RNA expression of <i>N. vectensis</i> glutamate transporter (GLUT)-like genes.

<p>(a) H&E staining of <i>Nemtostella</i> longitudal section- head area. (b) <i>Nematostella</i> NV_173595 expression in the head and part of the body. (c, f) <i>Nematostella</i> NV_173595 and NV_138860 expression in the endoderm around the pharynx. (d) NV_173595 expression around the testis. (e) NV_173595 expression in the endoderm of the head body wall. (g) NV_138860 expression around the testis and in the endoderm of the head body wall. (h) NV_138860 sense control with no staining. ph – pharynx, ts – testis, bw- body wall, ms – mesenteries, tl – tentacle. Scale bars: 100 μm.</p

Studied <i>N. vectensis</i> Genes.

<p>Studied <i>N. vectensis</i> Genes.</p

<i>Nematostella</i> FNGs expression illustration.

<p>Yellow and Red color represent localization as indicated in the legend.</p

RNA expression of <i>N. vectensis</i> monoamine oxidase (MO) and nicotinamide N-methyltransferase (NNMT).

<p>(a, d) <i>Nematostella</i> NV_94865 (MO) and NV_209258 (NNMT) expression in the endoderm around the pharynx. (b, e) NV_94865 and NV_209258 expression in the endoderm around testis. Body wall endoderm is not stained. (c) NV_94865 expression in testis and in body wall endoderm. (f) NV_209258 expression in young testis. ph – pharynx, ts – testis, bw- body wall, yt – young testis. Scale bars: 100 μm.</p

RNA expression of <i>N. vectensis</i> human glutamate decarboxilase (GAD) closely and distantly related genes.

<p>(a) <i>Nematostella</i> NV_70014 expression – whole animal longitudal section. (b) NV_70014 expression - longitudal section in a closed position. No expression is observed in tentacles. (c, g) <i>Nematostella</i> NV_70014 and NV_224555 expression showing the same localization in the endoderm around the pharynx. (d, h) NV_70014 and NV_224555 expression around the testis. (e, i) NV_70014 and NV_224555 expression in young testis. (f) NV_70014 expression - enlargement of the pharynx area showing the link between the pharyngeal expressing tissue ring and the expressing tissue surrounding gonads (marked with asterisk). (j) NV_224555 sense control with no staining. ph – pharynx, ts – testis, bw- body wall, yt – young tesis, ms – mesenteries, tl – tentacle. Scale bars: 100 μm.</p

Opposite localizations of <i>Nematostella</i> GLUT-like (NV_173595) compared to <i>Nematostella</i> GRP75 (NV_86017) control.

<p>(a–c) H&E stained histology of the expressing tissues. (a) Pharynx: The tested FNGs were expressed in the pharyngeal endoderm. (b) Testis: FNGs were expressed in the endoderm surrounding the testis with same morphology and cellular composition as in a. (c) Body wall and a fragment of a mesentery in the head area: FNGs were expressed in the body wall endoderm (d–f) typical expressions of <i>Nematostella</i> FNGs as appeared for <i>Nematostella</i> GLUT (NV_173595). (d) part of the expressing endodermal tissue around the pharynx, no expression in tentacles (e) expressing endodermal tissue around the gonads (e) expressing endodermal tissue of the head body wall (g–i) expression patterns of <i>Nematostella</i> GRP75 (NV_86017). (g) No expression in pharynx and some expression in tentacles ectoderm as opposed to d. (h) GRP75 expressing gametes inside the gonads. No expression in the surrounding tissue as opposed to b. (c) strong body wall ectodermic expression as opposed to C. ec – ectoderm, en - endodem, ts – testis, cn - cnidocyte, mg- mesoglea, me-mesentery, in – interior, out – exterior. Scale bars: 40 μm.</p

Different localization of RFamide neuropeptide and <i>Nematostella</i> vGLUT.

<p>(a–e) RFamide immunolocalization (Cy-3; red). (f–j) <i>Nemtostella</i> vGlut (NV_138860) mRNA expression (Texas red; red). (a) RFamide localization in the body wall, mostly in the basal side of the endoderm (toward the mesoglea). Cell nuclei are stained with DAPI (blue). (b) confocal microscope picture (stacking) of the body wall showing endodermal nerve-cells connected between them by a common endodermal thread. (c) Very few RFamide-positive cells (white arrow heads) were identified in the male gonads. (d) RFamide positive nerve cells scattered mostly in the ectoderm, but also in and endoderm of the pharyngeal ring. Tissue is visible in white light background. (e) Corresponding picture to d without white-light superimposing. (f) <i>Nemtostella</i> NV_138860 expression in an apical endodermal layer of the body wall. Cell nuclei are stained with DAPI (blue). (g) Corresponding picture to f without DAPI staining. (h) <i>Nemtostella</i> NV_138860 expression in the tissue surrounding the testis. Gametes are stained with DAPI (blue) (i) <i>Nemtostella</i> NV_138860 mRNA expression in the endodermal pharyngeal layer and in the epical side area of the body wall. Cell nuclei are stained with DAPI (blue). (j) corresponding picture to i without DAPI staining. ec – ectoderm, en – endoderm, ts – testis, bw – body-wall, mg – mesoglea, ph – pharynx. Scale bars: 20 μm.</p

RNA expression of <i>N. vectensis</i> acetylcholinesterase (AChE) and butyrylcholinesterase (BChE).

<p>(a, d) <i>Nematostella</i> NV_209664 (AChE) and NV_119959 (BChE) expression in the endoderm around the pharynx and in the endoderm of the head body wall. (b, e) NV_209664 and NV_119959 expression in the endoderm around the testis. (c) NV_209664 expression in young testis. (f) NV_119959 sense control with no staining. ph – pharynx, ts – testis, bw- body wall, yt – young testis. Scale bars: 100 μm.</p

MCT8 regulates axon branching in the Rohon-Beard sensory neurons.

<p><b>A</b>. A representative scheme of the Rohon-Beard (RB) sensory neuron location in zebrafish larvae. <b>B–D</b>. Double fluorescent ISH in 33 hpf embryos revealed co-localization of <i>p2rx3.1</i> (green) and <i>mct8</i> (red) in RB cell bodies. <b>E–F</b>. Whole mount ISH showed the spatial expression of <i>p2rx3.1</i> in the dorsal spinal cord of 2 dpf WT-sibling (<b>E</b>) and <i>mct8−/−</i> larvae (<b>F</b>). <b>G–I</b>. Whole-mount ISH and immunofluorescence revealed co-localization of EGFP (green) and <i>p2rx3.1</i> (red) in the cell body of an RB neuron in 2 dpf <i>huc:GAL4+uas:memYFP</i>-injected embryos. <b>J</b>. The percentages of embryos that express <i>memYFP</i> in single arborized RB neurons in the tail (black bars), are shown in 2 dpf WT-sibling, <i>mct8−/−</i> and <i>mct8</i> mRNA-injected <i>mct8−/−</i> embryos. Statistical significance was determined by the Chi square test. Different letters indicate significant difference. <b>K</b>. The percentages of embryos that express <i>memYFP</i> in single arborized RB neurons in the tail (black bars), are shown in 2 dpf WT-sibling, <i>mct8−/−</i>, WT-sibling treated with 0.5 nM TRIAC and <i>mct8−/−</i> treated with 0.5 nM TRIAC. Statistical significance was determined by the Chi square test. Different letters indicate significant difference. <b>L, M</b>. Lateral view of arborized RB-neuron that projects toward the tail in 2 dpf live <i>mct8−/−</i> and WT-sibling embryos, which are transiently expressed <i>huc:GAL4</i> and <i>uas:memYFP</i> constructs. <b>N</b>. Schematic illustration of arborized RB sensory neuron. Each color represents a single branch that was subjected to ImageJ software analysis. Filopodia are colored in black. The total length (<b>O</b>), average length (<b>P</b>), and number of branches (<b>Q</b>) measured in <i>mct8−/−</i> and WT-sibling embryos. Scale bar = 30 µm. Values represented as means±SEM (standard error of the mean). Statistical significance determined by <i>t</i>-test: Two-sample assuming unequal variances followed by one-sample Kolmogorov-Smirnov test, to assume normal distribution (*<i>p<0.05</i>).</p

Loss of MCT8 reduces synaptic density in axonal arbors of the motor neuron.

<p><b>A–C</b>. Confocal imaging of a 2 dpf live <i>tg</i>(<i>mct8:EGFP</i>) embryo co-injected with <i>huc:GAL4</i> and <i>uas:tRFP</i> constructs revealed co-localization of <i>mct8</i> (green) and the <i>huc</i> pan-neural marker (red) in a motor neuron. <b>D</b>. Schematic illustration of an axonal arbor in a motor neuron. Each color represents a single branch that was subjected to ImageJ software analysis. <b>E</b>. Lateral view of a 3 dpf <i>tg(huc:GAL4Xuas:memYFP)</i> embryo. memYFP expression driven by the <i>huc</i> promoter is observed in the spinal cord (SC) and in descending motor neurons. The dashed frame marks a single motor neuron that was selected for further comparative studies. High magnification of the framed area is shown in the trunk of 6 dpf <i>tg(huc:GAL4Xuas:memYFP)/mct8+/−</i> and <i>tg(huc:GAL4Xuas:memYFP)/mct8−/−</i> representative larvae (<b>F</b> and <b>G</b>, respectively). <b>H</b>. Lateral view of a 30 hpf <i>tg(mct8:GAL4Xuas:SYP-EGFP)</i> embryo. SYP-EGFP expression driven by the <i>mct8</i> promoter is observed in the spinal cord (SC) and in descending motor neurons. In order to compare the number of synapses in <i>mct8+/−</i> and <i>mct8−/−</i> larvae, single motor-neuron arbors were selected (dashed frame). High magnification of the dashed frame is shown in 6 dpf <i>tg(mct8:GAL4)/(uas:SYP-EGFP)/mct8+/−</i> and <i>tg(mct8:GAL4)/(uas:SYP-EGFP)/mct8−/−</i> representative larvae (<b>I</b> and <b>J</b>, respectively). The total arbor length (<b>K</b>) and the number of branches (<b>L</b>) were measured in 3 and 6 dpf <i>mct8+/−</i> larvae and in 3 and 6 dpf <i>mct8−/−</i> larvae. <b>M</b>. Synapse density in the axons of the motor-neurons was measured along the last 50 µm of a single branch in 3 and 6 dpf <i>mct8+/−</i> larvae and in 3 and 6 dpf <i>mct8−/−</i> larvae. <b>N</b>. The total number of synapses was measured in the motor-neuron arbor of 6 dpf <i>mct8+/−</i> and <i>mct8−/−</i> larvae. Scale bar = 30 µm. Values represented as means±SEM (standard error of the mean). Statistical significance determined by <i>t</i>-test: Two-sample assuming unequal variances followed by one-sample Kolmogorov-Smirnov test, to assume normal distribution (*<i>p<0.05</i>).</p