca 2+ sensors (ARL13B-GFP-RGECO1; 5-HT6R-RGECO1) were custom synthesized (VectorBuilder) into the lentiviral cassettes (pLV[Exp]-puro-CMV) to generate lentivirus (10^8 titer) solutions encoding for each reporter or sensor.

Techniques: Cell Culture, Transduction, Microscopy, Control, Fluorescence

Journal: bioRxiv

Article Title: Methods to assess neuronal primary cilia electrochemical signaling

doi: 10.1101/2025.04.01.646689

Figure Lengend Snippet: 5-HT6R-RGECO1 alone or in combination with the cilia cAMP sensor 5-HT6R-cADDis. Hippocampi was dissected from P0 Arl13B-EGFP tg ( A, B ) or WT ( C-F ) mice and cells were seeded in glass bottom dishes. On DIV 2-3, hippocampal cultured cells were transduced overnight with the lentivector 5-HT6R-RGECO1 (MOI= 1-2) and 5 days after transduction, ratiometric experiments were performed under a confocal microscope. For the 5-HT6R-cADDis, cells were transduced one day before the experiments. Ciliated neurons were then filmed (1 frame/sec) before and after the application of solutions containing ionomycin alone (1 μM) (n= 9) or with calcium (10 μM) (n= 9) ( B ), 5-HT6R agonist WAY 181187 (1 μM) (n= 5) ( D ), or with both WAY 181187 (1 μM) followed by ionomycin (1 μM) + Ca 2+ (10 μM) (n= 13) (F). Fluorescence intensity was calculated by the ratio of RGEGO1 (red)/GFP (green) and normalized by the F0 (fluorescence average of the initial 30 sec of filming) ( B ) or by the ΔF/F0 ( D, F ). Scale bar: 10 μm. Unpaired t test for B, * p< 0.05.

Article Snippet: Cilia-targeted identification reporters (ARL13B-EGFP; ARL13B-mCherry); Ca 2+ sensors (ARL13B-GFP-RGECO1; 5-HT6R-RGECO1) were custom synthesized (VectorBuilder) into the lentiviral cassettes (pLV[Exp]-puro-CMV) to generate lentivirus (10^8 titer) solutions encoding for each reporter or sensor.

Techniques: Cell Culture, Transduction, Microscopy, Fluorescence