Patterning mechanisms diversify neuroepithelial domains in the Drosophila optic placode

Abstract

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 Drosophila, 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.

Fig 1. The Eya-positive domain of the optic placode is subdivided at stage 11.

(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). ato is not required for restricting Tll expression (I). Scale bars represent 20 μm.

Fig 2. tll regulates Ato-dependent PR precursor cell fate in the embryo.

(A, B) Ato (green) and Eya (red) expression in the optic placode in wildtype and tll^49l mutants at embryonic stage 11. The tll 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 tll^49l mutant stage 15 embryos. FasII (green) marks differentiated PR neurons in the embryo. The number of Sal-expressing primary precursors is increased in tll^49l mutants (D’) as compared to wildtype (C’). (E, E’, F, F’) Svp (red) expression in wildtype and tll^49l mutant stage 15 embryos. Kr (green) marks PR precursors in the embryo. The number of Svp-expressing secondary precursors is also increased in tll^49l mutants (F’) compared to wildtype (E’). (G, H) All PR numbers (G) and percentages (H) were analyzed in wildtype as well as in tll mutants. Also, the cell number and percentage of Sal- and Svp-positive cells in wildtype and tll^49l 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 tll^49I p<0.001, t = -4.731; Number of all Sal-positive cells: Anova: p<0.001 F(5,44) = 104.2; wildtype vs tll^49I p = 0.0302, t = -2.851; Number of all Svp-positive cells: Anova: p<0.001 F(5,48) = 70.63; wildtype vs tll^49I p = 0.0009, t = -4.063 (G). Ratio of Sal-positive cells: Anova: p<0.001 F(5,44) = 114.3; wildtype vs tll^49I p = 0.978, t = -0.569; Ratio of Svp-positive cells: Anova: p<0.001 F(5,48) = 59.64; wildtype vs tll^49I p = 0.995, t = 0.402 (H). n = 14 (wildtype), 8 (tll^49I) (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.

Fig 3. tll is required for primary PR precursor cell fate.

(A, A’, B, B’) Sal (red) expression in the primary PR precursors in control (so>UAS-GFP) and tll overexpression (so>UAS-GFP;UAS-tll) in embryos at stage 15. GFP (green) marks so-expressing cells whereas Kr (blue) marks PR precursors in the embryo. Sal-expressing primary PR precursors are significantly reduced in tll overexpressing embryos (B’), indicating that Tll is sufficient for repressing primary PR precursor cell fate in the embryo. (C, C’, D, D’) Svp (red) expression in the secondary PR precursors in control (so>UAS-GFP) and tll-overexpressing (so>UAS-GFP;UAS-tll) embryos at stage 15. Svp-positive secondary PR precursors are also reduced (D’) potentially as a result of the decrease in the number of primary PR precursors (B’); Maximum intensity projections of confocal sections. (E) Schematic representation of Tll mediated neuroepithelial patterning and regulation of PR versus non-PR cell fate in the embryonic optic placode of tll^49l mutants. We show that Tll represses Ato expression and thereby regulates primary PR precursor cell fate. (F, G) We also quantified cell numbers (F) and percentages (G) in so>tll embryos. Tll overexpression leads to a reduction in the number of PR precursors that are formed (counted at stages 14–16). Interestingly, so>tll animals possess a ratio of Svp- vs Sal-positive PRs which is similar to that of wildtype. Number of all PRs: wildtype vs so>tll p<0.001, t = 4.439; Number of all Sal-positive cells: wildtype vs so>tll p = 0.3358, t = 1.729; Number of all Svp-positive cells: wildtype vs so>tll p = 0.009863, t = 3.250 (F). Ratio of Sal-positive cells: wildtype vs so>tll p = 0.612, t = -1.305; Ratio of Svp-positive cells: wildtype vs so>tll p = 0.203, t = -2.000 (G). n = 14 (wildtype), 18 (so>tll; all PRs), 7 (so>tll; Sal-positive cells), 11 (so>tll; Svp-positive cells) (F, G). 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 and ns = not significant (F, G). Scale bars represent 20 μm.

Fig 4. Hh regulates Ato- and Sens-dependent PR cell fate in the embryo.

(A, B) Ato (blue) expression in the optic placode in wildtype and ptc⁹ 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 ptc⁹ mutants (B) as compared to wildtype (A). (C, D) Sens (red) expression in the PR precursors of wildtype and ptc⁹ mutant stage 12 embryos. FasII (green) marks differentiated PR neurons at this stage. The number of Sens expressing cells is also increased in ptc⁹ mutants (D). (E, E’, F, F’) Sal (red) expression in the PR precursors in wildtype and ptc⁹ mutant stage 15 embryos. Kr (green) marks PR precursors at this stage. The number of Sal-expressing primary PR precursors is significantly increased in ptc⁹ mutants (F’) compared to wildtype (E’). (G, G’, H, H’) Svp (red) expression in the PR precursors in wildtype and ptc⁹ 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 ptc⁹ mutants (J). In ptc 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 ptc mutants and in wildtype. Number of all PRs: wildtype vs ptc⁹ p<0.001, t = -14.768; Number of all Sal-positive cells: wildtype vs ptc⁹ p<0.001, t = -6.608; Number of all Svp-positive cells: wildtype vs ptc⁹ p<0.001, t = -14.428 (I). Ratio of Sal-positive cells: wildtype vs ptc⁹ p = 0.876, t = 0.887; Ratio of Svp-positive cells: wildtype vs ptc⁹ p = 0.967, t = -0.627 (J). n = 14 (wildtype), 6 (ptc⁹) (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.

Fig 5. Hh signaling controls cell number in the optic placode.

Tll (blue) expression in wildtype and ptc⁹ 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 ptc⁹ mutants (B). Tll is expressed in the optic lobe precursors in wildtype (A’) and its expression is unchanged in ptc⁹ mutant embryos (B’). (C, C’, D, D’, E) Analysis of cell proliferation at stage 11 in the optic placode in wildtype and ptc⁹ 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 ptc⁹ mutants is not significantly different from that of wildtype p = 0.4655, t(18) = -0.7457 (E). n = 10 (wildtype), 10 (ptc⁹) (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 ptc⁹ mutants) show an increased number of Ato expressing cells in the optic placode [6]. Scale bars represent 20 μm.

Fig 6. Notch regulates Ato-dependent PR cell fate in the embryo.

(A-E) Notch activity in the optic placode at stages 10–14 determined by using the E(spl)mγ-HLH::GFP 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 N^55e11 mutant stage 11 embryos. The number of Ato expressing cells is significantly increased in N^55e11 mutants (G). Sal (red) and Svp (blue) expression in the PR precursors at embryonic stage 15 in wildtype (H, H’, H”), N^55e11 (I, I’, I”) and in the activated Notch overexpression (so>UAS-N^intra) (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 N^55e11 mutants the number of Sal expressing PRs is increased (I’), whereas in N^intra overexpression embryos they are absent (J’). In N^55e11 mutants Svp expressing PRs are significantly reduced or absent (I”) while N^intra 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, N^55e11 mutant and so>UAS-N^intra overexpression embryos. In N^55e11 mutants a higher number of PR precursors is specified compared to wildtype control (counted at stage 14–16). N^55e11 mutants possess more Sal-positive primary PR precursors than wildtype, whereas no Sal-positive cells were found in N^intra overexpressing embryos. Conversely, Svp-positive secondary PR precursors are severely reduced in N^55e11 mutants whereas in N^intra overexpressing embryos they represent 100% of the PR precursors. Number of all PRs: wildtype vs N^55e11 p<0.001, t = -8.203; wildtype vs so>N^Intra p = 0.048, t = 2.634; Number of all Sal-positive cells: wildtype vs N^55e11 p<0.001, t = -19.020; wildtype vs so>N^Intra p = 0.0312, t = 2.838; Number of all Svp-positive cells: wildtype vs N^55e11 p = 0.0028, t = 3.692; wildtype vs so>N^Intra p = 0.6041, t = 1.308 (K). Ratio of Sal-positive cells: wildtype vs N^55e11 p<0.001, t = -17.594; wildtype vs so>N^Intra p<0.001, t = 8.764; Ratio of Svp-positive cells: wildtype vs N^55e11 p<0.001, t = 12.425; wildtype vs so>N^Intra p<0.001, t = -6.189 (L). n = 14 (wildtype), 7 (N^55e11), 8 (so>N^Intra) (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.

Fig 7. Notch represses sens-mediated binary cell fate decision and controls primary versus secondary PR precursor cell fate.

(A, B) Tll (blue) expression analysis in the optic placode in wildtype and N^55e11 mutant stage 12 embryos. Hazy (green) marks all PR precursors and FasII (red) labels larval eye and optic lobe primordium at this stage. Tll is expressed in the optic lobe primordium but not in the PR precursors in wildtype (A). In N^55e11 mutant embryos Tll expression is completely absent (B). (C, C’ D, D’, E, E’) Sens (red) expression in wildtype and in embryos overexpressing a dominant negative form of Kuzbanian (Kuz^DN) or the intracellular domain of Notch (N^intra) in the optic placode by using a so-Gal4, UAS-mCD8::GFP recombinant transgenic line. Sens is normally expressed in four PR precursors in wildtype (C’), which adopt the primary PR precursor cell fate. Overexpression of Kuz^DN in the optic placode leads to an increase in the number of Sens expressing cells (D’), whereas overexpressing N^intra results in loss of Sens expression from the presumptive primary PR precursors (E’); z-projections of confocal sections. (F) Schematic representation of Notch mediated neuroepithelial patterning and regulation of cell fates in the embryonic optic placode. Notch activity is required for tll expression, and thus, indirectly and negatively regulates ato expression. Notch also represses sens expression. In N^55e11 mutant this repression is removed and ectopic Sens induces primary PR precursor cell fate while repressing secondary PR precursor cell fate. Scale bars represent 20 μm.

Fig 8. Model of neurogenic optic placode patterning and control of cell fate choices in the wildtype embryo.

Schematic representation of neuroepithelial patterning and regulation of optic lobe neuroepithelium versus PR cell fate in the optic placode in Drosophila. Regulation of ato and tll expression by Hh and Notch signaling in the embryo is required for the acquisition of cell fate identity in the primordium of optic lobe and larval eye. Temporal expression of ato (early and late ato) in the larval eye primordium marks PR cell fate whereas tll expression in the optic lobe primordium marks optic lobe neuroepithelial cell fate. Tll represses ato expression and maintains a balance between PR and non-PR cells in the optic placode. Hh signaling promotes Ato-dependent primary PR precursor specification and controls cell number in the optic placode possibly by regulating cell proliferation. Notch activity maintains optic lobe neuroepithelial cell fate in the optic lobe by indirectly repressing ato activity in a Tll-dependent manner. In addition, at later stages Notch represses Sens and thereby promotes secondary PR precursor formation at the expense of primary PR precursors.