Journal: bioRxiv

Article Title: Generation of human hindlimb/genital tubercle progenitors from pluripotent stem cells

doi: 10.64898/2026.04.14.718471

Figure Lengend Snippet: (A) Schematics showing the location and signalling requirements of NMP and pLPM progenitors in the mouse E8.5 embryo (left) and the treatment conditions used to induce NMPs and pLPM cells from hPSCs (right). (B) RT-qPCR-based expression analysis of key NMP and pLPM markers after 3 days of iPSC differentiation toward NMPs in the presence and absence of BMP4 ( n = 3 independent experiments). Relative expression levels to iPSCs were normalised to GAPDH gene expression (* p<0.05, ** p<0.01, *** p<0.001, mean ±SD) (paired, two-tailed t -test). (C) Immunofluorescence analysis of the expression of TBXT and ISL1 on day 3 of differentiation. Scale bars represent 100 µm. Image analysis of the percentage of nuclei positive for TBXT and ISL1 protein expression is also shown. Graph shows mean values ( n = 3 independent experiments) (* p<0.05, ** p<0.01, *** p<0.001, mean ±SD) (unpaired t -test with Welch’s correction). (D) Heatmap of differentially expressed genes showing key NMP and pLPM/HGTp markers from bulk RNA-seq analysis on day 3. Expression values are shown as Z-scores across samples. (E) Heatmap of differentially expressed genes showing KEGG signalling components on day 3. Expression values are shown as Z-scores across samples.

Article Snippet: Following primary antibodies were used; TBXT (Abcam, ab209665, 1:1000), ISL1 (DSHB, 39.4D5, 1:200), HAND1 (R&D Systems, AF3168, 1:100), PITX1 (Novus Bio, NBP1-88644, 1:500), HOXC9 (Abcam, ab50839, 1:200), KDR (R&D Systems, AF357, 1:1000), TP63 (Abcam, ab124762, 1:200).

Techniques: Quantitative RT-PCR, Expressing, Gene Expression, Two Tailed Test, Immunofluorescence, RNA Sequencing

Journal: bioRxiv

Article Title: Generation of human hindlimb/genital tubercle progenitors from pluripotent stem cells

doi: 10.64898/2026.04.14.718471

Figure Lengend Snippet: (A) UMAP visualisation of sc-RNA seq data from late RA treated cells collected on day 10 of differentiation. Cells were divided into 3 clusters indicated by different colours. Below: Bar graph showing the percentage of total cells per cluster. (B) Stacked violin plots showing the expression (log-normalized) of key lineage markers enriched across identified cell clusters. (C) Dot plot showing the proportion of cells per cluster expressing the indicated marker genes. The colour bar indicates the average log-normalized expression values. (D, E) Feature UMAPs showing the expression of representative marker genes (log-normalized). (F) UMAP showing the proportions of ISL+/TBX5+/TBX4+ cells across the identified cell clusters. Below: Bar graph showing the percentage of cells per cluster calculated for each cluster. (G) Dot plot showing the percentage expression of key surface ectoderm markers for each cell cluster. The colour bar indicates the average log-normalized expression values. (H) Immunofluorescence analysis of the expression of TP63 and ISL1 on day 10 HGTps. Sequential z-stacks are shown (z=1 bottom, z=20 top). Scale bars represent 100 µm.

Article Snippet: Following primary antibodies were used; TBXT (Abcam, ab209665, 1:1000), ISL1 (DSHB, 39.4D5, 1:200), HAND1 (R&D Systems, AF3168, 1:100), PITX1 (Novus Bio, NBP1-88644, 1:500), HOXC9 (Abcam, ab50839, 1:200), KDR (R&D Systems, AF357, 1:1000), TP63 (Abcam, ab124762, 1:200).

Techniques: RNA Sequencing, Expressing, Marker, Immunofluorescence

Journal: bioRxiv

Article Title: Dyrk1a gene dosage controls bipolar cell development and retinal connectivity

doi: 10.64898/2026.03.15.710015

Figure Lengend Snippet: (A – C) Representative coronal sections taken from the retina of Lhx2-Cre (A), Lhx2-Cre:Dyrk1a +/f (B) and Lhx2-Cre:Dyrk1a f/f (C) at E14.5 to illustrate the presence of Isl1/2 + neurons and cleaved Caspase3 + apoptotic cells (A – C, arrowheads in inset images). (D – F) Representative coronal sections taken from the retina of Lhx2-Cre (D), Lhx2-Cre:Dyrk1a +/f (E) and Lhx2-Cre:Dyrk1a f/f (D) at P0 to illustrate the presence of Isl1/2 + neurons and cleaved Caspase3 + apoptotic cells (D – F, arrowheads in inset images). (G – H) Quantitative analysis of Lhx2-Cre ( n = 5), Lhx2-Cre:Dyrk1a +/f ( n = 5) and Lhx2-Cre:Dyrk1a f/f ( n = 5) animals at E14.5 (G) and P0 (H) demonstrates a significant reduction in the number of amacrine and RGCs in the retinas of homozygous animals at both ages. (I – J) Quantitative analysis of Lhx2-Cre ( n = 5), Lhx2-Cre:Dyrk1a +/f ( n = 5) and Lhx2-Cre:Dyrk1a f/f ( n = 5) mice at E14.5 (I) and P0 (J) demonstrates a significant increase the number of apoptotic cells in the retinas of Lhx2-Cre:Dyrk1a f/f animals at both ages. All data represents the mean ± SEM values. Statistical differences were calculated using one-way ANOVA followed by a Tukey’s multiple comparison test. p-values are denoted as follows: *p ≤ 0.05, **p ≤ 0.01 and ****p ≤ 0.0001. Scale bars: (A – F) 100 μm. Abbreviations: Casp3, cleaved Caspase3; CM, ciliary margin; D, dorsal; LE, lens; NR, neural retina; RPE, retinal pigment epithelium; V, ventral.

Article Snippet: The following primary antibodies and dilutions were used in this study: Calbindin (1:300, Sigma-Aldrich, #C9848); Calretinin (1:1000, Swant, #CG1); cleaved Caspase3 (Asp175) (1:500, Cell Signalling, #9661S); chAT (1:100, Millipore, #AB144P); Chx10 (1:50, SCBT, #SC365519); Dyrk1a (1:100, Novus, #NBP1-84032); Isl1/2 (recognises both Isl1 and Isl2 proteins) (1:500; DSHB, #39.4D5); M-opsin (1:500, Proteintech, #30975-1-AP); Pax6 (1:100, DSHB, #Pax6); Pikachurin (1:200, Proteintech, #14578-1-AP); Rhodopsin (1:500, Millipore, #MABN15); S-opsin (1:500, Proteintech, #24660-1-AP); TH (1:300, Millipore, #AB152); vGlut1 (1:2000, Millipore, #AB5905).

Techniques: Comparison

Journal: bioRxiv

Article Title: Central amygdala Isl1 neurons control biting by integrating sensory and motivational signals

doi: 10.64898/2026.02.03.703447

Figure Lengend Snippet: A . Representative histological image of EGFP expression in the CeA. Scale bar, 100 µm. B . Representative image showing Isl1 immunostaining (green) and Ai9 reporter expression (red) with DAPI (blue) in an Isl1-CreER: Ai9 mouse. The CeA subregions are outlined. Scale bar, 30 µm. C . Quantification of the proportion of Ai9 + neurons expressing Isl1 and the proportion of Isl1 + neurons expressing Ai9 (n = 3 mice). D , Heatmap of marker gene expression for CeA neuronal subpopulations. Cells are grouped and color-coded by transcriptionally defined clusters (top), as described in a previous study . Red outlines and red text highlight the Isl1-positive population. E . Quantification of mean number of biting bouts for objects with varying physical properties (n=10 mice; N.S.; one-way ANOVA test) F . Quantification of bite duration for objects with varying physical properties (n=10 mice; ***P <0.001, **P <0.01; one-way ANOVA test). G . Average responses of the z-scored calcium activity for each condition: softwood, Styrofoam, sponge, cricket, and chow. Shaded areas represent s.e.m. H . Representative z-scored calcium trace from a single neuron aligned to behavioral events: first bite (red), eating (blue), and chewing (green). I-M , Feeding behavior. I . Mean neuronal activity during gnawing and chewing epochs (n=10 mice; **P < 0.01; Wilcoxon signed-rank test). J . Mean neuronal activity during gnawing versus non-gnawing epochs (n=10 mice; **P < 0.01; Wilcoxon signed-rank test). K . Mean neuronal activity during chewing versus non-chewing epochs (n=10 mice; N.S.; Wilcoxon signed-rank test). L . Proportion of neurons up-modulated, down-modulated, and non-modulated during gnawing. M . Proportion of neurons modulated during chewing. N-R . Cricket hunting behavior. N . Mean neuronal activity during gnawing and pursuit epochs (n=10 mice; N.S.; Wilcoxon signed-rank test). O . Mean neuronal activity during gnawing and holding epochs (n=10 mice; N.S.; Wilcoxon signed-rank test). P . Proportion of neurons up-modulated, down-modulated, and non-modulated during gnawing. Q . Proportion of neurons modulated during pursuit. R . Proportion of neurons modulated during holding.

Article Snippet: The Isl1-CreER transgenic line (Isl1 tm1 (cre/Esr1*) Krc /SevJ) was purchased from Jackson Laboratories.

Techniques: Expressing, Immunostaining, Marker, Gene Expression, Activity Assay

Journal: bioRxiv

Article Title: Central amygdala Isl1 neurons control biting by integrating sensory and motivational signals

doi: 10.64898/2026.02.03.703447

Figure Lengend Snippet: A . Schematic of optic-fiber placement above CeA Isl1 neurons expressing either ChR2 or EGFP. B . Representative frame from video showing a mouse grasping and biting linguine. C . Mean number of biting bouts across stimulation conditions (n= 12 mice per group; **p < 0.01, two-way ANOVA with Tukey’s post hoc test). D . Biting bout duration during 20 Hz stimulation (*p < 0.05, unpaired t-test). E . Weight of linguine consumed by 4 h fasted animals (N.S.; unpaired t-test). F . Schematic of a mouse freely biting pieces of chow/Styrofoam. G . Raster plot showing biting bouts (blue) and fictive feeding (orange) across individual fed ChR2 mice during alternating light OFF and light ON epochs in biting food assay. H-K . Quantification of behaviors during ON and OFF periods for both ChR2 mice and EGFP mice in biting food assay. H . Percentage of time spent biting food. I . Mean number of food-biting bouts. J . Percentage of time engaged in fictive feeding. K . Mean number of fictive feeding bouts. N= 13 mice per group. Two-way ANOVA with Tukey’s post hoc test, * P1<10.05, **P1<10.01, ***P1<10.001, ****P1<10.0001. L . Same as in panel G but for Styrofoam pieces. M-P . Quantification of behaviors during ON and OFF periods for both ChR2 mice and EGFP mice in biting Styrofoam assay. M . Percentage of time spent biting Styrofoam. N . Mean number of Styrofoam-biting bouts. O . Percentage of time engaged in fictive feeding. P . Mean number of fictive feeding bouts. N= 13 mice per group. Two-way ANOVA with Tukey’s post hoc test, * P ⍰ <10.05, **P ⍰ <10.01, ***P ⍰ <10.001,****P ⍰ <10.0001. Q . Probability density distributions of distances of the animal to the food source during biting (blue) and fictive feeding (orange) events. R . Quantification of mean distances for each behavior across animals (n = 7 mice, ****p < 0.0001, two-tailed unpaired t-test). S . Schematic of optic-fiber placement above CeA Isl1 neurons expressing either Arch3.0 or EGFP. T . Raster plots showing eating bouts (grey for eating bouts and green for eating bouts longer than 130 s) across light OFF and ON epochs in Arch3.0and EGFP groups during linguine consumption assays. U . Duration of individual feeding bouts across light OFF and ON epochs in Arch3.0 and EGFP groups during linguine consumption assays (n = 7 mice per group; * p < 0.05, **p < 0.01; two-way ANOVA with Tukey’s post hoc test). V . Schematic of Myomatrix microarrays (bottom), each containing eight electrode contacts on a flexible substrate, implanted into the masseter and temporalis muscles for electromyographic (EMG) recordings. W . Representative EMG traces from the right masseter and temporalis muscles of a freely behaving Isl1-CreER mouse expressing the inhibitory DREADD hM4Di in the central amygdala. Traces show activity during food biting after intraperitoneal injection of saline or CNO (2 mg kg −1 ). X . EMG amplitude measurements from the masseter muscle during food biting after saline or CNO (0.4 mg kg −1 ) injection in Isl1-CreER; hM4Di mice. n=4; ****p < 0.0001. Y . EMG amplitude measurements from the temporalis muscle during food biting after saline or CNO (0.4 mg kg −1 ) injection in Isl1-CreER; hM4Di mice. n=4; ****p < 0.0001.

Article Snippet: The Isl1-CreER transgenic line (Isl1 tm1 (cre/Esr1*) Krc /SevJ) was purchased from Jackson Laboratories.

Techniques: Expressing, Two Tailed Test, Muscles, Activity Assay, Injection, Saline

Journal: bioRxiv

Article Title: Central amygdala Isl1 neurons control biting by integrating sensory and motivational signals

doi: 10.64898/2026.02.03.703447

Figure Lengend Snippet: A . Raster plot showing biting bouts (blue) and fictive feeding (orange) across individual 4-h food-deprived ChR2 mice during alternating light OFF and light ON epochs in biting food assay. B-E . Quantification of behaviors during light ON and OFF periods for both ChR2 mice (n = 14) and EGFP mice (n = 4) in food biting assay. B . Percentage of time spent biting food. C . Mean number of f food-biting bouts. D . Percentage of time engaged in fictive feeding. E . Mean number of fictive feeding bouts. Two-way ANOVA with Tukey’s post hoc test, **P < 0.01, ****P < 0.0001. Blue is ChR2 group, and grey is EGFP. F . Schematic of the paradigm for testing the effects of photoactivation on feeding behavior. G . Food intake by fed ChR2 mice (n = 16) and EGFP mice (n = 9). N.S.; two-way ANOVA with Tukey’s post hoc test. H . Schematic of CeA Isl1 neurons expressing either the excitatory DREADD hM3Dq or control mCherry. I . Cumulative food intake of fed Isl1-CreER animals that expressing the excitatory DREADD hM3Dq or control mCherry in the CeA, after i.p. injections of saline and CNO (2 mg/Kg). N = 7 mice per group; N.S.; two-way ANOVA with Tukey’s post hoc test. J-O . Quantification of different behavioral epochs during light ON and OFF period for both ChR2 mice (n = 13) and EGFP mice (n = 7) in cricket hunting tasks. J . Time spent investigating crickets. K . Time spent pursuing crickets. L . Time spent capturing crickets. M . Time spent on fictive feeding. N . Time spent eating crickets. O . Number of crickets killed. Two-way ANOVA with Tukey’s post hoc test, *P < 0.05, ***P < 0.0001, ****P < 0.0001. P . Schematic of the paradigm for testing the effects of photoactivation on novel object exploration. Q . Time spent in the object area for both ChR2 mice (n = 8) and EGFP mice (n = 3); N.S.; two-way ANOVA with Tukey’s post hoc test. R . Mean number f entering the object area for both ChR2 mice (n = 8) and EGFP mice (n = 3) N.S.; two-way ANOVA with Tukey’s post hoc test.

Article Snippet: The Isl1-CreER transgenic line (Isl1 tm1 (cre/Esr1*) Krc /SevJ) was purchased from Jackson Laboratories.

Techniques: Expressing, Control, Saline

Journal: bioRxiv

Article Title: Central amygdala Isl1 neurons control biting by integrating sensory and motivational signals

doi: 10.64898/2026.02.03.703447

Figure Lengend Snippet: A . Mean number of feeding bouts across light OFF and ON epochs in Arch3.0 and EGFP groups during linguine consumption assays (N.S.; two-way ANOVA with Tukey’s post hoc test). B . Cumulative consumption of the first, second, and third linguine pieces across light OFF and ON epochs in Arch3.0 and EGFP groups (N.S.; two-way ANOVA with Tukey’s post hoc test). C . Linguine intake by 4-h food-deprived Arch3.0 mice (n = 7) and EGFP mice (n = 5) during alternating light OFF and light ON epochs. N.S.; two-way ANOVA with Tukey’s post hoc test. D . Example frame of a mouse consuming spaghetti. E . Raster plots showing eating bouts (black) and consumption duration of the second spaghetti piece (green) across light OFF and ON epochs in Arch3.0 and EGFP groups during spaghetti consumption assays. F . Duration of second spaghetti consumption across light OFF and ON epochs in Arch3.0 and EGFP groups (n = 7 mice per group; *P < 0.05, **P < 0.01; Two-way ANOVA with Tukey’s post hoc test). G . Duration of first spaghetti consumption across light OFF and ON epochs in Arch3.0 (n = 8) and EGFP (n = 5) groups. N.S.; two-way ANOVA with Tukey’s post hoc test. H . Duration of third spaghetti consumption across light OFF and ON epochs in Arch3.0 (n = 8) and EGFP (n = 5) groups. N.S.; two-way ANOVA with Tukey’s post hoc test. I . Duration of individual feeding bouts across light OFF and ON epochs in Arch3.0 (n = 8) and EGFP (n = 5) groups during spaghetti consumption assays. N.S.; two-way ANOVA with Tukey’s post hoc test. J . Raster plots showing eating bouts (orange for Arch3.0 and grey for EGFP) across light OFF and ON epochs in Arch3.0 and EGFP groups during chow consumption assays. K . Duration of individual feeding bouts across light OFF and ON epochs in Arch3.0 and EGFP groups during chow consumption assays (n = 7 mice per group; N.S.; Two-way ANOVA with Tukey’s post hoc test). L . Mean number of feeding bouts across light OFF and ON epochs in Arch3.0 and EGFP groups during chow consumption assays (n = 7 mice per group; N.S.; Two-way ANOVA with Tukey’s post hoc test). M . Cumulative consumption of the chow across light OFF and ON epochs in Arch3.0 and EGFP groups (n = 7 mice per group; N.S.; Two-way ANOVA with Tukey’s post hoc test). N . Food intake by 20-h food-deprived Arch3.0 mice (n = 7) and EGFP mice (n = 5). N.S.; two-way ANOVA with Tukey’s post hoc test. O . Food intake by 4-h food-deprived Arch3.0 mice (n = 7) and EGFP mice (n = 5). N.S.; two-way ANOVA with Tukey’s post hoc test. P . EMG amplitude measurements from the masseter muscle during softwood biting after saline or CNO (0.4 mg kg −1 ) injection in Isl1-CreER; hM4Di mice. n=4; **P < 0.01; paired t-test. Q . EMG amplitude measurements from the temporalis muscle during softwood biting after saline or CNO (0.4 mg kg −1 ) injection in Isl1-CreER; hM4Di mice. n=4; ***P < 0.001; paired t-test. R . EMG amplitude measurements from the masseter muscle during Styrofoam biting after saline or CNO (0.4 mg kg −1 ) injection in Isl1-CreER; hM4Di mice. n=4; **P < 0.01; paired t-test. S . EMG amplitude measurements from the temporalis muscle during Styrofoam biting after saline or CNO (0.4 mg kg −1 ) injection in Isl1-CreER; hM4Di mice. n=4; ****P < 0.0001; paired t-test. T . EMG amplitude measurements from the masseter muscle during soft silicone biting after saline or CNO (0.4 mg kg −1 ) injection in Isl1-CreER; hM4Di mice. n=4; N.S; paired t-test. U . EMG amplitude measurements from the temporalis muscle during soft silicone biting after saline or CNO (0.4 mg kg −1 ) injection in Isl1-CreER; hM4Di mice. n=4; ***P < 0.001; paired t-test. V . EMG amplitude measurements from the masseter muscle during sponge biting after saline or CNO (0.4 mg kg −1 ) injection in Isl1-CreER; hM4Di mice. n=4; *P < 0.05; paired t-test. W . EMG amplitude measurements from the temporalis muscle during sponge biting after saline or CNO (0.4 mg kg −1 ) injection in Isl1-CreER; hM4Di mice. n=4; N.S; paired t-test.

Article Snippet: The Isl1-CreER transgenic line (Isl1 tm1 (cre/Esr1*) Krc /SevJ) was purchased from Jackson Laboratories.

Techniques: Saline, Injection

Journal: bioRxiv

Article Title: Central amygdala Isl1 neurons control biting by integrating sensory and motivational signals

doi: 10.64898/2026.02.03.703447

Figure Lengend Snippet: A . Representative images of brain areas innervated by CeA Isl1 neurons. Scale bar, 100 µm. B . Major brain regions innervated by CeA Isl1 neurons depicted as percentage (mean ± s.e.m.) of total outputs (n = 3 mice). C . Schematic showing viral targeting of CeA Isl1 neurons and terminal stimulation in the PCRt using 473 nm light in Isl1-CreER mice. D . Raster plot showing biting bouts (pink) and fictive feeding bouts (orange) across individual ChR2 mice during alternating light OFF and light ON epochs in biting Styrofoam and food assay. E-G . Quantification of behaviors during ON and OFF periods for both ChR2 (pink) mice and EGFP (grey) mice in biting assay. E . Duration of biting Styrofoam. F . Duration of biting food. G . Duration of fictive feeding. N = 5 mice per group. Two-way ANOVA with Tukey’s post hoc test, *P1<10.05, ***P1<10.001, ****P1<10.0001. H . Representative heatmap showing occupancy plots of CeA Isl1 → PCRt ChR2 mice. I . Quantification of time spent in the light-paired (ON) and non-paired (OFF) compartments (n = 5 mice per group; N.S., two-tailed unpaired t-test). J . Average velocity in the light-paired (ON) and non-paired (OFF) compartments (n = 5 mice per group; N.S., two-tailed unpaired t-test). K . Total distance moved in the light-paired (ON) and non-paired (OFF) compartments (n = 5 mice per group; N.S., two-tailed unpaired t-test). L . Schematic showing viral targeting of CeA Isl1 neurons and terminal stimulation in the PPTg using 473 nm light in Isl1-CreER mice. M . Raster plot showing biting bouts (green) and fictive feeding bouts (orange) across individual ChR2 mice during alternating light OFF and light ON epochs in biting Styrofoam and food assay. N-P . Quantification of behaviors during ON and OFF periods for both ChR2 (green) mice and EGFP (grey) mice in biting assay. N . Duration of biting Styrofoam. O . Duration of biting food. P . Duration of fictive feeding. N = 5 mice per group. Two-way ANOVA with Tukey’s post hoc test, *P ⍰< ⍰ 0.05, **P ⍰ < ⍰ 0.01. Q . Representative heatmap showing occupancy plots of CeA Isl1 → PPtg ChR2 mice. R . Quantification of time spent in the light-paired (ON) and non-paired (OFF) compartments (n = 5 mice per group; *p < 0.05, two-tailed unpaired t-test). S . Average velocity in the light-paired (ON) and non-paired (OFF) compartments (n = 5 mice per group; **p < 0.01, two-tailed unpaired t-test). T . Total distance moved in the light-paired (ON) and non-paired (OFF) compartments (n = 5 mice per group; N.S., two-tailed unpaired t-test). Abbreviations: BNST, the bed nucleus of the stria terminalis; MiTg, microcellular tegmental nucleus; NTS, nucleus of the solitary tract; PAG, periaqueductal gray; PBN, parabrachial nucleus; PCRt; parvicellular reticular nucleus; PP, peripeduncular nucleus; PPTg, pedunculopontine tegmental nucleus; PIL, posterior intralaminar thalamic nucleus; PoT, posterior thalamic nuclear group, triangular Part; Rt, reticular thalamic nucleus; SNL, substantia nigra, lateral part; SNC, substantia nigra, compact part; STh, subthalamic nucleus; Su5, supratrigeminal nucleus; scp superior cerebellar peduncle (brachium conjunctivum); VPL, ventral posterolateral thalamic nucleus; VPM, ventral posteromedial thalamic nucleus.

Article Snippet: The Isl1-CreER transgenic line (Isl1 tm1 (cre/Esr1*) Krc /SevJ) was purchased from Jackson Laboratories.

Techniques: Two Tailed Test