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Figure 2. Differential contributions of Mesp1 and Pax3 lineages to neck and shoulders. ; Immunostainings on coronal cryosections of E18.5 Mesp1Cre;R26tdTomato and Pax3Cre;R26tdTomato mice for the myofibre Tnnt3 and Tomato markers at levels indicated in Figure 1. Higher magnifications of selected areas in (A–J) are shown in Figure 2—figure supplement 2; (n = 2 for each condition). See also the atlas of neck musculature in Figure 2—figure supplement 1. (A–H) Mesp1Cre labeled all neck muscles including branchiomeric (myh, esm, phm and ilm), cucullaris (stm, atp), somitic epaxial (epm) and hypaxial (tg, lcp, lcl, ifh) muscles. Pax3Cre marked somitic muscles. (I–J) At shoulder level, Mesp1-derived cells did not contribute to posterior somitic myofibres including scapular muscles (scp) compared to that observed in Pax3Cre embryos. ac, arytenoid cartilage; acp, scapular acromion process; atp, acromiotrapezius; cc, cricoid cartilage; epm, epaxial musculature; esm, esophagus striated muscle; hh, humeral head; ifh, infrahyoid muscles; ilm, intrinsic laryngeal muscles; lcl, longus colli; lcp, longus capitis; myh, mylohyoid; ob, occipital bone; oc, otic capsule; phm, pharyngeal muscles; stm, sternocleidomastoid; scp, scapular musculature; tc, thyroid cartilage; tg, tongue. Scale bars: in J for A-B 200 µm, for C-J 400 µm.

2018

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Figure 7—figure supplement 1. EPM metrics. ; (A) CNO developmental controls displayed increased open-arm preference, commitment index, exploration entrance, and distance traveled compared to untreated controls in distributions of the three z-score-normalized control groups. (B) In EPM, no differences in distance traveled were detected for acutely perturbed mice. Lobule VII and crus II perturbations displayed reduced distance traveled compared to controls.

2018

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Figure 3—figure supplement 2. Neural crest contribution to neck and pectoral structures. ; Contribution of Wnt1-derived NCCs to connective tissue of branchiomeric (mas, myh, esm) and somitic muscles (tg, ifh, ptm, epm, scp) (refer to Figure 3, n = 2) (A–H). Note that the esophagus (esm) and infrahyoid (thy, ohy, shy) muscles show little or no NCC contribution to connective tissue (C–D’). Only few Wnt1-derived cells are present at connection sites of cucullaris-derived muscles on the pectoral girdle (E–H). atp, acromiotrapezius; cl, clavicle; clm, cleidomastoid; clo, cleido-occipitalis; epm; epaxial neck musculature; esm, esophagus striated muscle; hy, hyoid bone; ifh, infrahyoid muscles; lcl, longus colli; mas, masseter; md; mandible; myh, mylohyoid; nl, nuchal ligament; ohy, omohyoid; ptm, pectoralis muscles; shy, sternohyoid; spn, scapular spine; scp, scapular muscles; st, sternum; std, sternomastoid; tc, thyroid cartilage; thy, thyrohyoid; tg, tongue; tr, tracheal ridge. Scale bars: in H for A-C, H 100 µm, for A’-C’, D-G 200 µm; for D’ 400 µm.

2018

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Figure 5—figure supplement 2. Viral-mediated expression of GPR158 in mPFC of Gpr158−/− mice rescues the antidepressant-like phenotype. ; (A) mPFC of Gpr158−/− mice was injected bilaterally with Adenovirus expressing either EGFP or GPR158. Western blot showing the expression of GPR158 only in mice injected with GPR158-Ad (right panel). Gpr158−/− mice injected with EGFP-Ad or GPR158-Ad into the mPFC underwent the (B) marble burying test, (C) EPM, (D) TST and (E) FST. (F) Emotionality score was calculated using the individual behavioral measures from the MB, EPM, TST and FST. Data shown as means ± SEM (n = 7–8 mice/group, Student’s t test,*p

2018

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Figure 1. Genetic lineage tracing of neck muscle progenitors. ; Whole-mount X-gal stainings of Mef2c-AHFCre;R26R, Islet1Cre;Pax7GPL, Mesp1Cre;Pax7GPL and Pax3Cre;Pax7GPL mice at E10.5 (A–D), E11.75 (E–H) and E18.5 (I–L’) (n = 3 for each condition). See associated Figure 1—supplements 1–3. (A–H) Note labeling of mesodermal core of pharyngeal arches (PAs) and cucullaris anlage (ccl) by Mef2c-AHF, Islet1 and Mesp1 lineage reporters; β-gal+ cells in anterior somites of Mesp1Cre embryos and in the clp anlagen of Islet1Cre embryos. Pax3 lineage marked somitic mesoderm. (I–L’) Mef2c-AHF, Islet1 and Mesp1 lineages marked branchiomeric (mas, tpr, dg) and cucullaris muscles (stm, atp and stp). Pax3Cre and Mesp1Cre labeled somitic epaxial neck muscles (epm). atp, acromiotrapezius; ccl, cucullaris anlage; clp, cutaneous maximus/latissimus dorsi precursor; dg, digastric; epm, epaxial musculature; h, heart; hc, hypoglossal cord; lbm, limb muscle anlagen and limb muscles; ltd, latissimus dorsi; mas, masseter; nc, nasal capsule; nt, neural tube; PA1-2, pharyngeal arches 1–2; S3, somite 3; stm, sternocleidomastoid; stp, spinotrapezius; tpr; temporal. Scale bars: in D for A-D and in H for E-H, 1000 µm; in L for I-L’, 2000 µm.

2018

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Figure 7—figure supplement 2. Shisa7 KO mice display no abnormalities in shock sensation, locomotor activity or anxiety-related behavior. ; (a) Mean velocity in the 3 minutes prior to shock (left), or during the 2 s 0.7 mA shock (right) was not different between Shisa7 KO (n = 10) and WT littermates (n = 9), indicating no effect on basal locomotor activity (unpaired t-test p=0.245) or the perception of the shock (unpaired t-test p=0.573). (b–d) A set of anxiety tests (open field, elevated plus maze (EPM), and dark-light box (DL-box)) was carried out sequentially in a batch of Shisa7 KO (n = 8) and WT littermates (n = 8) (cf. Supplementary Figure 2). (b) Open field exploratory behavior (total distance moved) or the anxiety-related component (time spent in the center) was not different between genotypes (MWU tests, p=0.798; p=0.878, respectively). (c) EPM anxiety parameters were similar for Shisa7 and WT mice for the percentage of visits to the open arms (MWU test, p=0.105), and time spent in the open arms (MWU test, p=0.234). Only in this test, one Shisa7 KO mouse was identified as outlier, and when removed this made the difference even smaller (p=0.189, p=0.397). (d) None of the anxiety-motivational parameters measured in the DL-box showed a genotype difference, with time spent in the light compartment, visits to the light compartment and latency to visit to the light compartment being non-significant (MWU tests, p=1.000; p=1.000; p=0.121, respectively). Only for latency, one WT mouse was identified as outlier, and when removed this made the difference even smaller (p=0.209).

2017

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Figure 1. Anxiety-related behavior in Cln3Δex1-6 mice. ; (A) Escape time to cross the rim barrier of a petri dish was increased in Cln3Δex1-6 mice compared to wt littermates indicating reduced explorative behavior (wt: n = 15/24 [7 month/14 month]; Cln3Δex1-6: n = 14/18; Mann-Whitney Test). (B) Increased anxiety-related behavior of Cln3Δex1-6 mice at the age of 14 months in the open field (OF) test. Representative tracks and data plots for OF related to genotypes. Note that Cln3Δex1-6 mice visited the center area in the OF less frequently and spent more time in the peripheral areas (n = 18 vs. 20; Mann-Whitney test). (C) Increased anxiety-related behavior in the elevated plus maze (EPM) test at the age of 14 months. In the EPM Cln3Δex1-6 mice showed less visits and reduced travel distance in the open arms and preferred to stay in the closed arms indicating increased anxiety-related behavior. Microbehavior analysis revealed reduced stretchings confirming reduced explorative drive in both test paradigms (n = 18 vs. 20; open arm distance: Mann-Whitney, all other Student`s t-test). Exact values of N, dispersion and precision measures are provided in Supplementary file 2.

2017

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Figure 2—figure supplement 2. Mesp1 and Pax3 lineage contributions to neck and shoulder muscles. ; High magnifications of selected areas of panels in Figure 2 at levels indicated in Figure 1. (A–J’) Immunostainings are indicated (n = 2 each condition). ac, arytenoid cartilage; atp, acromiotrapezius; epm, epaxial musculature; hpm, hypaxial musculature; ilm, intrinsic laryngeal muscles; myh, mylohyoid; phm, pharyngeal muscles; scp, scapular muscles; tc, thyroid cartilage; tg, tongue. Scale bars: in J’ for A-J’ 50 µm.

2018

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Supplementary file 1. Statistical results for Figure 1—figure supplement 1. ; Abbreviations: CMS, chronic mild unpredictable stress; EPM, elevated plus maze; FST, forced swimming test; NSF, novelty-suppressed feeding; OFT, open field test; SPT, sucrose preference test

2018

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Figure 4—figure supplement 2. Prx1 lineage contribution to neck and limbs. ; (A–C) Immunostainings on coronal cryosections (n = 2). Note Prx1-derived cells contributing to a great extent to form MCT, tendons and skeletal components of limb and shoulder (A–B); less β-gal+ cells are seen in acromiotrapezius (B); no contribution in neck epaxial muscles (C). (D–D”) Some Prx1-derived cells contribute to MCT fibroblasts of spinotrapezius, but not to myofibres. acp, scapular acromion process; atp, acromiotrapezius; ccl, cucullaris; epm, epaxial neck musculature; lbm; limb muscles; scp, scapular musculature; stp, spinotrapezius; uln, ulna. Scale bars: in C for A-C 200 µm, for D’ for D-D’ 50 µm.

2018

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Figure 1—figure supplement 3. Mesp1 and Pax3 lineage tracings in somitic neck muscles using the Pax7GPL reporter. ; (A–D) Immunostainings for β-gal and Pax7 on coronal cryosections of Mesp1Cre;Pax7GPL and Pax3Cre;Pax7GPL at E18.5 (n = 2 each condition). Note the white/yellow cells indicating colocalization of both markers. epm, epaxial muscle; tg, tongue. Scale bar in D for A-D 100 µm.

2018

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Figure 1—figure supplement 1. Ontogenetic analysis of Myf5 muscle progenitors at the head-trunk interface. ; (A–F’) Temporal X-gal stainings of Myf5Cre;Pax7GPL embryos (n = 2–4 each condition). (G–G’’) Immunostaining for GFP and Tuj1 in E10.5 Myf5Cre;R26mTmG embryo (n = 2). Ventral 3D projection of cranial region (G) and two sections 60 µm apart (dorso-ventral direction) in region indicated in (G) are shown. The cucullaris is innervated by the accessory nerve XI. V, trigeminal nerve; VII, facial nerve; IX, glossopharyngeal nerve; X, vagal nerve; XI, accessory nerve; XII, hypoglossal nerve; atp, acromiotrapezius; ccl, cucullaris anlage; clp, cutaneous maximus/latissimus dorsi precursor; dg, digastric muscles; epm, epaxial neck musculature; fmp, facial muscle precursors; h, heart; hc, hypoglossal cord; ifh, infrahyoid muscles; lb, limb bud; lbm, limb muscle anlagen and limb muscles; ltd, latissimus dorsi; lvs, levator scapula; mas, masseter; mmp, masticatory muscle precursor; nlb, nasolabialis muscles; oca, occipito/cervico-auricularis anlagen and muscles; PA1-6, pharyngeal arches 1–6; ptm, pectoralis muscles; S3-S4, somites 3–4; stm, sternocleidomastoid; stp, spinotrapezius; tgp, tongue primordia; tpr; temporal. Scale bars: in A-E’, 1000 µm; in F-F’, 2000 µm, in G for G, 400 µm for G-G’, 200 µm.

2018

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Figure 7. Lobule VII-dependent developmental and acute impairment of exploratory behavior. ; (A) Elevated plus-maze (EPM). Right, schematic view. (B) Entrance analysis. Entrances were defined as either full entries or jittery entries. (C) Opposite effects on open-arm preference were found between developmental and acute groups. Developmental perturbation of lobule VII led to reduced open-arm preference. Acute perturbation of lobule VII led to increased open-arm preference (one-way ANOVA, p

2018

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Figure 3. Neural crest contribution to neck muscle-associated tissue. ; Immunostainings on coronal cryosections of E18.5 Wnt1Cre;R26tdTomato mice at levels indicated in Figure 1. Tnnt3/Tomato immunostainings are shown in (A–D) and immunostainings for Tuj1/Tomato on selected areas of (A–D) are shown with higher magnifications in (A’–D’). See associated Figure 3—figure supplement 1–4; (n = 2). (A–A’) Note high Wnt1 contribution in the acromiotrapezius but not in epaxial muscles where Wnt1-derived cells marked neuronal cells. (B–C’) Wnt1-derived cells marked differentially the distinct muscles composing the sternocleidomastoid and laryngeal musculatures. (D–D’) At shoulder level, the Wnt1 cells did not contribute to attachment of acromiotrapezius to scapula. ac, arytenoid cartilage; acp, scapular acromion process; atp, acromiotrapezius; cc, cricoid cartilage; clm, cleidomastoid; clo, cleido-occipitalis; ct, cricothyroid; epm, epaxial musculature; hh, humeral head; ifh, infrahyoid muscles; lca, lateral cricoarytenoid; MCT, muscle-associated connective tissue; pca, posterior cricoarytenoid; phm, pharyngeal muscles; scp, scapular musculature; std, sternomastoid; tam, thyroarytenoid muscle; tc, thyroid cartilage; vm, vocal muscle. Scale bars: in D’ for A-D 400 µm for A’-D’ 200 µm.

2018

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Figure 6. Gpr158−/− mice are resilient to unpredictable chronic mild stress (UCMS). ; Individual behavioral measures were integrated to calculate the emotionality score for each animal. Behavioral analysis of Gpr158+/+ and Gpr158−/− mice subjected to UCMS in the (A) marble burying test, (B) elevated plus maze (EPM), (C) tail suspension test (TST) and (D) forced swim test (FST). (E) Emotionality score and (F) sucrose preference test for Gpr158+/+ and Gpr158−/− mice subjected to UCMS (n = 15–20 mice/group, Two-way ANOVA with Bonferroni post hoc test). Data shown as means ± SEM (n = 15–20 mice/group; Two-way ANOVA with Bonferroni post hoc; *p

2018

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Figure 5—figure supplement 1. Depressive- and anxiety-related behaviors are altered in both male and female Gpr158−/− mice. ; (A) Locomotor activity in 10 min bins (n = 5–6 mice/genotype). No significant difference between genotypes were found. (B) Effects of Gpr158−/− and Grp158+/+ mice on the accelerated rotarod test show no difference between genotype over six trials (n = 7 mice/genotype). (C) Female Gpr158−/− mice buried fewer marbles compared to Gpr158+/+ mice in the marble burying test. (D) Female Gpr158−/− mice spent significantly more time in the open arm and entered more frequently in the open arm during the elevated plus maze (EPM) test. Female Gpr158−/− mice displayed decreased immobility time in the (E) tail suspension test (TST) and (F) forced swim test (FST). (G) Emotionality score for Gpr158+/+ and Gpr158−/− female mice. Individual behavioral measures were integrated to calculate the emotionality score for each animal (top panel) (n = 10 mice/genotype). Data shown as means ± SEM (Student’s t test, *p

2018

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Figure 1—figure supplement 1. C57BL/6J and DAT-tdTomato mice do not significantly differ in body weight or behaviors at baseline or in response to CMS. ; Figure 1 was reanalyzed to compare C57BL/6J (C57) and DAT-tdTomato (tdTomato) reporter mice for control and CMS-induced effects on body weight (A) and behavior in OPT (B) SPT (C) EPM (D) NSF (E) and FST (F). Detailed statistical analysis is presented in Supplementary file 1. Control-C57, n = 7 mice; control-tdTomato, n = 6 mice; CMS-C57, n = 7 mice; CMS-tdTomato, n = 6 mice.

2018

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Figure 8. Increase in both norepinephrine and dopamine contribute to mania-like behaviors. ; (A), (B) Locomotor activity (A) and speed (B) of Th-Cre;Erbb4loxp/loxp mice treated with saline (sal), prazosin, or SCH23390 in open field test. n = 17 (Erbb4loxp/loxp + sal); n = 12 (Erbb4loxp/loxp + prazosin); n = 11 (Erbb4loxp/loxp + SCH23390); n = 11 (Th-Cre;Erbb4loxp/loxp + sal); n = 10 (Th-Cre;Erbb4loxp/loxp + prazosin); n = 9 (Th-Cre;Erbb4loxp/loxp + SCH23390). (C), (D) Distance (C) and duration (D) at HS in open field test. (E) Immobility time in open field test. (F), (G) Time (F) and entries (G) in open arms in EPM test. n = 11 (Erbb4loxp/loxp + sal); n = 12 (Erbb4loxp/loxp + prazosin); n = 11 (Erbb4loxp/loxp + SCH23390); n = 14 (Th-Cre;Erbb4loxp/loxp + sal); n = 11 (Th-Cre;Erbb4loxp/loxp + prazosin); n = 8 (Th-Cre;Erbb4loxp/loxp + SCH23390). (H), (I) Immobility time (H) and latency to first surrender (I) in forced swim tests. n = 12 (Erbb4loxp/loxp + sal); n = 10 (Erbb4loxp/loxp + prazosin); n = 10 (Erbb4loxp/loxp + SCH23390); n = 16 (Th-Cre;Erbb4loxp/loxp + sal); n = 12 (Th-Cre;Erbb4loxp/loxp + prazosin); n = 12 (Th-Cre;Erbb4loxp/loxp + SCH23390). (J) Sucrose preference of Th-Cre;Erbb4loxp/loxp mice after prazosin or SCH23390 treatment. n = 11 (Erbb4loxp/loxp + sal); n = 9 (Erbb4loxp/loxp + prazosin); n = 10 (Erbb4loxp/loxp + SCH23390); n = 11 (Th-Cre;Erbb4loxp/loxp + sal); n = 12 (Th-Cre;Erbb4loxp/loxp + prazosin); n = 12 (Th-Cre;Erbb4loxp/loxp + SCH23390). One-way ANOVA and Tukey's multiple comparison test. Data are expressed as means ± s.e.m. *p

2018

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Figure 7. Lithium rescued the behavioral, molecular, and electrophysiological phenotypes of Th-Cre;Erbb4loxp/loxp mice. ; (A), (B) Locomotor activity (A) and speed (B) in the open field test. n = 24 (Erbb4loxp/loxp), n = 10 (Erbb4loxp/loxp + lithium), n = 22 (Th-Cre;Erbb4loxp/loxp), n = 13 (Th-Cre;Erbb4loxp/loxp + lithium). (C–E) Distance (C) and duration (D) traveled at HS and immobility time (E) after lithium treatment. (F), (G) Percentage of time (F) and entries (G) into open arms by Th-Cre;Erbb4loxp/loxp mice with and without lithium in the EPM test. n = 23 (Erbb4loxp/loxp); n = 8 (Erbb4loxp/loxp + lithium); n = 24 (Th-Cre;Erbb4loxp/loxp); n = 18 (Th-Cre;Erbb4loxp/loxp + lithium). (H), (I) Immobility time (H) and latency to first surrender (I) in forced swim test. n = 20 (Erbb4loxp/loxp); n = 8 (Erbb4loxp/loxp + lithium); n = 16 (Th-Cre;Erbb4loxp/loxp); n = 20 (Th-Cre;Erbb4loxp/loxp + lithium). (J) Sucrose preference of Th-Cre;Erbb4loxp/loxp mice treated with lithium. n = 19 (Erbb4loxp/loxp); n = 12 (Erbb4loxp/loxp + lithium); n = 13 (Th-Cre;Erbb4loxp/loxp); n = 16 (Th-Cre;Erbb4loxp/loxp + lithium). (K) Western blots of LC samples from Th-Cre;Erbb4loxp/loxp mice with and without lithium treatment. (L) Protein level of NR2B and TH-Ser40 in the LC after lithium treatment. Protein levels of TH, s-COMT, and MB-COMT were not significantly changed in the LC after lithium treatment. TH, tyrosine hydroxylase; S-COMT, soluble catechol-o-methyltransferase; MB-COMT, membrane-binding form of COMT. n = 4 (Erbb4loxp/loxp); n = 4 (Erbb4loxp/loxp + lithium); n = 4 (Th-Cre;Erbb4loxp/loxp); n = 4 (Th-Cre;Erbb4loxp/loxp + lithium). (M), Spontaneous firing of LC-NE neurons after lithium treatment. n = 13 from three mice (Th-Cre;Erbb4loxp/loxp); n = 15 from three mice (Th-Cre;Erbb4loxp/loxp + lithium). (N) Interspike intervals after lithium treatment. n = 13 from three mice (Th-Cre;Erbb4loxp/loxp); n = 15 from three (Th-Cre;Erbb4loxp/loxp + lithium). Two-way ANOVA. Data are expressed as means ± s.e.m. Two-sample Kolmogorov-Smirnov test and data in (N) are presented as a cumulative frequency plot. *p

2018

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Figure 6. 3D reconstructions of neck musculoskeletal system in Tbx1 and Pax3 mutants. ; See interactive 3D PDFs in Supplementary file 1–3; control n = 1; mutants n = 2. (A–C) Branchiomeric and cucullaris-derived muscles marked by Mef2c-AHF/Islet1/Mesp1 lineages are indicated in pink. (D–F) Anterior somitic muscles (Mesp1, Pax3 lineages), in red. (G–I) Scapular muscles from more posterior somites (Pax3 lineage), in violet. atp, acromiotrapezius; cc, cricoid cartilage; dg, digastric muscles; epm, epaxial musculature; ifh, infrahyoid muscles; ilm, intrinsic laryngeal muscles; lcl, longus colli; lcp, longus capitis; myh, mylohyoid; sc, scapula; scp, scapular muscles; stm, sternocleidomastoid; tc, thyroid cartilage; tg, tongue.

2018

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