ICC hyperplasia in the antrum of 3-month-old <i>Spry4</i> KO and <i>Kit</i>^{<i>WT/K641E</i>}.

<p>A) Widefield microscopy acquisitions. PDE3A immunoreactivity (-ir) highlights ICC in the antrum of 3-month-old WT, <i>Spry4</i> KO and <i>Kit</i>^{<b><i>WT/K641E</i></b>} mice. B) Ratio of PDE3A-ir ICC area in the antrum muscularis propria. Abbreviations: LM: longitudinal muscle layer, CM: circular muscle layer, *: location of myenteric plexus, scale bar: 100μm. P-values (Kruskal-Wallis with Dunn’s post hoc) *: p<0.05, **: p<0.01</p

Primary and secondary antibodies used for immunofluorescence.

<p>Primary and secondary antibodies used for immunofluorescence.</p

No detectable pAKT-ir in ICC of WT, <i>Spry4</i> KO and <i>Kit</i>^{<i>WT/K641E</i>} antrum.

<p>Widefield microscopy, sequential channels acquisitions. <i>Left column</i>: PDE3A immunoreactivity (-ir) ICC in WT, <i>Spry4</i> KO and <i>Kit</i>^{<b><i>WT/K641E</i></b>}. <i>Middle column</i>: pAKT-ir in the 3 genotypes. <i>Right column</i>: merged images: PDE3A-ir and pAKT displayed in green and in red, respectively. pAKT (red) was consistently detected in myenteric plexus and nerve fibers in the muscularis propria but not in PDE3A-ir ICC (green). Abbreviations: LM: longitudinal muscle layer, CM: circular muscle layer, *: myenteric plexus, scale bar: 100μm.</p

No compensation mechanism detected in <i>Spry4</i> KO animals.

<p>A) qPCR of WT and <i>Spry4</i> KO P10 antrum for <i>Spred1</i>, <i>Spry1</i> and <i>Spry2</i>. B) Widefield microscopy, sequential channels acquisitions. Immunofluorescence of 3-month-old antrum. <i>Left column</i>: PDE3A-ir ICC in WT, <i>Spry4</i> KO and <i>Kit</i>^{<b><i>WT/K641E</i></b>}. <i>Middle column</i>: SPRY2-ir in in the 3 genotypes. <i>Right column</i>: merged images: PDE3A-ir and SPRY2-ir displayed in green and in red, respectively. SPRY2-ir was consistently detected in smooth muscle cells of the muscularis propria but not in PDE3A-ir ICC. Abbreviations: LM: longitudinal muscle layer, CM: circular muscle layer, *: location of myenteric plexus, scale bar: 100μm.</p

pERK-ir in nerve fibers but undetectable in ICC of 3-month-old WT, <i>Spry4</i> KO and <i>Kit</i>^{<i>WT/K641E</i>} antrum.

<p>Confocal microscopy, sequential channels acquisitions. <i>Left column</i>: grey scale images of PDE3A immunoreactivity (-ir) ICC. <i>Middle column</i>: grey scale images of pERK-ir. <i>Right column</i>: merged images. PDE3A-ir and pERK-ir are displayed in green and in red, respectively, with nuclear counterstain (DAPI) in blue. pERK-ir (red) was consistently detected in myenteric plexus and in nerve fibers in the muscularis propria but not in PDE3A-ir ICC (green). Abbreviations: LM: longitudinal muscle layer, CM: circular muscle layer, scale bar: 20μm</p

No detectable pSTAT5a/b in ICC of antrum of WT, <i>Spry4</i> KO or <i>Kit</i>^{<i>WT/K641E</i>}.

<p>Widefield microscopy, sequential channels acquisitions. <i>Left column</i>: PDE3A immunoreactivity (-ir) ICC in WT, <i>Spry4</i> KO and <i>Kit</i>^{<b><i>WT/K641E</i></b>} antrum. <i>Middle column</i>: pSTAT5a/b-ir for each genotype. <i>Right column</i>: merged images: PDE3A-ir and pSTAT5a/b-ir displayed in green and red, respectively. pSTAT5a/b was consistently detected in myenteric plexus and nerve fibers in the muscularis propria but not in PDE3A-ir ICC. Abbreviations: LM: longitudinal muscle layer, CM: circular muscle layer, *: location of myenteric plexus, scale bar: 100μm</p

SPRY4-ir in the ICC of 3-month-old <i>Kit</i>^{<i>WT/K641E</i>} antrum.

<p>Widefield microscopy, sequential channels acquisitions. Left column: KIT immunoreactivity (-ir) ICC in WT, Spry4 KO and KitWT/K641E. Middle column: SPRY4-ir in the 3 genotypes. Right column: merged images: KIT-ir and SPRY4-ir displayed in green and in red, respectively. SPRY4-ir was detected in the KIT-ir ICC solely in KitWT/K641E mice. Abbreviations: LM: longitudinal muscle layer, CM: circular muscle layer, *: location of myenteric plexus, scale bar: 100μm.</p

No detectable pp70S6 in ICC of antrum of WT, <i>Spry4</i> KO or <i>Kit</i>^{<i>WT/K641E</i>}.

<p>Widefield microscopy, sequential channels acquisitions. <i>Left column</i>: PDE3A immunoreactivity (-ir) ICC in WT, <i>Spry4</i> KO and <i>Kit</i>^{<b><i>WT/K641E</i></b>} antrum. <i>Middle column</i>: pp70S6-ir for each genotype. <i>Right column</i>: merged images: PDE3A and pp70S6-ir displayed in green and red, respectively. pp70S6 was consistently detected in myenteric plexus and nerve fibers in the muscularis propria but not in PDE3A-ir ICC. Abbreviations: LM: longitudinal muscle layer, CM: circular muscle layer, *: location of myenteric plexus, scale bar: 100μm</p

Decrease in myenteric neurons area in antrum of <i>Spry4</i> KO and <i>Kit</i>^{<i>WT/K641E</i>} mice.

<p>A) Widefield microscopy acquisitions. HuC/D-ir highlights soma of myenteric neurons in antrum of 3-month-old WT, <i>Spry4</i> KO and <i>Kit</i>^{<b><i>WT/K641E</i></b>} mice. B) Ratio of HuC/D-ir area in antrum muscularis propria. Abbreviations: LM: longitudinal muscle layer, CM: circular muscle layer, scale bar: 100μm. P-values (Kruskal-Wallis with Dunn’s post hoc), *: p<0.05</p

Primers used for genotyping.

<p>Primers used for genotyping.</p