The Eya-positive domain of the optic placode is subdivided at stage 11.

<p>(A-F) Eya (red), Tll-GFP (green) and Ato (blue) expression patterns in the embryonic optic placode at stages 9–13. At stage 10 a patch of Eya-positive cells is detected within the ventral most region of the optic placode (B, outlined in yellow). Some of these cells start expressing Ato during stage 11 and will form the Bolwig's organ (C, outlined in magenta), whereas other cells expressing Tll will form the optic neuropile. Ato expression is progressively restricted during stages 11 and 12 (D, E) and is no longer detectable at stage 13 (F). The retinal determination network transcription factor So is co-expressed with Eya in the optic placode (G, H). <i>ato</i> is not required for restricting Tll expression (I). Scale bars represent 20 μm.</p

Hh signaling controls cell number in the optic placode.

<p>Tll (blue) expression in wildtype and <i>ptc</i>^<i>9</i> mutants (stage 12–13 embryos). Hazy (green) marks all PR precursors whereas FasII (red) labels larval eye and optic lobe primordium. (A, B) In the larval eye precursors, Tll is neither expressed in wildtype (A) nor in <i>ptc</i>^<i>9</i> mutants (B). Tll is expressed in the optic lobe precursors in wildtype (A’) and its expression is unchanged in <i>ptc</i>^<i>9</i> mutant embryos (B’). (C, C’, D, D’, E) Analysis of cell proliferation at stage 11 in the optic placode in wildtype and <i>ptc</i>^<i>9</i> mutant embryos by staining with anti-pH3 (green) antibody. So (red) was used to mark the area of the optic placode, from where pH3-positive cells were counted (white outline in C and D). The number of pH3-positive cells in the Eya-positive domain of <i>ptc</i>^<i>9</i> mutants is not significantly different from that of wildtype p = 0.4655, t(18) = -0.7457 (E). n = 10 (wildtype), 10 (<i>ptc</i>^<i>9</i>) (E). Data is shown as mean and error bars as standard deviation. Circles represent numbers of individual samples. ns = not significant (E). (F) Schematic representation of Hh mediated optic placode patterning and acquisition of PR versus non-PR cell fate in the embryo. Hh signaling promotes Ato expression and Hh gain-of-function (in <i>ptc</i>^<i>9</i> mutants) show an increased number of Ato expressing cells in the optic placode [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007353#pgen.1007353.ref006" target="_blank">6</a>]. Scale bars represent 20 μm.</p

Hh regulates Ato- and Sens-dependent PR cell fate in the embryo.

<p>(A, B) Ato (blue) expression in the optic placode in wildtype and <i>ptc</i>^<i>9</i> mutant stage 11 embryos. So (red) marks cells of the entire optic placode at this stage. The number of Ato-expressing cells is increased in <i>ptc</i>^<i>9</i> mutants (B) as compared to wildtype (A). (C, D) Sens (red) expression in the PR precursors of wildtype and <i>ptc</i>^<i>9</i> mutant stage 12 embryos. FasII (green) marks differentiated PR neurons at this stage. The number of Sens expressing cells is also increased in <i>ptc</i>^<i>9</i> mutants (D). (E, E’, F, F’) Sal (red) expression in the PR precursors in wildtype and <i>ptc</i>^<i>9</i> mutant stage 15 embryos. Kr (green) marks PR precursors at this stage. The number of Sal-expressing primary PR precursors is significantly increased in <i>ptc</i>^<i>9</i> mutants (F’) compared to wildtype (E’). (G, G’, H, H’) Svp (red) expression in the PR precursors in wildtype and <i>ptc</i>^<i>9</i> mutant stage 15 embryos. The number of Svp expressing secondary PR precursors is also increased (H’), probably as a result in the increase of primary PR precursors (F’). (I, J) Quantification of PR cell number (I) and percentage of Sal- and Svp-positive PRs in the wildtype and <i>ptc</i>^<i>9</i> mutants (J). In <i>ptc</i> mutants more PR precursors are formed compared to wildtype control. Analyzing the subtype identity of these PR precursors, we found that the ratio of Svp- vs Sal-positive cells is the same in <i>ptc</i> mutants and in wildtype. Number of all PRs: wildtype vs <i>ptc</i>^<i>9</i> p<0.001, t = -14.768; Number of all Sal-positive cells: wildtype vs <i>ptc</i>^<i>9</i> p<0.001, t = -6.608; Number of all Svp-positive cells: wildtype vs <i>ptc</i>^<i>9</i> p<0.001, t = -14.428 (I). Ratio of Sal-positive cells: wildtype vs <i>ptc</i>^<i>9</i> p = 0.876, t = 0.887; Ratio of Svp-positive cells: wildtype vs <i>ptc</i>^<i>9</i> p = 0.967, t = -0.627 (J). n = 14 (wildtype), 6 (<i>ptc</i>^<i>9</i>) (I, J). Data is shown as mean and error bars as standard deviation. Circles represent numbers or percentages of individual samples. *** p<0.001and ns = not significant (I, J). Scale bars represent 20 μm.</p

Patterning mechanisms diversify neuroepithelial domains in the <i>Drosophila</i> optic placode

<div><p>The central nervous system develops from monolayered neuroepithelial sheets. In a first step patterning mechanisms subdivide the seemingly uniform epithelia into domains allowing an increase of neuronal diversity in a tightly controlled spatial and temporal manner. In <i>Drosophila</i>, neuroepithelial patterning of the embryonic optic placode gives rise to the larval eye primordium, consisting of two photoreceptor (PR) precursor types (primary and secondary), as well as the optic lobe primordium, which during larval and pupal stages develops into the prominent optic ganglia. Here, we characterize a genetic network that regulates the balance between larval eye and optic lobe precursors, as well as between primary and secondary PR precursors. In a first step the proneural factor Atonal (Ato) specifies larval eye precursors, while the orphan nuclear receptor Tailless (Tll) is crucial for the specification of optic lobe precursors. The Hedgehog and Notch signaling pathways act upstream of Ato and Tll to coordinate neural precursor specification in a timely manner. The correct spatial placement of the boundary between Ato and Tll in turn is required to control the precise number of primary and secondary PR precursors. In a second step, Notch signaling also controls a binary cell fate decision, thus, acts at the top of a cascade of transcription factor interactions to define PR subtype identity. Our model serves as an example of how combinatorial action of cell extrinsic and cell intrinsic factors control neural tissue patterning.</p></div

<i>tll</i> regulates Ato-dependent PR precursor cell fate in the embryo.

<p>(A, B) Ato (green) and Eya (red) expression in the optic placode in wildtype and <i>tll</i>^<i>49l</i> mutants at embryonic stage 11. The <i>tll</i> mutant placode is bigger, and, as a consequence, the number of Ato-expressing cells is increased (B). Ato-expression extends within the posterior region of the optic placode. (C, C’, D, D’) Sal (red) expression in wildtype and <i>tll</i>^<i>49l</i> mutant stage 15 embryos. FasII (green) marks differentiated PR neurons in the embryo. The number of Sal-expressing primary precursors is increased in <i>tll</i>^<i>49l</i> mutants (D’) as compared to wildtype (C’). (E, E’, F, F’) Svp (red) expression in wildtype and <i>tll</i>^<i>49l</i> mutant stage 15 embryos. Kr (green) marks PR precursors in the embryo. The number of Svp-expressing secondary precursors is also increased in <i>tll</i>^<i>49l</i> mutants (F’) compared to wildtype (E’). (G, H) All PR numbers (G) and percentages (H) were analyzed in wildtype as well as in <i>tll</i> mutants. Also, the cell number and percentage of Sal- and Svp-positive cells in wildtype and <i>tll</i>^<i>49l</i> mutant embryos were quantified, and the ratio of Sal- and Svp-positive PRs is not significantly changed in these two genotypes (G, H). Number of all PRs: Anova: p<0.001 F(5,55) = 92.92; wildtype vs <i>tll</i>^<i>49I</i> p<0.001, t = -4.731; Number of all Sal-positive cells: Anova: p<0.001 F(5,44) = 104.2; wildtype vs <i>tll</i>^<i>49I</i> p = 0.0302, t = -2.851; Number of all Svp-positive cells: Anova: p<0.001 F(5,48) = 70.63; wildtype vs <i>tll</i>^<i>49I</i> p = 0.0009, t = -4.063 (G). Ratio of Sal-positive cells: Anova: p<0.001 F(5,44) = 114.3; wildtype vs <i>tll</i>^<i>49I</i> p = 0.978, t = -0.569; Ratio of Svp-positive cells: Anova: p<0.001 F(5,48) = 59.64; wildtype vs <i>tll</i>^<i>49I</i> p = 0.995, t = 0.402 (H). n = 14 (wildtype), 8 (<i>tll</i>^<i>49I</i>) (G, H). Data is shown as mean and error bars as standard deviation. Circles represent numbers or percentages of individual samples. *** p<0.001, *p<0.05 and ns = not significant (G, H). Scale bars represent 20 μm.</p

Notch regulates Ato-dependent PR cell fate in the embryo.

<p>(A-E) Notch activity in the optic placode at stages 10–14 determined by using the <i>E(spl)mγ-HLH</i>::<i>GFP</i> reporter line and staining embryos with anti-GFP (green), anti-Eya (red) and anti-Ato (blue) antibodies. Notch activity is dynamic: it is initially expressed in most cells in the placode early during stage 11 (outlined in yellow), and then it becomes excluded from the patch of Ato-expressing cells, which later will develop as PR precursors (outlined in purple). (F, G) Ato (blue, outlined in white) expression in the optic placode in wildtype and <i>N</i>^<i>55e11</i> mutant stage 11 embryos. The number of Ato expressing cells is significantly increased in <i>N</i>^<i>55e11</i> mutants (G). Sal (red) and Svp (blue) expression in the PR precursors at embryonic stage 15 in wildtype (H, H’, H”), <i>N</i>^<i>55e11</i> (I, I’, I”) and in the activated <i>Notch</i> overexpression (<i>so>UAS-N</i>^<i>intra</i>) (J, J’, J”). Kr (green) marks all PR precursors at this stage. Four Sal expressing primary PR precursors (H’) and 8–10 Svp expressing secondary PR precursors (H”) are seen in wildtype. In <i>N</i>^<i>55e11</i> mutants the number of Sal expressing PRs is increased (I’), whereas in <i>N</i>^<i>intra</i> overexpression embryos they are absent (J’). In <i>N</i>^<i>55e11</i> mutants Svp expressing PRs are significantly reduced or absent (I”) while <i>N</i>^<i>intra</i> overexpression does not affect the number of Svp expressing PRs (J”). (K, L) Quantification of PR number (K) and percentage of Sal- and Svp-positive PRs (L) in wildtype, <i>N</i>^<i>55e11</i> mutant and <i>so>UAS-N</i>^<i>intra</i> overexpression embryos. In <i>N</i>^<i>55e11</i> mutants a higher number of PR precursors is specified compared to wildtype control (counted at stage 14–16). <i>N</i>^<i>55e11</i> mutants possess more Sal-positive primary PR precursors than wildtype, whereas no Sal-positive cells were found in <i>N</i>^<i>intra</i> overexpressing embryos. Conversely, Svp-positive secondary PR precursors are severely reduced in <i>N</i>^<i>55e11</i> mutants whereas in <i>N</i>^<i>intra</i> overexpressing embryos they represent 100% of the PR precursors. Number of all PRs: wildtype vs <i>N</i>^<i>55e11</i> p<0.001, t = -8.203; wildtype vs <i>so>N</i>^<i>Intra</i> p = 0.048, t = 2.634; Number of all Sal-positive cells: wildtype vs <i>N</i>^<i>55e11</i> p<0.001, t = -19.020; wildtype vs <i>so>N</i>^<i>Intra</i> p = 0.0312, t = 2.838; Number of all Svp-positive cells: wildtype vs <i>N</i>^<i>55e11</i> p = 0.0028, t = 3.692; wildtype vs <i>so</i>><i>N</i>^<i>Intra</i> p = 0.6041, t = 1.308 (K). Ratio of Sal-positive cells: wildtype vs <i>N</i>^<i>55e11</i> p<0.001, t = -17.594; wildtype vs <i>so>N</i>^<i>Intra</i> p<0.001, t = 8.764; Ratio of Svp-positive cells: wildtype vs <i>N</i>^<i>55e11</i> p<0.001, t = 12.425; wildtype vs <i>so>N</i>^<i>Intra</i> p<0.001, t = -6.189 (L). n = 14 (wildtype), 7 (<i>N</i>^<i>55e11</i>), 8 (<i>so</i>><i>N</i>^<i>Intra</i>) (K, L). Data is shown as mean and error bars as standard deviation. Circles represent numbers or percentages of individual samples. *** p<0.001, **p<0.01, *p<0.05 and ns = not significant (K, L). Scale bars represent 20 μm.</p

Underlying data main and supplementary figures

2 Excel files with underlying data of Figs. 1CEFGH, 2AD, 3AGH, 4AB, 5BEGI, 6F, 7F, 9BE, S1ABC, S2BCD, S3ABCDE, S5, S6A, S7BC, S17BE, S18, and 2 video for Fig 1

Detached paranodal loops and wider nodes in dKO.

<p>Electron micrographs of ultrathin longitudinal control and dKO sciatic nerve sections at 8 wk post-tamoxifen showing in (A) paranodal loops attached to the axolemma and septate-like junctions (arrows) in control nerves, and detached paranodal loops devoid of septate-like junctions (arrows) in dKO nerves. In some dKO nodes, microvilli (highlighted in blue, image on the right) invaded the space between paranodal loops and the axolemma. Images on the right are magnifications of white boxes depicted on the left images. The graph representing the percentage of paranodes with detached loops in control and dKO demonstrates frequent occurrence of these defects in dKO sciatic nerves. Three animals per genotype were used, 11 to 38 paranodes were counted per animal, and 56 to 72 were counted per genotype. In (B), electron micrographs represent nodes of control (Ctr in the graph) and dKO nerves, and the quantification of nodal widths in the graph shows significant widening of the nodal region in dKO sciatic nerves. Three animals per genotype were used for quantification. The average width of 7 to 17 nodes of Ranvier was calculated per animal (<i>n</i> = 3), a total of 32 to 42 nodes were measured per genotype. Scale bars = 1 μm. In (A), error bar = SEM. In (B), the graph is a box plot where the lower box (Median − Quartile 1) and the upper box (Quartile 3 − Median) are separated by the Median value and flanked by top and bottom Whiskers. <i>P</i>-values (unpaired two-tailed Student's <i>t</i> test): *** = <i>p</i> < 0.001, <i>n</i> = 3.</p

In contrast to the S49L P0 mutant, D32G and H52Y P0 mutants rescue myelination of HDAC1/2 <i>plp</i>-dKO DRG but not paranodal/nodal integrity.

<p>Coimmunofluorescence of MBP (red) and (A) neurofilament (NF, green), and Myc or GFP fluorescence (blue), or (C) neurofascins (NFasc, green), or (D) Caspr (green) in myelinated HDAC1/2 <i>plp</i>-dKO DRG cultures transduced with lentiviruses expressing either GFP, H52Y-myc, D32G-myc, S49L-myc or P0-myc, and treated with tamoxifen for 10 d after completion of myelination. A–B show that H52Y and D32G but not S49L P0 mutants are able to rescue myelination of <i>plp</i>-dKO DRG cultures, similarly to P0-myc, and C–D show that S49L, but not H52Y or D32G, P0 mutant is able to partially rescue paranodal/nodal defects of <i>plp</i>-dKO DRG cultures. In (C), pictures on the right are magnifications of the white boxes depicted on left images. Arrows indicate paranodes/nodes. In (B), quantification of MBP fluorescence intensity normalized to NF and compared to GFP or P0-myc (set to 1). DRG of six <i>plp</i>-dKO embryos were quantified (three <i>plp</i>-dKO embryos per graph, four coverslips per <i>plp</i>-dKO). In (C,D), DRG of three <i>plp</i>-dKO embryos were analyzed and representative pictures are shown. In (E), the graph represents the percentage of intact (Caspr-positive or high NFasc levels) nodes and heminodes. DRG of three <i>plp</i>-dKO embryos were quantified, four coverslips per <i>plp</i>-dKO, 80 to 300 nodes/heminodes counted per <i>plp</i>-dKO per virus. <i>P</i>-values (paired (B) and unpaired (E) two-tailed (unless stated otherwise in the figure) Student's <i>t</i> test): * = <i>p</i> < 0.05, ** = <i>p</i> < 0.01, error bars = SEM.</p

The four P0 mutations D6Y, D32G, H52Y and S49L result in three different binding profiles to neurofascins: preserved (S49L), impaired binding to NFasc155 (D32G), impaired binding to both NFasc (D6Y and H52Y), while binding to P0 is maintained for all mutants.

<p>Adhesion assay in HEK293T cells. Confocal images of P0-Fc, P0-D6Y-Fc, P0-D32G-Fc, P0-H52Y-Fc, P0-S49L-Fc or control-Fc (Neg-Fc) particles (green) and neurofascins or Myc (red) coimmunofluorescence in HEK293T cells expressing P0-myc (A), NFasc155 (B) or NFasc186 (C), indicated by arrows. Overlays appear yellow. Nuclei are labeled in blue with DAPI. Single optical sections are shown. At least three independent experiments were analyzed for each panel and representative pictures are shown.</p