Journal: The Journal of the Acoustical Society of America

Article Title: Individual predictors of response to biofeedback training for second-language production

doi: 10.1121/1.5139423

Figure Lengend Snippet: (Color online) Examples of visual-acoustic biofeedback displays generated with KayPentax Sona-Match software (PENTAX MEDICAL). Incorrect (top) and correct (bottom) productions of the vowel /y/ contrast in the frequency location of the second formant peak in a Linear Predictive Coding (LPC) spectrum.

Article Snippet: For this purpose, we used the PENTAX Medical Sona-Match real-time Linear Predictive Coding (LPC) spectrum module on the CSL system. (Note that the visual-acoustic biofeedback display was used in the process of selecting a target speaker even for participants assigned to the ultrasound biofeedback condition.)

Techniques: Generated, Software

Journal: Nature ecology & evolution

Article Title: A multifunctional Wnt regulator underlies the evolution of rodent stripe patterns

doi: 10.1038/s41559-023-02213-7

Figure Lengend Snippet: a and b, Whole-mount in situ hybridization for Dkk4 in wild-type and Sfrp2 knockout E16.5 embryos (a) and corresponding width measurements of dorsal regions 1 and 3 (that is, R1 and R3) (b). Note that Dkk4 expression diminishes in response to Sfrp2 knockout. c, Hair length measurements in postnatal day 3 wild-type and Sfrp2 knockout individuals. In b and c, n = 3 biologically independent samples for each Sfrp2 knockout and Sfrp2 wild-type individuals.

Article Snippet: To generate LV- SFRP2-GFP , we amplified the Sfrp2 coding sequence from an Sfrp2 ( NM_009144 ) Mouse Tagged ORF Clone (Origene catalogue no. MR204070) using CloneAmp HiFi PCR Premix (Takara 639298) and cloned it (in frame) into the linearized LV- GFP plasmid using In-Fusion Snap Assembly Master Mix (Takara no. 638947).

Techniques: In Situ Hybridization, Knock-Out, Expressing

Journal: Nature ecology & evolution

Article Title: A multifunctional Wnt regulator underlies the evolution of rodent stripe patterns

doi: 10.1038/s41559-023-02213-7

Figure Lengend Snippet: a, Schematic of skin regions (R1–R3) dissected for scRNA-seq (left) and cell-type clustering (right). b, Dermal fibroblasts (green, left) cluster with Sfrp2-expressing cells (green, right). c, Fibroblasts sorted by high (>2 UMI) and low (<2 UMI) Sfrp2 expression. d, Ontology analysis of genes downregulated in Sfrp2High cells, show Wnt signalling as the main enriched pathway (P values corrected for multiple testing (Padj = 0.00406 (P00057); Padj = 0.0068 (P006959); Padj = 0.0073 (P00025); Padj = 0.0081 (P00017); Padj = 0.0194 (P00005); Padj = 0.0196 (P04380); Padj = 0.0354 (P00058); Padj = 0.04123 (P00027); Padj = 0.0416 (P04398); Padj = 0.04166 (P04398; one-sided hypergeometric test)). NS, not significant. e, Lef1 and Ctnnb1 show upregulation in Sfrp2low cells (P = 2.2 × 10−16 (Lef1); P = 2.2 × 10−16 (Ctnnb1); two-sided, non-parametric Wilcoxon rank sum test)). f–i, In situ hybridization for Sfrp2 coupled with immunofluorescence (IF) for KRT14 in stage-matched striped mouse (f) and laboratory mouse (h) embryos. IF for CTNNB1 in stage-matched striped mouse (g) and laboratory mouse (i) embryos. Red boxes denote zoomed-in regions. At least three samples per stage were analysed in f–i. j,k, Comparative scRNA-seq analysis shows both the percentage of fibroblasts that express Sfrp2 (left, Spearman’s correlation, R = 0.79) and the expression levels of Sfrp2 within striped mouse fibroblasts (j) increase in the days before visible placode formation while, in laboratory mice (k), both the percentage of fibroblasts expressing Sfrp2 (left, Spearman’s correlation, R = −0.78) and Sfrp2 expression levels within those fibroblasts (right) decrease in the days before visible placode formation. l,m, Representative transverse sections showing that double-transgenic mice have elevated levels of GFP expression (l) and lower numbers of hair follicles (l,m), compared to controls (P = 0.0002; two-sided t-test; n = 4). Panel l shows absence of transgene expression in wild type (left) and robust expression of transgene in double-transgenic (Dermo-Cre;RosaSfrp2-GFP) animals (right). Since the vector used contains the Sfrp2 cDNA cloned in frame with GFP, GFP is a readout of SFRP2 distribution. Bars in c, e, j and k represent average expression levels. Scale bars, 200 μm (zoomed out) and 100 μm (zoomed in) f and h; 200 μm (zoomed out) and 50 μm (zoomed in) in g and i; and 50 μm in l. NT, neural tube. Embryo schematic in a created with BioRender.com.

Article Snippet: To generate LV- SFRP2-GFP , we amplified the Sfrp2 coding sequence from an Sfrp2 ( NM_009144 ) Mouse Tagged ORF Clone (Origene catalogue no. MR204070) using CloneAmp HiFi PCR Premix (Takara 639298) and cloned it (in frame) into the linearized LV- GFP plasmid using In-Fusion Snap Assembly Master Mix (Takara no. 638947).

Techniques: Expressing, In Situ Hybridization, Immunofluorescence, Transgenic Assay, Plasmid Preparation, Clone Assay

Journal: Nature ecology & evolution

Article Title: A multifunctional Wnt regulator underlies the evolution of rodent stripe patterns

doi: 10.1038/s41559-023-02213-7

Figure Lengend Snippet: a, Sfrp2 expressing fibroblasts are expressed primarily in the reticular (lower) dermis. Papillary (upper) and reticular (lower) dermis fibroblasts were defined based on previously established markers3; Papillary dermis: Ntn1, Pdpn, Ackr4, Lrig1, Apcdd1; Reticular dermis: Tgm2, Cnn1, Cdh2, Mgp, Dlk1. b, At E16.5, expression levels of Sfrp2 and the percentage of fibroblasts expressing Sfrp2 are highest in Region 1 (R1) and lowest in Region 3 (R3), in agreement with the dorsoventral gradient revealed by the bulk RNA-seq data. In b, n = 3 biologically independent samples. Left panel: bars represent average expression levels. Right panel: mean values (+/− SEM).

Article Snippet: To generate LV- SFRP2-GFP , we amplified the Sfrp2 coding sequence from an Sfrp2 ( NM_009144 ) Mouse Tagged ORF Clone (Origene catalogue no. MR204070) using CloneAmp HiFi PCR Premix (Takara 639298) and cloned it (in frame) into the linearized LV- GFP plasmid using In-Fusion Snap Assembly Master Mix (Takara no. 638947).

Techniques: Expressing, RNA Sequencing

Journal: Nature ecology & evolution

Article Title: A multifunctional Wnt regulator underlies the evolution of rodent stripe patterns

doi: 10.1038/s41559-023-02213-7

Figure Lengend Snippet: a, In situ hybridization in striped mouse E16.5 embryos shows that Sfrp2 is primarily expressed in the reticular dermis. Right side image shows expression of Sfrp2 at subcellular resolution. b-c, LEF1 immunostaining in staged matched striped (b) and laboratory (c) mouse embryos. Red boxes denote zoomed-in regions. Scale bars: 200 μm (zoomed out) and 100 μm (zoomed in) in a; 200 μm (zoomed out) and 50 μm (zoomed in) in b and c. NT = neural tube. For a-c, three different individuals were analysed.

Article Snippet: To generate LV- SFRP2-GFP , we amplified the Sfrp2 coding sequence from an Sfrp2 ( NM_009144 ) Mouse Tagged ORF Clone (Origene catalogue no. MR204070) using CloneAmp HiFi PCR Premix (Takara 639298) and cloned it (in frame) into the linearized LV- GFP plasmid using In-Fusion Snap Assembly Master Mix (Takara no. 638947).

Techniques: Expressing, In Situ Hybridization, Immunostaining

Journal: Nature ecology & evolution

Article Title: A multifunctional Wnt regulator underlies the evolution of rodent stripe patterns

doi: 10.1038/s41559-023-02213-7

Figure Lengend Snippet: A Dermo-Cre mouse was used to drive Cre expression in dermal fibroblasts. As illustrated above, a subset of Dermo1 expressing fibroblasts express Sfrp2. Thus, this mouse strain is adequate for driving expression of Cre in cells expressing Sfrp2.

Article Snippet: To generate LV- SFRP2-GFP , we amplified the Sfrp2 coding sequence from an Sfrp2 ( NM_009144 ) Mouse Tagged ORF Clone (Origene catalogue no. MR204070) using CloneAmp HiFi PCR Premix (Takara 639298) and cloned it (in frame) into the linearized LV- GFP plasmid using In-Fusion Snap Assembly Master Mix (Takara no. 638947).

Techniques: Expressing

Journal: Nature ecology & evolution

Article Title: A multifunctional Wnt regulator underlies the evolution of rodent stripe patterns

doi: 10.1038/s41559-023-02213-7

Figure Lengend Snippet: a, Schematic showing the role of Sfrp2 as an inhibitor of Wnt signalling. b, Gradient steepness increases central stripe width independent of model. Each row depicts a schematic and equations governing a particular variant of our modulator-activator-inhibitor system (left) and the resulting simulations of stripe spacing for different gradient steepness values using these models (right). In all cases, gradient steepness affects stripe spacing. c, Predictions from an alternative model of positional information. Patterning based on positional information is inconsistent with our experimental results. We illustrate this by considering two standard paradigms for stripe patterning by positional information. Under a classic ‘French Flag’ model (left, top), each stripe (marked in grey) is assigned to a region of space in which a single morphogen gradient exists between two pathway-specific threshold concentrations (horizontal red lines). (top, left) Under such a paradigm, a substantial reduction in morphogen expression, in this case by 80 percent, makes it impossible for the gradient to reach certain thresholds entirely, leading to stripe loss. (bottom, left) Alternatively, stripes are frequently determined via an ‘opposing gradients’ motif via the interaction of multiple gradients. We depict one example, in which each stripe is determined by two opposite facing gradients, such that a stripe forms in the region where each gradient exceeds a morphogen-specific threshold. (right, bottom) Major reduction of a single morphogen eliminates one stripe while leaving the other unperturbed.

Article Snippet: To generate LV- SFRP2-GFP , we amplified the Sfrp2 coding sequence from an Sfrp2 ( NM_009144 ) Mouse Tagged ORF Clone (Origene catalogue no. MR204070) using CloneAmp HiFi PCR Premix (Takara 639298) and cloned it (in frame) into the linearized LV- GFP plasmid using In-Fusion Snap Assembly Master Mix (Takara no. 638947).

Techniques: Variant Assay, Expressing

Journal: Nature ecology & evolution

Article Title: A multifunctional Wnt regulator underlies the evolution of rodent stripe patterns

doi: 10.1038/s41559-023-02213-7

Figure Lengend Snippet: a, Schematic of the in vivo gene editing method used. b, P5 Tyrosinase wild-type (Tyr+/+) and knockout (Tyr−/−) striped mouse littermates. c,d, Sfrp2 regulates stripe pattern width. Shown are representative images (c) and measurements (d) revealing differences in stripe width between wild-type (Sfrp2+/+) and Sfrp2 knockout (Sfrp2−/−) pups (P = 0.000075 (R1′), P = 0.025105 (R1′′), P = 0.401739 (R2), P = 0.000008 (R3); ANOVA test; Sfrp2+/+; n = 10; Sfrp2−/−, n = 7). e, Representative differences in coat colour between Sfrp2+/+ and Sfrp2−/− adult mice taken as a single image. f, Quantification of pigmentation between Sfrp2+/+ and Sfrp2−/− adult mice reveal stripe-specific changes in colour (P = 0.011884 (R1′), P = 0.493389 (R1’′), P = 0.000719 (R2), P = 0.835735 (R3); ANOVA test; Sfrp2+/+, n = 3; Sfrp2−/−, n = 3). g, Model describing the role of Sfrp2 in establishing (top) and implementing (bottom) the coat pattern in striped mice. Scale bar in c, 500 pixels. Schematic in a created with BioRender.com.

Article Snippet: To generate LV- SFRP2-GFP , we amplified the Sfrp2 coding sequence from an Sfrp2 ( NM_009144 ) Mouse Tagged ORF Clone (Origene catalogue no. MR204070) using CloneAmp HiFi PCR Premix (Takara 639298) and cloned it (in frame) into the linearized LV- GFP plasmid using In-Fusion Snap Assembly Master Mix (Takara no. 638947).

Techniques: In Vivo, Knock-Out

Journal: Nature ecology & evolution

Article Title: A multifunctional Wnt regulator underlies the evolution of rodent stripe patterns

doi: 10.1038/s41559-023-02213-7

Figure Lengend Snippet: a, Schematic of the Sfrp2 locus (exons in red) showing the transcriptional start site (TSS), protospacer adjacent motif (PAM) short guide RNA (sgRNA) target/sequence. Four types of deletions were achieved: 2 bp, 13 bp, 466 bp 527 bp (white boxes). All mutations are predicted to cause frameshift mutations. b, Representative western blot of individuals carrying different combinations of wild-type and a 13 bp deleted allele (wild type: Sfrp2+/+; heterozygous: Sfrp2+/−; homozygous: (Sfrp−/−). Sfrp−/− have no detectable SFRP2 Protein (green). Bands ~30 kDa correspond to SFRP2 protein. b-TUBULIN (~50 kDa, red) was used as a loading control. In b, two different individuals from each genotype were analysed.

Article Snippet: To generate LV- SFRP2-GFP , we amplified the Sfrp2 coding sequence from an Sfrp2 ( NM_009144 ) Mouse Tagged ORF Clone (Origene catalogue no. MR204070) using CloneAmp HiFi PCR Premix (Takara 639298) and cloned it (in frame) into the linearized LV- GFP plasmid using In-Fusion Snap Assembly Master Mix (Takara no. 638947).

Techniques: In Vivo, Sequencing, Western Blot, Control

Journal: Nature ecology & evolution

Article Title: A multifunctional Wnt regulator underlies the evolution of rodent stripe patterns

doi: 10.1038/s41559-023-02213-7

Figure Lengend Snippet: In situ hybridization showing specific Sfrp2 expression in the dermal papilla of P4 striped mouse hair follicles. b, Melanocytes were stably transduced with either a control (LV-GFP) or an experimental (LV-Sfrp2GFP) lentivirus and expression of Wnt targets and melanogenesis genes in stably transduced control and experimental cells, as determined was determined via qPCR (P = 0.12026 (Axin); P = 0.001816 (C-myc); P = 0.006739 (CyclinD); P = 0.001040 (Mitf); P = 0.010712 (Tyr); ANOVA test; N = 4). c, Quantitative PCR (qPCR) showing Sfrp2 mRNA fold change levels along different dorsal skin regions in embryonic and postnatal stages (E16.5: P = 0.0283 (R1vsR2); P = 0.0062 (R1vsR3); P = 0.3959 (R2vsR3); E19.5: P = 0.8685 (R1vsR2); P = 0.6319 (R1vsR3); P = 0.9015 (R2vsR3); P0: P = 0.9724 (R1vsR2); P = 0.8207 (R1vsR3); P = 0.6971 (R2vsR3); P4: P = 0.0003 (R1vsR2); P = 0.0022 (R1vsR3); P = 0.0001 (R2vsR3); ANOVA test; N = 3 for E16.5, E19.5 P0, N = 4 for P4). Scale bars in a: 100 μm (left) and 25 μm (right). In a, three different individuals were analysed. In b and c, data are presented as mean values +/− SEM.

Article Snippet: To generate LV- SFRP2-GFP , we amplified the Sfrp2 coding sequence from an Sfrp2 ( NM_009144 ) Mouse Tagged ORF Clone (Origene catalogue no. MR204070) using CloneAmp HiFi PCR Premix (Takara 639298) and cloned it (in frame) into the linearized LV- GFP plasmid using In-Fusion Snap Assembly Master Mix (Takara no. 638947).

Techniques: In Situ Hybridization, Expressing, Stable Transfection, Transduction, Control, Real-time Polymerase Chain Reaction

Journal: Nature ecology & evolution

Article Title: A multifunctional Wnt regulator underlies the evolution of rodent stripe patterns

doi: 10.1038/s41559-023-02213-7

Figure Lengend Snippet: a, Phylogenetic tree of murid species used in our comparative genomic analyses. Phylogeny was redrawn from a previous study46. b, RERs in different murid species shows that Sfrp2 does not have an elevated evolutionary rate in the striped mouse (R. pumilio; red dot). c, The Sfrp2 locus contains five nearby CREs, whose synteny is conserved in laboratory mouse (M. musculus), striped mouse (R. pumilio) and African grass rat (A. niloticus). Figure illustrates examples of striped mouse-specific deletions and insertions leading to changes in transcription factor (TF) consensus binding motifs. Box colours illustrate whether a TF-binding motif is conserved across all three species or unique to striped mouse.

Article Snippet: To generate LV- SFRP2-GFP , we amplified the Sfrp2 coding sequence from an Sfrp2 ( NM_009144 ) Mouse Tagged ORF Clone (Origene catalogue no. MR204070) using CloneAmp HiFi PCR Premix (Takara 639298) and cloned it (in frame) into the linearized LV- GFP plasmid using In-Fusion Snap Assembly Master Mix (Takara no. 638947).

Techniques: Binding Assay