Deregulation of the protocadherin gene FAT1 alters muscle shapes: implications for the pathogenesis of facioscapulohumeral dystrophy.

International audienceGeneration of skeletal muscles with forms adapted to their function is essential for normal movement. Muscle shape is patterned by the coordinated polarity of collectively migrating myoblasts. Constitutive inactivation of the protocadherin gene Fat1 uncoupled individual myoblast polarity within chains, altering the shape of selective groups of muscles in the shoulder and face. These shape abnormalities were followed by early onset regionalised muscle defects in adult Fat1-deficient mice. Tissue-specific ablation of Fat1 driven by Pax3-cre reproduced muscle shape defects in limb but not face muscles, indicating a cell-autonomous contribution of Fat1 in migrating muscle precursors. Strikingly, the topography of muscle abnormalities caused by Fat1 loss-of-function resembles that of human patients with facioscapulohumeral dystrophy (FSHD). FAT1 lies near the critical locus involved in causing FSHD, and Fat1 mutant mice also show retinal vasculopathy, mimicking another symptom of FSHD, and showed abnormal inner ear patterning, predictive of deafness, reminiscent of another burden of FSHD. Muscle-specific reduction of FAT1 expression and promoter silencing was observed in foetal FSHD1 cases. CGH array-based studies identified deletion polymorphisms within a putative regulatory enhancer of FAT1, predictive of tissue-specific depletion of FAT1 expression, which preferentially segregate with FSHD. Our study identifies FAT1 as a critical determinant of muscle form, misregulation of which associates with FSHD

Fat1 regulates astrocyte maturation and angiogenesis in the retina

Angiogenesis is a stepwise process leading to blood vessel formation. In the vertebrate retina, endothelial cells are guided by astrocytes migrating along the inner surface, and the two processes are coupled by a tightly regulated cross-talk between the two cell types. Here, we investigated how the FAT1 Cadherin, a regulator of tissue morphogenesis governing tissue cross-talks, influences retinal vascular development. Through late-onset inactivation in the neural lineage in mice, we bypassed an early contribution of Fat1 to eye development, and assessed its requirement for postnatal retina angiogenesis. We found that neural Fat1 expression, by controlling the polarity of astrocyte progenitor migration, regulates astrocyte maturation. By interfering with astrocyte migration and maturation, neural Fat1 deletion deregulates the astrocyte/endothelial cell coupling, and delays retinal angiogenesis. Mice with neural-Fat1 ablation exhibit persistent abnormalities of the retinal vascular architecture, such as an increased vascular density in deep layers. Altogether, this study identifies Fat1 as a regulator of neurovascular communication, essential for retinal vascular development and integrity

Astrocyte-intrinsic and -extrinsic Fat1 activities regulate astrocyte development and angiogenesis in the retina

International audienceAngiogenesis is a stepwise process leading to blood vessel formation. In the vertebrate retina, endothelial cells are guided by astrocytes migrating along the inner surface, and the two processes are coupled by a tightly regulated cross-talks between the two cell types. Here, I have investigated how the FAT1 cadherin, a regulator of tissue morphogenesis that governs tissue cross-talk, influences retinal vascular development. Late-onset Fat1 inactivation in the neural lineage in mice, by interfering with astrocyte progenitor migration polarity and maturation, delayed postnatal retinal angiogenesis, leading to persistent vascular abnormalities in adult retinas. Impaired astrocyte migration and polarity were not associated with alterations of retinal ganglion cell axonal trajectories or of the inner limiting membrane. In contrast, inducible Fat1 ablation in postnatal astrocytes was sufficient to alter their migration polarity and proliferation. Altogether, this study uncovers astrocyte-intrinsic and -extrinsic Fat1 activities that influence astrocyte migration polarity, proliferation and maturation, disruption of which impacts retinal vascular development and maintenance

Tissue-specific activities of the Fat1 cadherin cooperate to control neuromuscular morphogenesis

International audienceMuscle morphogenesis is tightly coupled with that of motor neurons (MNs). Both MNs and muscle progenitors simultaneously explore the surrounding tissues while exchanging reciprocal signals to tune their behaviors. We previously identified the Fat1 cadherin as a regulator of muscle morphogenesis and showed that it is required in the myogenic lineage to control the polarity of progenitor migration. To expand our knowledge on how Fat1 exerts its tissue-morphogenesis regulator activity, we dissected its functions by tissue-specific genetic ablation. An emblematic example of muscle under such morphogenetic control is the cutaneous maximus (CM) muscle, a flat subcutaneous muscle in which progenitor migration is physically separated from the process of myogenic differentiation but tightly associated with elongating axons of its partner MNs. Here, we show that constitutive Fat1 disruption interferes with expansion and differentiation of the CM muscle, with its motor innervation and with specification of its associated MN pool. Fat1 is expressed in muscle progenitors, in associated mesenchymal cells, and in MN subsets, including the CM-innervating pool. We identify mesenchyme-derived connective tissue (CT) as a cell type in which Fat1 activity is required for the non-cell-autonomous control of CM muscle progenitor spreading, myogenic differentiation, motor innervation, and for motor pool specification. In parallel, Fat1 is required in MNs to promote their axonal growth and specification, indirectly influencing muscle progenitor progression. These results illustrate how Fat1 coordinates the coupling of muscular and neuronal morphogenesis by playing distinct but complementary actions in several cell types

Tissue cross talks governing limb muscle development and regeneration

International audienceFor decades, limb development has been a paradigm of three-dimensional patterning. Moreover, as the limb muscles and the other tissues of the limb’s musculoskeletal system arise from distinct developmental sources, it has been a prime example of integrative morphogenesis and cross-tissue communication. As the limbs grow, all components of the musculoskeletal system (muscles, tendons, connective tissue, nerves) coordinate their growth and differentiation, ultimately giving rise to a functional unit capable of executing elaborate movement. While the molecular mechanisms governing global three-dimensional patterning and formation of the skeletal structures of the limbs has been a matter of intense research, patterning of the soft tissues is less understood. Here, we review the development of limb muscles with an emphasis on their interaction with other tissue types and the instructive roles these tissues play. Furthermore, we discuss the role of adult correlates of these embryonic accessory tissues in muscle regeneration

<i>Fat1</i> is expressed in CM progenitors and the surrounding subcutaneous mesenchyme.

<p><b>(A)</b><i>Fat1</i> expression is visualized in an E12.5 <i>Fat1</i>^{<i>LacZ/+</i>} embryo by X-gal staining. Left panel: whole embryo picture; right panel: higher magnification of the forelimb and flank region in which the CM spreads. In the right panel, the approximate CM shape is highlighted by red dotted lines, and the level of sections shown in <b>(C)</b> is indicated by vertical lines. <b>(B)</b> <i>Gdnf</i> expression is visualized in an E12.5 <i>Gdnf</i>^{<i>LacZ/+</i>} embryo (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004734#pbio.2004734.s002" target="_blank">S1 Table</a>) by Salmon-Gal staining. Left panel: whole embryo left side view. Right panel: higher magnification of the upper forelimb and flank region, showing that the CM exhibits a high level of <i>Gdnf</i>^<i>LacZ+</i> expression (highlighted with red dotted lines). The level of sections shown in <b>(D)</b> is represented by vertical bars. (C) Cross sections of an E12.5 <i>Fat1</i>^{<i>LacZ/+</i>} embryo at anterior and posterior CM levels were immunostained with antibodies against Pax7 (red), Myh1 (green), and β-galactosidase (white). The right panels show neighboring sections of the same <i>Fat1</i>^{<i>LacZ/+</i>} embryo in which β-galactosidase activity was revealed by Salmon-Gal staining. <b>(D)</b> Comparison between expression of <i>Gdnf</i>^<i>LacZ</i> (visualized with an anti-β-galactosidase antibody [red]) and that of Fat1 (green, Ab FAT1-1869 Sigma) on two cross sections of an E12.5 <i>Gdnf</i>^{<i>LacZ/+</i>} mouse embryo at middle and posterior CM levels, as indicated in <b>(B)</b>. Fat1 protein is detected both within and around the <i>Gdnf</i>^{<i>LacZ/+</i>} CM progenitors. Scale bars (A, B): 1 mm (left), 500 μm (right); (C, D): 200 μm (low magnification), 50 μm (high magnification). β-Gal, β-galactosidase; CM, cutaneous maximus.</p

Mesenchymal <i>Fat1</i> is required for expansion of the myogenic component of <i>Gdnf</i> expression domain but dispensable for <i>Gdnf</i> expression in plexus mesenchyme.

<p><b>(A)</b> Top: principle of the genetic paradigm used to follow the <i>Prx1-cre</i> lineage, using the <i>R26</i>^{<i>Lox-STOP-Lox-YFP</i>} reporter line combined with <i>Prx1-cre</i>. In tissues in which cre is not expressed, YFP expression is prevented by the STOP cassette. In CRE-expressing mesenchymal cells, STOP cassette excision allows YFP expression. Bottom: scheme of a cross section of a <i>Prx1-cre</i>; <i>R26</i>^<i>YFP/+</i>; <i>Gdnf</i>^{<i>LacZ/+</i>} embryo, highlighting in green the cells in which YFP expression is activated, in red, the cells expressing <i>Gdnf</i>^<i>LacZ</i>, and in white or gray, the other non-recombined tissues. <b>(B, C)</b> Cross sections of E12.5 <i>Prx1-cre; Fat1</i>^{<i>Flox/+</i>}<i>; Gdnf</i>^{<i>LacZ/+</i>}; <i>R26</i>^<i>YFP/+</i> <b>(B)</b> and <i>Prx1-cre; Fat1</i>^{<i>Flox/Flox</i>}<i>; Gdnf</i>^{<i>LacZ/+</i>}; <i>R26</i>^<i>YFP/+</i> <b>(C)</b> embryos stained with antibodies against GFP (YFP; to reveal the domain of <i>Prx1-cre</i> activity, in green), with an anti-β-galactosidase antibody (for <i>Gdnf</i>^<i>LacZ</i>, red), visualized at four successive rostro-caudal positions spanning from the brachial plexus to the caudal half of the CM muscle. For each level, the inserts below represent a high-magnification view of the area indicated in the yellow dotted boxes, showing red only, green only, and overlay. <b>(D)</b> Visual summary of the two components of <i>Gdnf</i>^<i>LacZ</i> expression domain, spanning the sections shown in <b>(B)</b> and <b>(C)</b>: at the plexus level, <i>Gdnf</i>^<i>LacZ</i> is expressed in YFP⁺ cells derived from <i>Prx1-cre</i> mesenchyme, whereas in the CM and LD muscles (emerging from the plexus and extending dorsally and caudally), <i>Gdnf</i>^<i>LacZ</i>-positive cells do not express YFP, as they are from the myogenic rather than the mesenchymal lineage. At the point where the first myogenic patches emerge from the plexus, such myogenic patches (red only, yellow dotted line in <b>[B]</b>, second section) can be surrounded by mesenchymal-Gdnf cells (red + green = yellow, white dotted lines). The overall analysis shows that <i>Prx1-cre</i>-mediated <i>Fat1</i> ablation does not affect <i>Gdnf</i> expression in the plexus mesenchyme but causes non–cell-autonomous reduction in the myogenic component of <i>Gdnf</i> expression domain through a reduction of the number of <i>Gdnf</i>^<i>LacZ</i>-expressing myogenic progenitors. Scale bars: <b>(B, C)</b> low magnification: 200 μm; inserts: 20 μm. CM, cutaneous maximus; <i>cre</i>, cre recombinase; LD, latissimus dorsi; Lox, recombination sites for the CRE recombinate; Lox-STOP-Lox, cassette in which STOP signal for transcription/translation is flanked by Lox sites; <i>Prx1-cre</i>, transgene driving cre expression in the mesenchyme; <i>R26</i>, Rosa26 locus; YFP, yellow fluorescent protein.</p

Impact of mesenchyme-specific and motor neuron–specific <i>Fat1</i> ablation on adult CM muscle anatomy and grip strength.

<p>(<b>A–C</b>) Analysis of NMJ morphology (<b>A</b>) was performed by immunostaining with α-Bungarotoxin (green, detecting AchR), phalloidin (white, detecting F-actin), and anti-neurofilament antibodies on cryosections of the dCM in <i>Fat1</i>^{<i>Flox/Flox</i>}, in <i>Olig2</i>^<i>cre/+</i><i>; Fat1</i>^{<i>Flox/Flox</i>}, and in <i>Prx1-cre; Fat1</i>^{<i>Flox/Flox</i>} adult mice (about 1 year old). (<b>A</b>) Top pictures show low 20× magnifications, whereas bottom pictures show high magnification of individual synapses. (<b>B, C</b>) Histograms showing quantification of NMJ area (<b>B</b>) and fiber diameter (<b>C</b>) distributions in the dCM muscle of <i>Fat1</i>^{<i>Flox/Flox</i>} (<i>n</i> = 9), <i>Olig2</i>^<i>cre/+</i><i>; Fat1</i>^{<i>Flox/Flox</i>} (<i>n</i> = 11), and <i>Prx1-cre; Fat1</i>^{<i>Flox/Flox</i>} (<i>n</i> = 7) mice. Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004734#pbio.2004734.s001" target="_blank">S1 Data</a>. <b>(B)</b> Left plot shows the synapse area, with all synapses plotted (circles) for each genotype, as well as the median NMJ area per mouse (with each triangle representing one mouse). An average of 20 synapses per mouse were analyzed in the indicated number of mice. Total numbers of synapses and mice are indicated below the graph. Right plots: average distribution of synapse areas in <i>Fat1</i>^{<i>Flox/Flox</i>} (blue bars) and <i>Olig2-cre; Fat1</i>^{<i>Flox/Flox</i>} (red bars) mice (top) and <i>Fat1</i>^{<i>Flox/Flox</i>} (blue bars, same data as above) and <i>Prx1-cre; Fat1</i>^{<i>Flox/Flox</i>} (green bars) mice (bottom). <b>(C)</b> Left plots: average distribution of fiber diameters in the same samples. Right plots show the median fiber diameter in each mouse (one dot representing one mouse). Statistical significance: * indicates <i>p</i> < 0.01, ** indicates <i>p</i> < 0.001, Mann Whitney test. Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004734#pbio.2004734.s001" target="_blank">S1 Data</a>. (<b>D</b>) Measurements of grip strength in mice with the indicated genotypes. In the plots, each dot represents the average value for one mouse, assaying forelimb grip strength (left plot) or cumulative strength of forelimbs plus hind limbs (right). For each mouse, the measured strength was normalized to the mean strength of the control group of the same gender, so that males and females could be expressed as percentages and pooled on the same graph. Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004734#pbio.2004734.s001" target="_blank">S1 Data</a>. Statistical significance: <i>p</i>-values are indicated for the relevant comparisons, using unpaired student <i>t</i> test for values with normal distribution and equal variance, or Mann Whitney test, otherwise. Significance threshold: <i>p</i> < 0.01. Scale bars: (A) upper images: 50 μm; (A) lower images: 10 μm. AchR, acetylcholine receptor; CM, cutaneous maximus; <i>cre</i>, cre recombinase; dCM, dorsal cutaneous maximus; NMJ, neuromuscular junction; <i>Olig2-cre</i>, cre expression in MN progenitors; <i>Prx1-cre</i>, cre expression in the mesenchyme.</p

<i>Fat1</i> knockout alters motor innervation of the CM muscle.

<p><b>(A, B)</b> The nerve pattern was analyzed by IHC with antibodies against neurofilament in E12.5 <b>(A)</b> to E12.75 <b>(B)</b> wild-type and <i>Fat1</i>^<i>-/-</i> embryos. Embryos were cut in half, cleared in BB-BA, and flat-mounted. Upper panels are low-magnification images of the left flank, showing the whole trunk. Lower panels show high-magnification views of the area containing the CM muscle. The area covered by CM-innervated axons is highlighted in white (middle panels). Axons of vertically oriented thoracic spinal nerves have been manually removed by dissection in the lower panels to improve visibility of CM axons. Inserts in the lower panels represent higher magnification of the area in the yellow squares. <b>(C)</b> Quantifications of the relative expansion of the area covered by CM-innervating axons. Upper plot: for each embryo side, the area covered by CM-innervating axons is plotted relative to the length of a thoracic nerve (T10, from dorsal root origin to ventral tip). Arrows point the stages of representative examples shown in <b>(A)</b> and <b>(B)</b>. Bottom plot: for each embryo, the CM-innervated area/T10 length was normalized to the median ratio of control embryos, by size range. Blue dots: <i>Fat1</i>^<i>+/+</i> (<i>n</i> = 35, same sample set as in controls of <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004734#pbio.2004734.s006" target="_blank">S3 Fig</a>); red dots: <i>Fat1</i>^<i>-/-</i> (<i>n</i> = 12). Underlying data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004734#pbio.2004734.s001" target="_blank">S1 Data</a>. Scale bars: 500 μm (large images); 100 μm (inserts in lower panels). BB-BA, benzyl-benzoate/benzyl-alcohol mix; CM, cutaneous maximus; IHC, immunohistochemistry; T10, 10th thoracic nerve; WT, wild-type.</p