Journal: Molecular metabolism

Article Title: Inhibition of stearoyl-CoA desaturase 1 in the mouse impairs pancreatic islet morphogenesis and promotes loss of β-cell identity and α-cell expansion in the mature pancreas.

doi: 10.1016/j.molmet.2022.101659

Figure Lengend Snippet: Figure 2: Pancreatic islets in SCD1L/L mice exhibit alterations of the expression of TFs that are involved in maintaining a-cell and b-cell identity. mRNA levels of (A) b-cell-specific genes (Ins2, Pdx1, Nkx6.1, MafA), (B) a-cell-specific genes (Gcg, Arx, MafB), (C) both a-cell and b-cell signature genes (Isl1, Pax6, Nkx2.2, FoxO1, Neurod1, Ngn3), and dedifferentiation marker Sox9 in pancreatic islets from 10-week-old control (WT) and SCD1/ mice. (D) Immunoblot analysis of GCG, PDX1, FOXO1, NKX2.2, ISL1, PAX6, and ARX proteins in pancreatic islet lysates from WT and SCD1/ mice: representative immunoblots and quantitative, densitometric analysis of Western blot bands. b- actin was used as a loading control. The data are representative of n ¼ 6 mice/group. The data are expressed as mean SD. *p < 0.05, vs. WT. nd, not detected.

Article Snippet: The following primary antibodies were used for Western blot: PDX1 (catalog no. 5679, Cell Signaling, Hartsfordshire, UK), FOXO1 (catalog no. 2880, Cell Signaling), anti-Myc Taq (catalog no. 2272, Cell Signaling), NKX2.2 (catalog no. sc-398951, Santa Cruz Biotechnology, Santa Cruz, CA, USA), ISL1 (catalog no. sc-390793, Santa Cruz Biotechnology), PAX6 (catalog no. sc-81649, Santa Cruz Biotechnology), ARX (catalog no. sc-293449, Santa Cruz mbH.

Techniques: Expressing, Marker, Control, Western Blot

Journal: Molecular metabolism

Article Title: Inhibition of stearoyl-CoA desaturase 1 in the mouse impairs pancreatic islet morphogenesis and promotes loss of β-cell identity and α-cell expansion in the mature pancreas.

doi: 10.1016/j.molmet.2022.101659

Figure Lengend Snippet: Figure 3: SCD1 expression/activity determines the expression level of signature TFs in pancreatic INS-1E cells but does not affect the expression of signature TFs in pancreatic aTC1-6 cells. (A) Schematic design of the in vitro study of the effects of SCD1 inhibition/overexpression on the levels of a-cell- and b-cell-specific TFs. (BeD) mRNA levels of Arx (B) and Western blot analysis of the abundance of FOXO1 and PAX6 proteins (C, D) in aTC1-6 cells that were incubated with the SCD1 inhibitor (SCDi) and/or subsequently co-treated with 16:0. (EeG) Representative immunoblots that show levels of recombinant SCD1 protein fused with Myc-Taq (E) and content of FOXO1, PAX6, and NKX2.2 proteins (F, G) in aTC1-6 cells that transiently overexpressed SCD1. (H, J) Evaluation of PDX1, FOXO1, and ISL1 protein content in INS-1E cells that were treated with the SCD1 inhibitor and/or palmitic acid (16:0): (H) representative immunoblots, (J) quantitative, densitometric analysis of Western blot bands. *p < 0.05, vs. vehicle; ^p < 0.05, vs. SCD1i. (I, K, L) Level of recombinant SCD1 protein fused with Myc-Taq (I), PDX1, FOXO1, NKX2.2, ISL1, and PAX6 proteins (K), and densitometric validation of the abundance of these proteins (L) in INS-1E cells that transiently overexpressed SCD1 (transfected with pCMV6/hSCD1 plasmid) and control INS-1E cells (transfected with empty vector pCMV6). *p < 0.05, vs. empty pCMV6. b-actin was used as a loading control. The data are expressed as mean SD. n ¼ 3. ns, nonsignificant.

Article Snippet: The following primary antibodies were used for Western blot: PDX1 (catalog no. 5679, Cell Signaling, Hartsfordshire, UK), FOXO1 (catalog no. 2880, Cell Signaling), anti-Myc Taq (catalog no. 2272, Cell Signaling), NKX2.2 (catalog no. sc-398951, Santa Cruz Biotechnology, Santa Cruz, CA, USA), ISL1 (catalog no. sc-390793, Santa Cruz Biotechnology), PAX6 (catalog no. sc-81649, Santa Cruz Biotechnology), ARX (catalog no. sc-293449, Santa Cruz mbH.

Techniques: Expressing, Activity Assay, In Vitro, Inhibition, Over Expression, Western Blot, Incubation, Recombinant, Biomarker Discovery, Transfection, Plasmid Preparation, Control

Journal: Development (Cambridge, England)

Article Title: A non-autonomous function of the core PCP protein VANGL2 directs peripheral axon turning in the developing cochlea

doi: 10.1242/dev.159012

Figure Lengend Snippet: Vangl2 acts non-autonomously to guide type II SGN peripheral axon turning. (A,A′) Cross-section of an Emx2-Cre; RosatdTomato cochlea at P2 shows Cre-mediated recombination in the apical (a), middle (m) and basal (b) turns of the cochlear duct and organ of Corti (arrows). Limited recombination overlaps with ISL1-immunopositive neurons of the SGN (arrowheads) in a gradient along the length of the cochlea and is more frequent in the apical turn. (B) Quantification of tdTomato activation in the SGN shows a significant decrease from cochlear apex to base. (C) Schematic representation of Emx2-Cre-mediated recombination in the cochlear base. (D,E) In situ hybridization demonstrates reduction of Vangl2 mRNA in the Vangl2 CKO organ of Corti (square brackets) but not in the SGN. (F,G) Turning errors (arrowheads) are frequent in the basal turn of Emx2-Cre; Vangl2 CKOs. (H) Quantification of turning errors throughout the total length of Vangl2 CKO (n=5) cochlea relative to littermate controls (n=4), and comparison of turning errors between type II SGNs innervating the base versus apex. (I,J) In Fgfr3-iCreER; RosatdTomato cochlea at E18.5, recombination is restricted to supporting cells in the organ of Corti following tamoxifen induction initiated at E14.5. Dashed outlines indicate relative position of each supporting cell type. (K) Schematic representation of Fgfr3-iCreER-mediated recombination in the organ of Corti. (L) Incorrectly turned type II fibers (arrowheads) are readily detected in Fgfr3-iCreER; Vangl2 CKOs. (M) Quantification of turning errors in the middle turn of the Fgfr3-iCreER; Vangl2 CKO cochlea (n=3) relative to littermate controls (n=3). Data are mean±s.e.m. Asterisks show significant differences between genotypes using Student's t-test (**P<0.01). Scale bars: 100 µm in A,A′,I; 50 µm in D,E; 10 µm in F,G,J,L.

Article Snippet: Commercial antibodies used in this study were: rat anti-E-cadherin (Invitrogen, 13-1900, 1:1000), rat anti-CD44 (BD Biosciences, 550538, 1:750), mouse anti-islet 1 (DSHB, 39.3F7, deposited by T. M. Jessell and S. Brenner-Morton, 1:100), mouse anti-myosin 7a (DSHB, 138-1 deposited by D. J. Orten, 1:100), rabbit anti-NF200 (Millipore, AB1989, 1:1500), rat anti-tdTOMATO (Kerafast, EST203, 1:2000) and goat anti-SOX2 (Santa-Cruz, sc17320, 1:100).

Techniques: Activation Assay, In Situ Hybridization

Journal: Scientific Reports

Article Title: Onecut-dependent Nkx6.2 transcription factor expression is required for proper formation and activity of spinal locomotor circuits

doi: 10.1038/s41598-020-57945-4

Figure Lengend Snippet: Validation of the conditional motor neuron-specific Oc -null mice. ( A ) YFP-positive cells in a whole Olig2-Cre/Rosa26-YFP/Oc1 Δ/− Oc2 Δ/− embryo at e10.5, viewed from right side. The dotted line delineates the embryo, YFP fluorescence is observed along the spinal cord and in the encephalon. Scale bar = 1000 μm. ( B ) Immunostaining for YFP and Isl1 on a transverse section of control embryo at e10.5. Differentiating Isl1-positive MNs contained YFP. YFP fluorescence was also detected in cells ventral to MNs, likely corresponding to V3 INs. ( C-F ) Immunostaining for Isl1, OC-1, OC-2 and OC-3 at e10.5 in control or conditional double-mutant ( Oc1 −/− Oc2 −/− ) embryos. OC factor expression was lost in Isl1-positive MNs and Isl1 expression was decreased in the most laterally situated MNs (arrows). Scale bars = 20 μm.

Article Snippet: Primary antibodies against the following proteins were used: BetaGal (chicken; 1:2000; Abcam #ab9361), Chx10 (sheep; 1:500; Exalpha Biologicals #X1179P), Er81 (rabbit; 1:10000; Covance), Foxp1 (goat; 1:1000; R&D Systems #AF4534), Gata3 (rat; 1:50; Absea Biotechnology #111214D02), GFP (chick; 1:1000; Aves Lab #GFP-1020), OC-1 (guinea pig; 1:2000 ; or rabbit; 1:100; Santa Cruz #sc-13050; or sheep; 1:1000 R&D Systems #AF6277), Isl1 (goat; 1:3000; Neuromics #GT15051), Lhx3 (mouse; 1:1000, DSHB #67.4E12), MafA (guinea pig; 1:500; kindly provided by T. Müller), cMaf (rabbit; 1:5000; kindly provided by H. Wende), Nkx6.1 (mouse; 1:2000; DSHB #F55A10), nNOS (rabbit, 1:4000; Immunostar #24287), OC-2 (rat; 1:400 ; or sheep; 1:500; R&D Systems #AF6294), OC-3 (guinea pig; 1:6000), Olig2 (rabbit; 1:2000; Millipore #AB9610), RALDH2 (rabbit; 1:10000) and Shox2 (mouse; 1:500; Abcam #AB55740).

Techniques: Fluorescence, Immunostaining, Mutagenesis, Expressing

Journal: Scientific Reports

Article Title: Onecut-dependent Nkx6.2 transcription factor expression is required for proper formation and activity of spinal locomotor circuits

doi: 10.1038/s41598-020-57945-4

Figure Lengend Snippet: OC factors inversely regulate the expression of Nkx6.2 in motor neurons and in ventral interneurons. (A-I) In situ hybridization for Nkx6.2 on transverse sections at brachial, thoracic and lumbar levels of control or Oc constitutive double-mutant spinal cord at e11.5 or of conditional (cdKO) Oc mutant spinal cord at e12.5. ( D,H,L ) shows a summary of Nkx6.2 expression (blue) in each genotype. ( A-D ) In control embryos, Nkx6.2 was expressed in p1 progenitors (arrowheads) and in ventral INs (black arrows) at all 3 levels, and in some MNs (grey arrows) at brachial and lumbar levels. ( E-H ) In the constitutive mutant, Nkx6.2 was present in the p1 domain, lost in ventral INs (asterisks), but expanded in the brachial and lumbar MNs (grey arrows). ( I-L ) In the conditional mutant, Nkx6.2 was expressed in the p1 progenitors (arrowheads) and in ventral INs (black arrows), and expanded in brachial and especially lumbar MNs (grey arrows). ( M-T ) To determine if this expansion of Nkx6.2 expression was specific to MNs, we combined in situ hybridization for Nkx6.2 (Q-T) with immunofluorescence labeling for Isl1 (U-X) and RALDH2 (Y-BB) on transverse spinal cord sections at the brachial level of control or constitutive mutant at e12.5. RALDH2 labels all the limb-innervating LMC MNs. The Nkx6.2 in situ hybridization signal coincides with, and masks, RALDH2 immunostaining (which defines LMC MNs), indicating that Nkx6.2 expression in the Oc mutant embryos expanded to all the LMC cells. Scale bars = 100 μm.

Article Snippet: Primary antibodies against the following proteins were used: BetaGal (chicken; 1:2000; Abcam #ab9361), Chx10 (sheep; 1:500; Exalpha Biologicals #X1179P), Er81 (rabbit; 1:10000; Covance), Foxp1 (goat; 1:1000; R&D Systems #AF4534), Gata3 (rat; 1:50; Absea Biotechnology #111214D02), GFP (chick; 1:1000; Aves Lab #GFP-1020), OC-1 (guinea pig; 1:2000 ; or rabbit; 1:100; Santa Cruz #sc-13050; or sheep; 1:1000 R&D Systems #AF6277), Isl1 (goat; 1:3000; Neuromics #GT15051), Lhx3 (mouse; 1:1000, DSHB #67.4E12), MafA (guinea pig; 1:500; kindly provided by T. Müller), cMaf (rabbit; 1:5000; kindly provided by H. Wende), Nkx6.1 (mouse; 1:2000; DSHB #F55A10), nNOS (rabbit, 1:4000; Immunostar #24287), OC-2 (rat; 1:400 ; or sheep; 1:500; R&D Systems #AF6294), OC-3 (guinea pig; 1:6000), Olig2 (rabbit; 1:2000; Millipore #AB9610), RALDH2 (rabbit; 1:10000) and Shox2 (mouse; 1:500; Abcam #AB55740).

Techniques: Expressing, In Situ Hybridization, Mutagenesis, Immunofluorescence, Labeling, Immunostaining

Journal: Scientific Reports

Article Title: Onecut-dependent Nkx6.2 transcription factor expression is required for proper formation and activity of spinal locomotor circuits

doi: 10.1038/s41598-020-57945-4

Figure Lengend Snippet: Nkx6.2 is required for the maintenance of LMCl motor neurons. Immunostaining of transverse spinal cord sections of Nkx6.2 heterozygous control or Nkx6.2 -null mutant embryos at e12.5 ( A–J ) or e14.5 ( K–T ). ( A–J ) In the heterozygous control embryos at e12.5, β-galactosidase was absent from HMC (Isl1+), MMC (Isl1 + Lhx3 + ) and PGC (Isl1 + nNos + ) at thoracic level, but was detected in rare MNs of the LMCl (Foxp1 + Isl1-) at brachial level (arrow in C ) and in a greater number of LMCl cells at lumbar level (arrows in D ). In Nkx6.2 -null mutants, the β-galactosidase signal was observed in more LMCl MNs at brachial (arrows in G ) and at lumbar (arrows in H ) levels. The production of HMC, MMC and PGC MNs was not affected in the absence of Nkx6.2 . Similarly, no change was detected in the LMC at brachial or lumbar levels of the spinal cord. ( K-T ) At e14.5, β-galactosidase signal was only detected in some LMCl MNs at lumbar level (arrows in R ) in Nkx6.2 mutant mice. The production of HMC, MMC and PGC MNs at thoracic level was not changed by absence of Nkx6.2 . In contrast, the number of LMCl MNs at lumbar level was significantly decreased in the Nkx6.2 -null mice ( N,R,T ) with no perturbation of the LMC at brachial level and of the LMCm (Foxp1 + Isl1 + ) at lumbar level. n ≥ 3; * p < 0.05. Scale bar = 50 μm.

Article Snippet: Primary antibodies against the following proteins were used: BetaGal (chicken; 1:2000; Abcam #ab9361), Chx10 (sheep; 1:500; Exalpha Biologicals #X1179P), Er81 (rabbit; 1:10000; Covance), Foxp1 (goat; 1:1000; R&D Systems #AF4534), Gata3 (rat; 1:50; Absea Biotechnology #111214D02), GFP (chick; 1:1000; Aves Lab #GFP-1020), OC-1 (guinea pig; 1:2000 ; or rabbit; 1:100; Santa Cruz #sc-13050; or sheep; 1:1000 R&D Systems #AF6277), Isl1 (goat; 1:3000; Neuromics #GT15051), Lhx3 (mouse; 1:1000, DSHB #67.4E12), MafA (guinea pig; 1:500; kindly provided by T. Müller), cMaf (rabbit; 1:5000; kindly provided by H. Wende), Nkx6.1 (mouse; 1:2000; DSHB #F55A10), nNOS (rabbit, 1:4000; Immunostar #24287), OC-2 (rat; 1:400 ; or sheep; 1:500; R&D Systems #AF6294), OC-3 (guinea pig; 1:6000), Olig2 (rabbit; 1:2000; Millipore #AB9610), RALDH2 (rabbit; 1:10000) and Shox2 (mouse; 1:500; Abcam #AB55740).

Techniques: Immunostaining, Mutagenesis