Journal: Disease Models & Mechanisms

Article Title: Tfap2a -dependent changes in mouse facial morphology result in clefting that can be ameliorated by a reduction in Fgf8 gene dosage

doi: 10.1242/dmm.017616

Figure Lengend Snippet: Alterations in gene expression in Neo/Null mice. (A,B) Volcano plots from the (A) maxillary and (B) nasal microarrays. Orange shows all values called ‘above absent’, blue all values called ‘present’, with a P -value of 0.05 (adjusted) and a fold change >1.25. Positive fold change is increased expression in Neo/Null compared with Neo/Wt. (C) Fold change of Fgf pathway members in Neo/Null E10.5 nasal prominences compared with control embryos. (D–K) In situ hybridization for Fgf8 (D–G) and Dusp6 (H–K) expression in Neo/Wt (D,F,H,J) and Neo/Null (E,G,I,K) E10.5 embryos showing frontal (D,E,H,I) and lateral (F,G,J,K) views. Arrow in G shows increased Fgf8 expression at the lateral edge of nasal pit. (L,M) Fgf8 staining visualized by OPT in E10.5 Neo/Wt (L) and Neo/Null (M) samples. (N) qRT-PCR examination of Fgf8 and Wnt9b transcript levels from E10.5 facial prominences. * P <0.05.

Article Snippet: For real-time PCR, pre-validated probes and primers were used to examine Tfap2a levels targeting exons 2–3 and 6–7 (IDT PrimeTime assay Mm.PT.56a.1580695 and 12995135, Integrated DNA Technologies, Coralville, IA) as well as Fgf8 (TaqMan gene expression assay Mm00438922_m1, Applied Biosystems, San Francisco, CA) and Wnt9b (TaqMan gene expression assay Mm00457102_m1).

Techniques: Gene Expression, Expressing, Control, In Situ Hybridization, Staining, Quantitative RT-PCR

Journal: Disease Models & Mechanisms

Article Title: Tfap2a -dependent changes in mouse facial morphology result in clefting that can be ameliorated by a reduction in Fgf8 gene dosage

doi: 10.1242/dmm.017616

Figure Lengend Snippet: Reduced Fgf8 gene dosage leads to a rescue of bilateral cleft lip. (A) Numbers of various genotypes and phenotypes observed from the Tfap2a (neo/neo) × Tfap2a (+/−) ; Fgf8 (+/−) crosses (L and R are left- and right-sided cleft, respectively). (B,C) Frontal images of the heads of Neo/Null; Fgf8 wt (B) and Neo/Null; Fgf8 het (C) P0 pups. Black arrow shows the unilateral cleft line. (D–F) Ventral view of skulls using μCT for Neo/Wt; Fgf8 wt (D), Neo/Null; Fgf8 wt (E) and Neo/Null; Fgf8 het (F) P0 pups. Red arrows show clefting at points where the premaxilla and the palatal process of the premaxilla should be fused.

Article Snippet: For real-time PCR, pre-validated probes and primers were used to examine Tfap2a levels targeting exons 2–3 and 6–7 (IDT PrimeTime assay Mm.PT.56a.1580695 and 12995135, Integrated DNA Technologies, Coralville, IA) as well as Fgf8 (TaqMan gene expression assay Mm00438922_m1, Applied Biosystems, San Francisco, CA) and Wnt9b (TaqMan gene expression assay Mm00457102_m1).

Techniques:

Journal: Disease Models & Mechanisms

Article Title: Tfap2a -dependent changes in mouse facial morphology result in clefting that can be ameliorated by a reduction in Fgf8 gene dosage

doi: 10.1242/dmm.017616

Figure Lengend Snippet: Morphometric analysis of the genetic interactions between Tfap2a and Fgf8 in facial development. (A) Morphometric analysis of E10.5 embryos from Tfap2a (neo/neo) × Tfap2a (+/−) ; Fgf8 (+/−) crosses. CVA showing between-group differences. The wireframes visualize +10 of the axis in black and 0 in gray and the warp images show −6 and +6, respectively, along the axes. (B) PCA of E10.5 Neo/Null; Fgf8 het compared with Neo/Wt; Fgf8 wt. Wireframes and warp embryo images show −0.05 and +0.05 along PC1. (C) PCA of E10.5 Neo/Null; Fgf8 het compared with Neo/Null; Fgf8 wt. Wireframes and warp embryo images show −0.06 and +0.06 along PC1.

Article Snippet: For real-time PCR, pre-validated probes and primers were used to examine Tfap2a levels targeting exons 2–3 and 6–7 (IDT PrimeTime assay Mm.PT.56a.1580695 and 12995135, Integrated DNA Technologies, Coralville, IA) as well as Fgf8 (TaqMan gene expression assay Mm00438922_m1, Applied Biosystems, San Francisco, CA) and Wnt9b (TaqMan gene expression assay Mm00457102_m1).

Techniques:

Journal: Disease Models & Mechanisms

Article Title: Tfap2a -dependent changes in mouse facial morphology result in clefting that can be ameliorated by a reduction in Fgf8 gene dosage

doi: 10.1242/dmm.017616

Figure Lengend Snippet: Analysis of variance and integration of facial morphology associated with Tfap2a and Fgf8 mutations. (A) Trace of the variance-covariance matrix to measure variation in shape at E10.5. P -value was determined based on overlap between the curves based on 1000 repetitions of the analysis. (B) Two-block PLS analysis to determine integration at E10.5 between the maxilla and the nasal prominences, the two regions with the largest differences in shape. Statistics can be found in supplementary material Table S5 . The data concerning E10.5 C57Bl/6J is presented as an additional control for background or strain effects and was obtained by re-landmarking scans generated previously .

Article Snippet: For real-time PCR, pre-validated probes and primers were used to examine Tfap2a levels targeting exons 2–3 and 6–7 (IDT PrimeTime assay Mm.PT.56a.1580695 and 12995135, Integrated DNA Technologies, Coralville, IA) as well as Fgf8 (TaqMan gene expression assay Mm00438922_m1, Applied Biosystems, San Francisco, CA) and Wnt9b (TaqMan gene expression assay Mm00457102_m1).

Techniques: Blocking Assay, Control, Generated

Journal: Nature Methods

Article Title: A polarized FGF8 source specifies frontotemporal signatures in spatially oriented cell populations of cortical assembloids

doi: 10.1038/s41592-024-02412-5

Figure Lengend Snippet: a , Schematic summarizing factors and gene markers involved in human cortical area specification. b , Overview of the protocol used to treat neural organoids with patterning factors from 3 to 5 div. Diff, differentiation medium ± vitamin A. c , Percentage of organoids with detectable SP8 > GFP expression after treatment with rostralizing factors (left) or EMX1>mNeonGreen expression after treatment with caudalizing factors (right). CHRDL1, chordin like 1; FST, follistatin; SB, SB-431542 dual SMAD inhibitor; CER1, cerberus 1; CHIR, GSK-3β inhibitor CHIR99021; WNT1, Wnt family member 1. Data indicate the mean ± s.d. from three lines per treatment ( n = 15 organoids per condition and line; except n = 16 for FGF8 high and CHIR low; n = 20, n = 19 and n = 17 for Chir high, and n = 18, n = 19 and n = 17 for Wnt1 treatment for the three lines). P values from one-way analysis of variance (ANOVA; Tukey’s multiple-comparisons test) for comparisons to untreated conditions are provided. d , Experimental procedure for assembloid generation with the SP8 > GFP line and OrEBs. D, day after EB formation; Diff, differentiation medium ± vitamin A; ULA, ultra-low attachment plate . e , RT–qPCR analysis of FGF8 target genes expressed in the SP8 > GFP EBs severed from OrEB after 1 day of co-culture. Data are the log of expression over TBP , shown as the mean ± s.d. ( n = 6 EBs for 0%, n = 5 for 1%, n = 5 for 10% grown from three independent clones). P values are the results of one-way ANOVA. f , GFP intensity of the organoids from g . Whiskers are the minima to maxima, boxes represent the 25th to 75th percentiles (Q1 to Q3) and lines indicate the median ( n = 7 organoids for 0%, n = 11 for 1%, n = 19 for 10%, grown from three independent clones). P values are the results of one-way ANOVA. g , Images of organoids generated with the SP8 > GFP transgenic line and co-culture with OrEBs at 60 div (scale bars, 500 µm). **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.1. a.u., arbitrary units.

Article Snippet: Levels of FGF8 protein in cell extracts and supernatants were measured using an ELISA kit (Cusabio CSB-E15861h) according to the manufacturer’s instruction.

Techniques: Expressing, Quantitative RT-PCR, Co-Culture Assay, Clone Assay, Generated, Transgenic Assay

Journal: Nature Methods

Article Title: A polarized FGF8 source specifies frontotemporal signatures in spatially oriented cell populations of cortical assembloids

doi: 10.1038/s41592-024-02412-5

Figure Lengend Snippet: a ) Schematic diagram of CAG > FGF8 reporter (top). Genotyping of the selected clones (bottom) shows the three heterozygotes selected. The PCR was designed to amplify a wild type AAVS1 (WT) amplicon, a left homology arm amplicon (LA), a right homology arm amplicon (RA), and an inner part (IP). b ) RT-qPCR analysis of pluripotency markers and differentiation gene T/BRA in the selected CAG > FGF8 expressing clones and parental wild-type cells. Values are mean ± SD (n = 4 biological replicates for wt; n = 3 biological replicates, one for each CAG > FGF8 clone). P-values resulting from one-way ANOVA (Tukey’s multiple comparisons test) among the different lines are: OCT4 wt vs. OCT4 CAG > FGF8, p = 0.9166; NANOG wt vs. NANOG CAG > FGF8, p = 0.9841; SOX2 wt vs. SOX2 CAG > FGF8, p = 0.9997; TBRA wt vs. TBRA CAG > FGF8, p > 0.9999. c ) Box plots showing RT-qPCR analysis of FGF8 and WNT1 genes in the selected clones of the CAG > FGF8 line compared to wt. Whiskers are min to max, boxes represent the 25th to 75th percentiles (Q1 to Q3); n = 6 from 3 independent clones. One-way ANOVA analysis is p < 0.0001 for FGF8 expression and p > 0.9999 for WNT1 expression. d ) Quantification of FGF8 protein levels in cell lysates (CELLS) and supernatants (SUP) measured by ELISA assay. Values are mean ± SD, n = 6 from 3 independent clones; P-values from two-sided unpaired t-tests are: CELLS wt vs CELLS CAG > FGF8, p = 0.0004; SUP wt vs SUP CAG > FGF8, p = 0.0823.

Article Snippet: Levels of FGF8 protein in cell extracts and supernatants were measured using an ELISA kit (Cusabio CSB-E15861h) according to the manufacturer’s instruction.

Techniques: Clone Assay, Amplification, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nature Methods

Article Title: A polarized FGF8 source specifies frontotemporal signatures in spatially oriented cell populations of cortical assembloids

doi: 10.1038/s41592-024-02412-5

Figure Lengend Snippet: a ) Representative images of 4 div embryoid bodies co-cultured with OrEBs containing cells expressing FGF8 and tdTomato mixed at different percentage (0%, 1%, 10%), immunostained for the FGF8 downstream target ETV1 (green) (scale bars 50 µm, the SP8 > GFP organoid is outlined with a dashed contour). The experiment has been repeated 4 times with organoids derived from three independent clones. b ) Immunostaining of 60 div organoids generated with the SP8 > GFP transgenic line and co-cultured with OrEBs in Fig. , stained for the rostral marker LMO4 (scale bars 500 µm in the main panels, 50 µm for the insets). The SP8 > GFP organoid is outlined with a dashed contour. c ) Box plots showing the fraction of LMO4+ cells normalized to total DAPI+ cells in 0%, 1% and 10% FGF8 conditions at 60 div. Whiskers are min to max, boxes represent the 25th to 75th percentiles (Q1 to Q3); n = 8 organoids. P-values from one-way ANOVA (Tukey’s multiple comparisons tests) are: 0% vs 1%, p = 0.0019, 0% vs 10%, p < 0.0001, 1% vs 10%, p < 0.0001. d ) Immunostaining of organoids generated with the SP8 > GFP transgenic line and co-cultured with OrEBs as described in Fig. for 60 days, stained for the neural marker Sox1 and DAPI. Scale bars 500 µm. e ) Quantification of the number of Sox1+ cells over total (DAPI + ) cells. Data show mean ± SD (n = 2 assembloids for 0%, n = 4 assembloids for 1%, n = 3 assembloids for 10%). P-values for each comparison resulting from one-way ANOVA (Tukey’s multiple comparisons tests) are: 0% vs 1%, p = 0.113; 0% vs 10%, p = 0.2171; 1% vs 10%, p = 0.8893. f ) RT-qPCR analysis of anterior ( PAX6, SIX3 ) and optic cup ( SNAI2, PAX3, OTX2, LHX2, RAX ) markers from SP8 > GFP organoids severed from the co-culture experiment at 60 div. Data is Log of expression over TBP, shown as mean ± SD; n = 6 for 0% condition, n = 5 for 1% and 10% condition, from 3 independent clones. P-values from one-way ANOVA (Tukey’s multiple comparisons tests) are: RAX 0% vs. RAX 1%, p = 0.0718; RAX 0% vs. RAX10%, p = 0.0003; RAX 1% vs. RAX 10%, p = 0.0263; all the other comparisons are p < 0.0001. g ) GFP intensity per segment (P, proximal; M, medial; D, distal) in the 1% FGF8 condition from experiments shown in panel b . Each segmented line represents an individual organoid. n = 7 from 3 clones; ns, p = 0.5783 one-way ANOVA. Ns, non-significant. a.u., arbitrary units.

Article Snippet: Levels of FGF8 protein in cell extracts and supernatants were measured using an ELISA kit (Cusabio CSB-E15861h) according to the manufacturer’s instruction.

Techniques: Cell Culture, Expressing, Derivative Assay, Clone Assay, Immunostaining, Generated, Transgenic Assay, Staining, Marker, Comparison, Quantitative RT-PCR, Co-Culture Assay

Journal: Nature Methods

Article Title: A polarized FGF8 source specifies frontotemporal signatures in spatially oriented cell populations of cortical assembloids

doi: 10.1038/s41592-024-02412-5

Figure Lengend Snippet: a , Experimental procedure for elongated assembloids using mosaic OrEBs containing CAG>tdTOMATO and non-fluorescent CAG > FGF8 -expressing cells. Diff, differentiation medium ± vitamin A. b , Representative images of elongated cortical assembloids at 1 div in the PDMS molds (indicated by white arrows in (i); scale bar, 500 µm), at 7 div after removal from the molds and before Matrigel embedding (ii) or after embedding in large Matrigel droplets ((iii); scale bar, 5 mm), at 120 div in the six-well plate ((iv); scale bar, 5 mm). c , Position of the OrEB on elongated cortical assembloids length (as a percentage) at 15, 60 and 120 div. Values are the mean ± s.d. ( n = 9 organoids for 15 div, n = 11 for 60 div and 120 div grown from three independent clones). P values for comparisons among time points (one-way ANOVA Tukey’s multiple-comparisons test) are: 15 div versus 60 div, P = 0.9455; 15 div versus 120 div, P = 0.9781; 60 div versus 120 div, P > 0.9999. d , Images of elongated cortical assembloids generated with the SP8 > GFP transgenic line and mosaic OrEBs at 60 div (scale bars, 500 µm). Right, SP8 > GFP intensity per segment (P, M and D) in individual assembloids. Each segmented line represents an individual elongated cortical assembloid ( n > 2 from at least 2 clones). P values are the results of one-way ANOVA among segments per condition (0%, P = 0.9045; 1%, P < 0.0001 and 10%, P = 0.9828). e , f , Images of proximal and distal CPNE8 ( e ) or NR2F1 ( f ) stainings (scale bars, 50 µm). Bottom, fraction of CPNE8 + ( e ) or NR2F1 + cells ( f ) normalized to total (DAPI + ) cells in proximal and distal insets of controls (conCAs) and polCAs at 60 div. Whiskers are min to max, boxes represent the 25th to 75th percentiles (Q1 to Q3) and lines indicate the median; CPNE8: n = 20 insets for P and D from 3 conCAs, n = 42 insets for P and n = 39 for D from 3 polCAs; NR2F1: n = 60 insets for P and D from 6 conCAs, n = 40 insets for P and D from 4 polCAs. P values from one-way ANOVA (Tukey’s multiple-comparisons test) are: P = 0.9988 in CPNE8 proximal conCA versus distal conCAs, P = 0.8509 in NR2F1 proximal conCAs versus distal conCAs, P < 0.0001 for other comparisons; NS, not significant. g , g ′, Images of 60 div polCA immunostained with tdTomato in red, DAPI in blue and NR2F1 in white ( g ) or intensity rainbow ( g ′). Scale bar, 500 µm.

Article Snippet: Levels of FGF8 protein in cell extracts and supernatants were measured using an ELISA kit (Cusabio CSB-E15861h) according to the manufacturer’s instruction.

Techniques: Expressing, Clone Assay, Generated, Transgenic Assay

Journal: Nature Methods

Article Title: A polarized FGF8 source specifies frontotemporal signatures in spatially oriented cell populations of cortical assembloids

doi: 10.1038/s41592-024-02412-5

Figure Lengend Snippet: a-b ) Representative images of elongated assembloids (8 div) properly embedded maintaining elongated shape (a) compared to elongated assembloids from the same batch without matrigel, shrinking without spatial constrain (b). c ) Length of elongated assembloids after different days in vitro (div, div1 n = 8, div7 n = 6, div30 n = 4, div60 n = 24, div120 n = 22 assembloids grown from 3 independent clones). P-values resulting from one-way ANOVA Tukey’s multiple comparisons tests) among the different lines are: div1 vs div7, p = 0.0045; div1 vs div30, p = 0.9999; div7 vs div30, p = 0.0366; div7 vs div60, p = 0.9998; div7 vs div120, p = 0.51; div30 vs div60, p = 0.0049; div60 vs div120, p = 0.1825; div1 vs div60, div1 vs div120, div30 vs div120, p < 0.0001. d ) RT-qPCR analysis of neural markers (SOX2, SOX1, SP8 and LMO4), ventral marker NXK2.1 and differentiation markers SNAI1 and T-BRA, in elongated assembloids (eOrg) compared to round organoids (rOrg), after treatment with high FGF8 as described in Fig. . Values are mean ± SD, n = 3 in three lines (hESC H9, H1 and iPSC 178/5). P-values resulting from one-way ANOVA (Tukey’s multiple comparisons tests) are: eORGSOX2 d10 vs. rORGSOX2 d10, p = 0.0024; eORGSOX2 d30 vs. rORGSOX2 d30, p = 0.3144; eORGSOX2 d60 vs. rORGSOX2 d60, p = 0.9434; eORGSOX1 d10 vs. rORGSOX1 d10, p = 0.0009; eORGSOX1 d30 vs. rORGSOX1 d30, p = 0.1573; eORGSOX1 d60 vs. rORGSOX1 d60, p = 0.7266; eORGSP8 d10 vs. rORGSP8 d10, p = 0.0006; eORGSP8 d30 vs. rORGSP8 d30, p = 0.1891; eORGSP8 d60 vs. rORGSP8 d60, p = 0.8004; eORGLMO4 d10 vs. rORGLMO4 d10, p = 0.0114; eORGLMO4 d30 vs. rORGLMO4 d30, p = 0.8378; eORGLMO4 d60 vs. rORGLMO4 d60, p = 0.9821; eORGNKX2.1 d10 vs. rORGNKX2.1 d10, p = 0.0008; eORGNKX2.1 d30 vs. rORGNKX2.1 d30, p = 0.1345; eORGNKX2.1 d60 vs. rORGNKX2.1 d60, p = 0.9036; eORGSNAI1 d10 vs. rORGSNAI1 d10, p = 0.0026; eORGSNAI1 d30 vs. rORGSNAI1 d30, p = 0.6125; eORGSNAI1 d60 vs. rORGSNAI1 d60, p = 0.96; eORGT-BRA d10 vs. rORGT-BRA d10, p = 0.6815; eORGT-BRA d30 vs. rORGT-BRA d30, p = 0.3026; eORGT-BRA d60 vs. rORGT-BRA d60, p = 0.1297. e ) Fraction of TUNEL+ cells normalized to total DAPI+ cells in elongated versus conventional round organoids (div, days in vitro ). N = 36 insets for 4 div elongated, n = 33 for 4 div Round, n = 122 for 7 div elongated, n = 37 for 7 div Round, n = 81 for 30 div elongated, n = 82 for 30 div Round organoids from 3 lines per condition. P-values resulting from one-way ANOVA (Tukey’s multiple comparisons tests) are: 4 div eOrg vs. 4 div rOrg, p < 0.0001; 7 div eOrg vs. 7 div rOrg, p = 0.31; 30 div eOrg vs. 30 div rOrg, p = 0.0115. f ) Box plots showing the fraction of TUNEL+ cells normalized to total DAPI+ cells in proximal and distal insets of elongated assembloids (n = 18 insets for 4 div Proximal and Distal, n = 32 for 7 div Proximal, n = 33 for 7 div Distal, n = 40 for 30 div Proximal, n = 42 for 30 div Distal; organoids are grown from 3 lines per condition). P-values resulting from one-way ANOVA (Tukey’s multiple comparisons tests) are: 4 div proximal vs. 4 div distal, p = 0.6097; 7 div proximal vs. 7 div distal, p = 0.6559; 30 div proximal vs. 30 div distal, p = 0.9897. g ) Representative images of the OrEB localization throughout elongated assembloids growth quantified in Fig. (scale bars 500 µm). h ) Top, representative images of proximal and distal CTIP2 staining (scale bars 50 µm). Bottom, fraction of CTIP2+ cells normalized to total DAPI+ cells in proximal and distal insets of controls (conCA) and polCAs at 60 div (n = 50 insets for P and D conCAs, n = 40 insets for P and D polCAs from 3 organoids). P-values resulting from one-way ANOVA (Tukey’s multiple comparisons tests) are: proximal conCA vs. distal conCA, p = 0.8204; proximal polCA vs. distal polCA, p < 0.0001. i ) Top, representative images of proximal and distal LMO4 staining (scale bars 50 µm). Bottom, fraction of LMO4+ cells normalized to total DAPI+ cells in proximal and distal insets of control(tdt) and polCAs at 60 div (n = 61 insets for P and D from 6 conCAs, n = 51 insets for P and D from 4 polCA). P-values resulting from one-way ANOVA (Tukey’s multiple comparisons tests) are: proximal conCA vs. distal conCA, p > 0.9999; proximal polCA vs. distal polCA, p < 0.0001. ns, non-significant. All whiskers are min to max, boxes represent the 25th to 75th percentiles (Q1 to Q3) and lines indicate median. j-j’ ) Images of immunostained longitudinal sections of elongated assembloids at 60 div (scale bars 500 µm).

Article Snippet: Levels of FGF8 protein in cell extracts and supernatants were measured using an ELISA kit (Cusabio CSB-E15861h) according to the manufacturer’s instruction.

Techniques: In Vitro, Clone Assay, Quantitative RT-PCR, Marker, TUNEL Assay, Staining, Control

Journal: Nature Methods

Article Title: A polarized FGF8 source specifies frontotemporal signatures in spatially oriented cell populations of cortical assembloids

doi: 10.1038/s41592-024-02412-5

Figure Lengend Snippet: a ) UMAP embedding for the scRNA-seq dataset containing cells derived from dissections of three segments of polCA and control counterparts annotated by stress levels based on Gruffi analysis. b ) Sankey plot showing similar mapping of cells from individual segments of both control and polCA to stressed cells according to Gruffi’s results. c ) Box plots showing the number of cells expressing FGF8 normalized by the total number of cells per segment for the three replicates. The box displays the median, the inter-quartile range, the minimum and maximum values for each segment. d ) Dot plot for the top five markers for each cluster, genes with logFC> 3 are shown. LogFCs colors are scaled by gene. e ) UMAP embedding plots colored by expression levels of markers of radial glia ( NES, SOX2, VIM ), cycling radial glia ( TOP2A, MKI67 ), Cajal-Retzious cells ( RELN ), neurons ( DCX, TUBB3 ), interneuron progenitor cells ( DLX6-AS1, GAD2 ), LGE-derived progenitors ( GSX2 ), MGE-derived progenitors ( NKX2.1 ), retinal progenitor cells ( RORB, VSX2 ), stress responsive cells ( GOLGA4 ), endothelial cells ( DCN, BGN, COL1A2 ), BMP responsive cells ( TTR, RSPO2, LMX1A, MSX1 ), cilium bearing cells ( PCP4, NPHP1 ), oligodendrocytes ( OLIG1, OLIG2 ), astrocytes ( GFAP, AQP4 ) and microglia ( AIF1, CD68 ).

Article Snippet: Levels of FGF8 protein in cell extracts and supernatants were measured using an ELISA kit (Cusabio CSB-E15861h) according to the manufacturer’s instruction.

Techniques: Derivative Assay, Control, Expressing

Journal: Development (Cambridge, England)

Article Title: Delta-like ligand 4-mediated Notch signaling controls proliferation of second heart field progenitor cells by regulating Fgf8 expression

doi: 10.1242/dev.185249

Figure Lengend Snippet: Dll4 expression in SHF cells is required to maintain expression of key SHF-related proteins. (A-B‴) Transverse sections were evaluated for Mef2c and Fgf8 transcript expression at E9 in control (A) and Mef2c-AHF-Cre,Dll4F/F mutant (B) by RNAscope. Higher magnification of the boxed areas in A and B are shown as Mef2c expression (A′,B′), Fgf8 expression (A″,B″) and merged images (A‴,B‴) to demonstrate the reduced expression of Fgf8 transcripts in the mutants compared with the controls in the pharyngeal mesoderm (PM). (C-D‴) Similarly, transverse sections were evaluated for Mef2c and Fgf10 transcript expression at E9 in control (C) and Mef2c-AHF-Cre,Dll4F/F mutant (D). Higher magnification of the boxed areas in C and D are shown as Mef2c expression (C′,D′), Fgf10 expression (C″,D″) and merged images (C‴,D‴) to demonstrate that the PM in mutants has decreased expression of Fgf10 transcripts. (E-F‴) Transverse sections of control (E) and Mef2c-AHF-Cre,Dll4F/F mutant (F) E9.5 embryos were co-stained for Islet1 and Fgf8 protein expression. Higher magnification of the boxed areas in E and F are shown as Islet1 expression (E′,F′), Fgf8 expression (E″,F″) and merged images (E‴,F‴), showing reduced expression of Fgf8 in the SHF region. (G-H″) Transverse sections of control (G) and Mef2c-AHF-Cre,Dll4F/F mutant (H) E11.5 embryos were stained for Hand2 protein expression. Higher magnification of the boxed areas in G and H show the RV and LV in control (G′,G″) and mutant (H′,H″) embryos. Hand2 expression is lost in the mutant RV compared with controls. There is no change in the low basal level expression seen in LV. Scale bars: 50 µm (E′-F‴); 100 µm (A′-D‴,E,F,G′-H″); 250 µm (A-D,G-H).

Article Snippet: Thoracic organs were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and varying doses of recombinant Fgf8 (Novus Biologicals, NBP2-35033) for 8 h. The organs were then cryoembedded.

Techniques: Expressing, Control, Mutagenesis, RNAscope, Staining

Journal: Development (Cambridge, England)

Article Title: Delta-like ligand 4-mediated Notch signaling controls proliferation of second heart field progenitor cells by regulating Fgf8 expression

doi: 10.1242/dev.185249

Figure Lengend Snippet: Dll4-mediated notch signaling regulates Fgf8 expression in SHF. (A) Schematic of the mouse chromosome 10 around the region of the Fgf8 gene (E, exon). Putative RBPjk binding sites are indicated with asterisks. Constructs cloned for luciferase assay are shown as black boxes. Promoter 3 and Enhancer 2 were used as negative controls. (B) 293T cells were treated with DAPT to quench basal Notch activity. They were then transfected with various luciferase expression vectors with (empty bars) or without (solid bars) the NICD expression vector. Luciferase activity was measured in triplicate wells (mean±s.e.m.) 24 h later with eight experimental repeats. (C) The experiment was then repeated in triplicate after mutating the putative RBPjk binding site of Promoter 1. Mutation of putative binding sites led to loss of luciferase activity. (D-F″) Thoracic regions were dissected in control (D-D″) and Mef2c-AHF-Cre,Dll4F/F mutant (E-F″) embryos at E9.5 and cultured in vitro. Mutant organs were cultured with (F-F″) or without (E-E″) exogenous recombinant Fgf8. Sections were then co-stained for Islet1 and pHH3 expression to study SHF proliferation. Representative images are shown as Islet1 expression (D′,E′,F′), pHH3 expression (D″,E″,F″) and merged images (D,E,F). (G) Double-positive cells were counted in multiple fields (23 untreated control, 23 Fgf8 100 ng/µl control, 40 Fgf8 500 ng/µl control, 37 Fgf8-untreated mutant, 7 Fgf8 100 ng/µL mutant and 14 Fgf8 500 ng/µl mutant sections; mean±s.e.m.) showing a significant reduction in SHF proliferation in mutant organs compared with control (P<0.0001 between Fgf8-untreated control and mutant, P>0.05 between Fgf8-untreated controls and Fgf8-treated mutants by two-tailed t-tests). For quantification purposes, the boxed regions in D′, E′ and F′ were used as the area occupied by SHF progenitor cells. Exogenous Fgf8 supplementation had no significant impact on control embryos, but fully rescued proliferation defects seen in mutant embryos. (H-L′) Compound heterozygotes were analyzed by H&E staining of transverse sections of E14.5 embryos to demonstrate genetic synergy between Dll4-mediated Notch and Fgf8 signaling in SHF maturation. Cre-negative control embryos showed fully septated ventricles (H) and an aortic valve normally aligned over the left ventricle (H′). Heterozygous knockdown of Dll4 driven by Mef2c-AHF-Cre (Mef2c-Cre,Dll4F/wt) also demonstrated a normal phenotype (I,I′). Heterozygous knockdown of Fgf8 driven by Mef2c-AHF-Cre (Mef2c-Cre,Fgf8F/wt) showed a low incomplete penetrance of cardiac defects. The majority of the embryos showed a normal phenotype (J,J′). A shallow VSD (arrow in K) and a slightly mal-aligned aorta mildly over-riding the ventricular septum (arrowhead in K′) was seen in 14% of the embryos. Double heterozygous knockdown of Dll4 and Fgf8 driven by Mef2c-AHF-Cre (Mef2c-Cre,Dll4F/wt,Fgf8F/wt) showed high penetrance of DORV, with 83% of the embryos studied showing VSD (arrow in L) and a prominent over-riding of aorta with greater than 50% aorta arising from the RV (arrowhead in L′). (M) Table indicates number and phenotypes of embryos recovered amongst the different genotypes shown. The number of embryos recovered, the percentage recovery and the expected percentage recovery are based on Mendelian inheritance. Scale bars: 50 µm (D-F″); 300 µm (H-L′).

Article Snippet: Thoracic organs were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and varying doses of recombinant Fgf8 (Novus Biologicals, NBP2-35033) for 8 h. The organs were then cryoembedded.

Techniques: Expressing, Binding Assay, Construct, Clone Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Control, Cell Culture, In Vitro, Recombinant, Staining, Two Tailed Test, Negative Control, Knockdown

Journal: Cancers

Article Title: Effect of Tasurgratinib as an Orally Available FGFR1–3 Inhibitor on Resistance to a CDK4/6 Inhibitor and Endocrine Therapy in ER + /HER2 − Breast Cancer Preclinical Models

doi: 10.3390/cancers17071084

Figure Lengend Snippet: Antitumor activity and gene expression alterations of FGF/FGFR with palbociclib + fulvestrant in five ER + breast cancer PDX models (OD-BRE-0438, OD-BRE-0704, OD-BRE-0450, OD-BRE-0188, IM-BRE-556). ( a ) Relative tumor volume (upper) and relative body weight (lower) after treatment with palbociclib (100 mg/kg, once daily [Q1D] × 21) in combination with fulvestrant (5 mg/mouse, once a week [Q7D] × 3). Data show the mean (no treatment groups: n = 2 per group) or the mean ± SEM (palbociclib + fulvestrant treatment groups: n = 3 per group). ( b ) Alterations of human mRNA expression in tumors collected on the day following the treatment with palbociclib (100 mg/kg, Q1D × 14) in combination with fulvestrant (250 mg/kg, Q7D × 2). Data show the geometric mean (OD-BRE-0450 and OD-BRE-0188: n = 2 per group) or the geometric mean ± geometric SD (OD-BRE-0438: n = 6 per group, OD-BRE-0704 and IM-BRE-556: n = 3 per group). The expressions of FGF1 in OD-BRE-0450 and IM-BRE-556, FGF7 in IM-BRE-556, FGF8 in OD-BRE-0704, and FGF10 in OD-BRE-0450 and OD-BRE-0188 were not detected in both baseline and post-treatment. * p < 0.05, ** p < 0.01 versus the no treatment group (unpaired t -test) in the OD-BRE-0438 and IM-BRE-556 models in which mouse numbers were more than two in both no treatment groups and treated groups. Values of Ct and delta Ct, p -values in unpaired t -test, and ratio to no treatment are shown in . mPFS: median progression-free survival; ns: not significant.

Article Snippet: TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) and TaqMan Gene Expression Assays (Hs99999909_m1 for HPRT1 , Hs01092738_m1 for FGF1 , Hs00940253_m1 for FGF7 , Hs00171832_m1 for FGF8 , Hs00610298_m1 for FGF10 , Hs00182599_m1 for FGF17 , Hs00241111_m1 for FGFR1 , Hs01552926_m1 for FGFR2 , and Hs00179829_m1 for FGFR3 ; Thermo Fisher Scientific) were used for quantitative RT-PCR (q-PCR), which was conducted on a QuantStudioTM 7 Flex Real-Time PCR System (Thermo Fisher Scientific).

Techniques: Activity Assay, Gene Expression, Expressing

Journal: bioRxiv

Article Title: Competition between stochastic neuropeptide signals calibrates the rate of satiation

doi: 10.1101/2023.07.11.548551

Figure Lengend Snippet: (A) Model: Bidirectional regulation of cAMP signaling in satiety-promoting PVH MC4R neurons by hunger and satiety peptides. (B-C) AAV expression of PDE4D3-Cat, a constitutive cAMP PDE, in PVH MC4R neurons (B) results in drastic weight gain when compared to mCherry controls (C; n = 9-15 mice, t-test). (D) Mice that express PDE4D3-Cat have elevated lean mass and fat mass (n = 6-7 mice, one-way ANOVA). (E) PDE4D3-Cat expression causes elevated 24-hr food intake (n = 6-8 mice, one-way ANOVA). (F-H) Over 24 hours, mice that express PDE4D3-Cat show a net energy surplus (F) that is driven by a large increase in energy intake (G) that is partially offset by elevated energy expenditure (H). n = 6-7 mice, t-test. (I) Correlation between energy expenditure and total mass in PDE4D3-Cat-expressing and control mice (n = 6-7 mice, Pearson correlation). (J) PDE4D3-Cat expression does not affect PVH MC4R cell counts (n = 6-7 mice, t-test). Model: cAMP signaling in PVH MC4R neurons is important for reducing feeding. n.s. not significant, *p < 0.05, **p < 0.01, ***p < 0.001 for all figures. See Table S1 for statistical details and sex-specific characterizations.

Article Snippet: For experiments that measure the effect of cAMP degradation on PVH MC4R neuron activity during feeding, AAV2/1-EF1a-DIO-mKate2-PDE4D3-Cat (Boston Children’s Hospital Vector Core; Addgene 169128) and AAV1-Syn-Flex-GCaMP6s-WPRE-SV40 (Addgene 100845-AAV1) were unilaterally injected into the PVH of MC4R-2A-Cre mice (1:1 mixture, 150 nl total; Bregma: AP -0.75, ML 0.2 mm, DV -4.8 mm).

Techniques: Expressing

Journal: bioRxiv

Article Title: Competition between stochastic neuropeptide signals calibrates the rate of satiation

doi: 10.1101/2023.07.11.548551

Figure Lengend Snippet: (A) AAV expression of PDE4D3-Cat in PVH MC4R neurons in adult mice results in ∼80% weight gain in the 4 weeks following surgery (n = 9-15 mice, t-test). (B) Mice that express PDE4D3-Cat have an elevated contribution of fat mass to their body composition (n = 6-7 mice, One-Way ANOVA). (C) PDE4D3-Cat expression in PVH MC4R neurons in adult mice results in ∼0.5 cm increase in axial length in the 4 weeks following surgery (n = 7-8 mice, t-test). (D-J) A panel of 24-hr recordings of metabolic parameters in 30-min bins: feeding rate (D), drinking rate (E), VO 2 (F), VCO 2 (G), respiratory exchange ratio (H), energy expenditure (I), locomotor activity (J). n = 6-7 mice. Gray shading: dark cycle. (K-M) Across individual mice, energy expenditure is well correlated with lean mass (K), fat mass (L), and change in mass (M), in both the presence or absence of PDE4D3-Cat expression. n = 6-7 mice. (N) Diagram of energy gain: PDE4D3-Cat expression results in increased energy intake (10 kCal/day) that is only partially offset by elevated energy expenditure (5 kCal/day), resulting in a net 5 kCal/day surplus which translates to 0.6 g/day of weight gain. Hence, mice become obese over time.

Article Snippet: For experiments that measure the effect of cAMP degradation on PVH MC4R neuron activity during feeding, AAV2/1-EF1a-DIO-mKate2-PDE4D3-Cat (Boston Children’s Hospital Vector Core; Addgene 169128) and AAV1-Syn-Flex-GCaMP6s-WPRE-SV40 (Addgene 100845-AAV1) were unilaterally injected into the PVH of MC4R-2A-Cre mice (1:1 mixture, 150 nl total; Bregma: AP -0.75, ML 0.2 mm, DV -4.8 mm).

Techniques: Expressing, Activity Assay

Journal: bioRxiv

Article Title: Competition between stochastic neuropeptide signals calibrates the rate of satiation

doi: 10.1101/2023.07.11.548551

Figure Lengend Snippet: (A-B) In a feeding assay where fasted mice lick during a cue to obtain a small bolus of milkshake at 1 trial/min, GCaMP6s signals in PVH MC4R neurons demonstrate gradual growth of feeding-related responses (red), which likely promote satiety. When biPAC is used to produce cAMP 5 s before each trial, calcium responses grow faster and to a greater amplitude (purple). Artifacts due to bleed-through of photo-stimulation light were blanked. Reduced and delayed growth in calcium responses is seen when PDE4D3-Cat is co-expressed to blunt natural and artificial increases in cAMP in PVH MC4R neurons (orange). In B, the first five trials of the biPAC stimulation group (purple) are plotted individually to capture the rapid growth in response magnitude in the presence of additional biPAC stimulation prior to each cue presentation. Peak calculations are from 5-8 s after cue onset. n = 8 mice per condition. (C-E) Brief 100-ms biPAC stimulation (example in C) potentiates amplitude (D) of spontaneous excitatory inputs, thereby sensitizing PVH MC4R neurons to excitatory inputs (E). n = 16 cells from 3 fed mice. (F-J) cAMP degradation by PDE4D3-Cat (F) reduces the firing rate (G-H) and spontaneous EPSC frequency (I) as well as EPSC amplitude (J) in PVH MC4R neurons in brain slices from fed mice (H-J: unpaired t-test; H: n = 19-23 cells from 4 mice each, I-J: 6-7 cells from 4 mice). mCh: mCherry control.

Article Snippet: For experiments that measure the effect of cAMP degradation on PVH MC4R neuron activity during feeding, AAV2/1-EF1a-DIO-mKate2-PDE4D3-Cat (Boston Children’s Hospital Vector Core; Addgene 169128) and AAV1-Syn-Flex-GCaMP6s-WPRE-SV40 (Addgene 100845-AAV1) were unilaterally injected into the PVH of MC4R-2A-Cre mice (1:1 mixture, 150 nl total; Bregma: AP -0.75, ML 0.2 mm, DV -4.8 mm).

Techniques: Feeding Assay

Journal: bioRxiv

Article Title: Competition between stochastic neuropeptide signals calibrates the rate of satiation

doi: 10.1101/2023.07.11.548551

Figure Lengend Snippet: (A) An example session from a fasted mouse that licks during the cue (blue) to obtain reward (orange). Panel shows a heatmap of RCaMP1a photometry signals (each row is a trial). There is a delayed increase in calcium activity during each trial (which peaks ∼4s after cue onset, and several seconds after consumption onset), potentially reflecting ingestion-related gastrointestinal signals. This increase in activity takes ∼10 trials to develop. (B) Photometry recording of spontaneous bulk calcium activity after a session suggests increases in ongoing PVH MC4R calcium activity after feeding. (C-F) In mice co-expressing biPAC and a calcium sensor (GCaMP6s or RCamp1a) in PVH MC4R neurons, briefly stimulating biPAC through a fiber does not result in noticeable calcium transients in either fed mice (D and E) or fasted mice (F). We tested RCaMP1a, a green-light sensitive calcium sensor, to avoid biPAC activation by photometry light. Blanking (2-4 s) in traces is done to remove temporary photobleaching due to optogenetic stimulation. n = 8 mice per panel. (G) Brief 100-ms biPAC stimulation did not increase the frequency of spontaneous excitatory inputs, thereby sensitizing PVH MC4R neurons to excitatory inputs. n = 16 cells from 3 fed mice. (H) In acute brain slices, brief biPAC activation (100 ms or 300 ms, 10 mW) resulted in changes in calcium activity that can be described in two categories: 41% of PVH MC4R neurons showed a persistent elevation in calcium activity, while a different 43% showed relatively transient (∼100 s) increases in calcium activity (n = 5 slices from 2 fed mice). Because the transient activation was not seen in vivo (see ), we did not pursue it further. (I) Example cell-attached recording shows elevated firing rate (negative deflections) for ∼2 min after each brief biPAC stimulation pulse (100 ms). The acute neuronal activation by biPAC stimulation was not seen in vivo (see ). Such a difference between slice and in vivo results could be due to lower extracellular calcium concentration in vivo , as the excitability effects in slice depended on extracellular calcium (right). (J-L) In acute brain slices, brief biPAC activation (2 s, 1 mW) induces cAMP increments in PVH MC4R neurons that gradually decay back to baseline. Co-expressing PDE4D3-Cat completely blocks biPAC-induced cAMP transients and therefore should also reduce feeding-related cAMP increments. Traces in L allow for longer time to visualize cAMP decay. n = 3-5 slices from 3 mice total. (M) In slices with both glutamate and GABA blockers, PDE4D3-Cat-expressing cells almost never show spontaneous spikes (n = 6-7 cells from 4 fed mice total).

Article Snippet: For experiments that measure the effect of cAMP degradation on PVH MC4R neuron activity during feeding, AAV2/1-EF1a-DIO-mKate2-PDE4D3-Cat (Boston Children’s Hospital Vector Core; Addgene 169128) and AAV1-Syn-Flex-GCaMP6s-WPRE-SV40 (Addgene 100845-AAV1) were unilaterally injected into the PVH of MC4R-2A-Cre mice (1:1 mixture, 150 nl total; Bregma: AP -0.75, ML 0.2 mm, DV -4.8 mm).

Techniques: Activity Assay, Expressing, Activation Assay, In Vivo, Concentration Assay