Absence of HNF-6 results in muscle hypotrophy or hypertrophy.

<p><b><i>A</i></b>, Tibialis anterior, gastrocnemius, extensor digitorium longus and soleus muscles from control (blue) or <i>Hnf6−/−</i> (red) mice were weighted. The tibialis anterior and the gastrocnemius are hypotrophied in <i>Hnf6−/−</i> mice, whereas the soleus is hypertrophied. <b><i>B</i></b><i>, </i><b><i>C</i></b>, Hematoxylin-eosin stainings of transverse sections in tibialis anterior from control (B) or <i>Hnf6−/−</i> (C) mice does not evidence any sign of muscle degeneration/regeneration but shows a reduction in fiber size. <b><i>D</i></b>, Distribution of fiber area in the tibialis anterior of control (blue) or <i>Hnf6−/−</i> (red) mice. The tibialis anterior fibers are smaller in the absence of HNF-6. <b><i>E</i></b><i>, </i><b><i>F</i></b>, Hematoxylin-eosin staining of transverse sections in the soleus of control (E) or <i>Hnf6−/−</i> (F) mice shows an increase in fiber size. <b><i>G</i></b><b>,</b> Distribution of fiber area in the soleus of control (blue) or <i>Hn6−/−</i> (red) mice. The soleus fibers are bigger in the absence of HNF-6. Gastro: gastrocnemius; EDL: extensor digitorium longus; TA: tibialis anterior. (n = 3) Student’s t-test (A), χ² Pearson’s test (D,G), * = p<0.05; *** = p<0.001. Scale bar = 50 µm.</p

HNF-6 is required for proper morphology of the NMJ.

<p><b><i>A–D”</i></b><b>,</b> Labeling of acetylcholine receptors by α-bungarotoxin (red) and immunofluorescence detection of synaptophysin (green) and of neurofilaments (blue) on hindlimb sections of control (A–A”) or <i>Hnf6−/−</i> (B–D”) mice. (A–A”) In control mice, NMJ display the expected “pretzel-like” shape, are innervated and show perfect apposition of the synaptophysin labeling to the motor endplate. (B–B”) Some NMJ in <i>Hnf6−/−</i> mice are very similar to that observed in control animals. (C–D”) However, a majority of the <i>Hnf6−/−</i> junctions are characterized by disorganized topology, fragmentation or absence of gutters (arrowheads). These junctions also show defective localization of synaptophysin, which is either absent (asterisk) or fragmented (blue arrows) and ectopically located. In contrast, all the junctions were innervated (white arrows). <b><i>E</i></b><b>,</b> Quantification of motor endplates that exhibit the expected “pretzel-like” unfragmented morphology in control (blue) and in <i>Hnf6−/−</i> (red) mice (n = 6). <b>F,</b> Quantification of defective synaptophysin localization in <i>Hnf6−/−</i> mice. Synaptophysin is either properly superposed to the α-bungarotoxin labeling, as observed in a vast majority of control junctions (blue), or fragmented (red), weak (green) or absent (purple) (n = 5). <b><i>G–N</i></b><b>,</b> Labeling of control (G,I,K,M) or <i>Hnf6−/−</i> (H,J,L,N) junctions with synaptophysin (G,H,K,L, green) or α-bungarotoxin (I,J,M,N, red) and VAChT (G–J, blue) or SV2 (K–N, blue). In <i>Hnf6−/−</i> mice, the VAChT and SV2 are properly localized at the motor terminal ends. <b><i>O, P</i></b><b>,</b> Ultrastructure of the NMJ observed by transmission electron microscopy in control (O) or <i>Hnf6−/−</i> (P) tibialis anterior muscles. Synaptic vesicles are present and normally localized (n = 3). <b><i>Q, R</i></b><b>,</b> Electromyogram recordings in control (Q) or <i>Hnf6−/−</i> (R) mice do not evidence any sign of denervation of the mutant hindlimb muscles (n = 3). BTX: α-bungarotoxin; PanNF: Pan-Neurofilament; SV2: synaptic vesicle 2; SYN: synaptophysine; VAChT: vesicular acetylcholine transporter. Student’s t-test; *** = p<0.001. Scale bar in A and G = 5 µm; scale bar in O = 0.4 µm.</p

HNF-6 is required for the formation of NMJ.

<p><b><i>A–F</i></b><b>,</b> Labeling of acetylcholine receptors by α-bungarotoxin (red) and immunofluorescence detection of synaptophysin (green) and of neurofilaments (blue) on hindlimb sections of control (A–C) or <i>Hnf6−/−</i> (D–F) mice at postnatal day 0 (A,D), 7 (B,E) or 14 (C,F). The progressive increase in apposition of the motor nerve terminals to the motor endplates is disrupted in the absence of HNF-6, and the maturation of the motor endplate is defective. <b><i>G</i></b><b>,</b> Quantification of NMJ maturation in control (blue) or <i>Hnf6−/−</i> (red) mice based on 4 morphological stages (M1: oval plaque; M2: single perforation; M3: multiple perforations; M4: mature junction). The maturation of the junctions is delayed in the absence of HNF-6 (n = 3). <b><i>H–K</i></b><b>,</b> Labeling of VAChT (H,I, red) or synaptophysin (J,K, green) and of neurofilaments (blue) in control (H,J) or <i>Hnf6−/−</i> (I,K) mice at postnatal day 14. VAChT and synaptophysin were present in the terminal portion of motor axons in <i>Hnf6−/−</i> mice (I,K) whereas they were restricted to nerve terminals in control mice (H,J). <b><i>L</i></b><b>,</b> Schematic representation of NMJ formation in control and in <i>Hnf6−/−</i> mice. BTX: α-bungarotoxin; PanNF: Pan-neurofilament; P: postnatal day; SYN: synaptophysin; VAChT: vesicular acetylcholine transporter. Student’s t-test, * = p<0.05, ** = p<0.01, *** = p<0.001. Scale bar = 5 µm.</p

Depletion of agrin and of neuregulin contributes to defective NMJ formation in <i>Hnf6</i> mutant mice.

<p><b><i>A–H</i></b>, NMJ-like structures obtained <i>in vitro</i> by co-culture of control myotubes with control (A,C,E,G) or <i>Hnf6−/−</i> (B,D,F,H) embryonic spinal cord slices in medium supplemented with vehicle only (A,B) or with recombinant agrin (C,D), neuregulin (E,F) or TGFß (G,H). <b><i>I</i></b><b>,</b><b><i>J,</i></b> Quantification of the length (I) and of the area (J) of the NMJ-like structures obtained with control (blue) or <i>Hnf6−/−</i> (red) spinal cord slices. The size and area deficits observed with <i>Hnf6−/−</i> slices are fully rescued by agrin and by neuregulin, but not by TGFß (n = 3). Nrg-1: neuregulin-1. Dunett’s (I) and Newman-Keuls (J) multiple comparison tests; * = p<0.05, ** = p<0.01, *** = p<0.001. Scale bar = 10 µm.</p

Absence of HNF-6 results in abnormal locomotion.

<p><b><i>A</i></b>, Newborn <i>Hnf6−/−</i> mice (right) are smaller than their control littermates (left) and show paresis of the hindlimbs (arrows). <b><i>B</i></b>, Neurological score of male or female control (blue) or <i>Hnf6−/−</i> (red) mice. The score is reduced in both male and female <i>Hnf6−/−</i> mice (n≥7). <b><i>C</i></b>, <b><i>D</i></b>, <i>Hnf6−/−</i> mice (D) exhibit a feet-clasping posture of the hindlimbs upon tail suspension, both for the legs (arrows) and for the feet (inset). <b><i>E</i></b>, The hindlimb muscular strength of male and of female control (blue) or <i>Hnf6−/−</i> (red) mice normalized to the body weight is reduced in <i>Hnf6−/−</i> mice (n≥4). <b><i>F–M</i></b><b>,</b> Catwalk analyses were performed for male (F,H,J,K) or female (G,I,L,M) control (F,G,J–M) or <i>Hnf6−/−</i> (H,I,J–M) mice. Locomotion alterations characterized by reduced overlap of fore paw (light colors) and of hind paw (dark colors) prints, reduced area of hind paw contacts, variability in the distance between the left (green) and the right (red) prints and less linear displacement, are observed in <i>Hnf6−/−</i> mice. Quantifications indicate that the intensity of the hind paw contacts is reduced (J,L) while the hindlimb base of support (the distance between the left and right hind paws) is increased (K,M). In contrast, these parameters are not affected for the forelimbs (n≥4 in each group). BOS: base of support; LF: left front; LH: left hind; RF: right front; RH: right hind. Student’s t-test; * = p<0.05; ** = p<0.01; *** = p<0.001.</p

Serotonin levels in ALS and control groups.

<p>Groups were compared using Mann-Whitney test and the p-value is shown.</p

Demographic parameters in ALS and control groups.

<p>Demographic parameters in ALS and control groups.</p

Serotonin levels in ALS patients as a function of site of onset.

<p>Groups were compared using Kruskal Wallis test. *, p<0.05; **, p<0.01.</p

Correlation between platelet serotonin levels and survival in patients with amyotrophic lateral sclerosis (ALS).

<p>Kaplan-Meyer survival curves of patients with ALS stratified according to their platelet serotonin levels. Survival was calculated as the time period between blood sampling and death. The black line represents patients with ALS having abnormal serotonin values (serotonin <50 ng/mL, n = 22 patients); light grey line represents patients with high normal values (serotonin >100 ng/mL i.e. median of control group, n = 25 patients), and light black line patients with borderline values (50–100 ng/mL, n = 35 patients). Group comparison shows a statistically significant difference (p = 0.024 by the log-rank).</p

Impact of contextual fear conditioning on histone acetylation in the rat hippocampus.

<p>(A) Experimental design. Three groups of rats (n = 16/group) were used. In one group, rats were kept in the context but received no shock (CX). Others received three immediate and consecutive shocks and were subsequently left in the context for 8 min (IS). In the last group, rats received three randomly-distributed shocks while being kept in the context as noted (CS). Animals (n = 10/group) were then either tested for freezing behavior after 24 h (probe) (B; n = 10/group<b>)</b> or euthanized after 1 h for tissue collection (dorsal hippocampus) and western blot analyses of acetylated histones (C; n = 6/group<b>)</b>. (B) Freezing levels at 24 h. Notice that marked freezing was observed only in the Context-shock group (CS), demonstrating that rats of this group were the only ones to have associated the shock with the context and memorized this association. (C) Comparison of acetylated and total histone levels in the three groups relative to their counterparts taken from the home cage (HC, n = 6). Lysine acetylations measured were H2BK5 (K5Ac, plain histograms), H2BK5K12K15K20 (Tetra Ac, stripped histograms), H4K12 (K12Ac) and H3K9K14 (K9K14Ac). Typical western blots are shown in duplicates. Quantified results are represented as % induction of the Acetylated/total ratio for each histone. The ratio obtained in the HC condition was arbitrarily set at 100%. Newman-Keuls multiple comparisons test: ***p<0.001**p<0.01, *p<0.05, as compared to HC group. Global H2B and H4 histone acetylation levels were clearly increased in the group exhibiting fear towards the context (CS) as compared to the other situations, while H3 acetylation levels were increased in CS and both controls (CX and IS ) as compared to rats completely naive to the test situation (HC).</p