Extended Data figure 8: Temporal expression of concentric genes in medulla neurons.

(a) Temporal expression of concentric genes in the medulla cortex.

(i-i’’’) Expression (from early to late born) of Bsh, Run, Vvl, Toy and Slp in medulla neurons at L3 stage. Bsh neurons are closer to the medulla neuropil (indicated with dashed line), while Slp neurons (arrowhead) are closer to the surface of the brain (NB layer). Anterior is shown to the right and posterior to the left throughout the Figure.

(ii-ii’’’) Expression of Hth, Svp and Toy in medulla neurons at L3 stage. Hth is expressed in the first-born neurons, located closer to the medulla neuropil. Svp is expressed in several laminae along the medulla cortex. Toy is expressed in mid-late born neurons.

(iii-iii’’’) Expression of Svp, Forkhead domain 59A (Fd59A) and Hbn in medulla neurons at L3 stage. Svp is expressed in several laminae along the medulla cortex. Fd59A is expressed in mid-late born neurons, some of them (magenta arrowheads) generated before Hbn neurons. Cyan arrowheads denote Lawf1-2 neurons, which have a different origin³⁸.

(iv-iv’’’) Expression of Tup, Knot (Kn), and Toy in medulla neurons at L3 stage. Tup is expressed in several laminae of early and late born neurons along the medulla cortex, some of them being born before Kn neurons (iv’). Tup+Toy neurons are the first-born Toy neurons in the ventral side of the medulla (iv’’, arrowhead). Kn neurons are generated before Kn+Toy neurons (iv’’’, arrowhead).

(v-v’’’) Expression of Toy, Fkh:GFP and Otd in medulla neurons at L3 stage. Some Toy neurons are generated before Fkh and Otd neurons (E’-E’’). Fkh+Otd neurons (arrowhead) are only found in the posterior tips of the main OPC (E’’’), mainly in the Dpp region, as previously described⁴⁰.

(vi-vi’’’) Expression of Toy, Fd59A and Ets65A:GFP in medulla neurons at L3 stage. Toy and Fd59A expressing neurons are intermingled in the medulla cortex, being coexpressed in specific subregions of the medulla cortex (vi’, arrowheads). Many Toy and Fd59A neurons are generated before Ets65A-expressing neurons (vi’’-vi’’’).

(vii) Expression of TfAP-2 and Run in medulla neurons at L3 stage. The first laminae of TfAP-2+Ap+ neurons (Notch^ON) are intermingled with Run neurons (Notch^OFF) (arrowheads), suggesting that they could be sister neurons.

(viii) Expression of TfAP-2 and Vvl in medulla neurons at L3 stage. The first laminae of TfAP-2 neurons are generated before Vvl neurons. The second laminae of TfAP-2 neurons are generated after some Vvl neurons.

(ix) Expression of TfAP-2 and Toy in medulla neurons at L3 stage. The first laminae of TfAP-2 neurons are generated before Toy neurons. The second laminae of TfAP-2 neurons are intermingled with some Toy neurons.

(x) Expression of TfAP-2 and Ets65A:GFP in medulla neurons at L3 stage. All TfAP-2 neurons are born before Ets65A-expressing neurons, except in the cases where both TFs are coexpressed (arrowhead). These neurons are very late born and are the only Notch^ON medulla neurons that are glutamatergic.

(xi) Expression of Vvl and Kn in medulla neurons at L3 stage. Vvl+Ap+ (Notch^ON) and Kn neurons (Notch^OFF) start to be generated at similar times, suggesting that they could be sister neurons.

(xii) Expression of Toy and Sox102F in medulla neurons at L3 stage. Toy and Sox102F neurons are mid-late born neurons intermingled in the medulla cortex. Toy and Sox102F are expressed individually or coexpressed in medulla neurons.

(xiii) Expression of Toy and Tj in medulla neurons at L3 stage. Toy and Tj neurons are expressed in mid-late born neurons intermingled in the medulla cortex. They are coexpressed in specific subregions of the medulla cortex (arrowhead).

(xiv) Expression of Toy and Hbn in medulla neurons at L3 stage. Hbn neurons are generated after some Toy neurons.

Scale bars: 10 μm

(b) Concentric genes expressed both early and late in medulla neurons.

(i) TFs Seven-Up (Svp), (ii) TfAP-2 and (iii) Tailup (Tup) are expressed in several laminae along the medulla cortex and are expressed in neurons generated in several temporal windows.

(c) Notch status of medulla neurons and concentric gene expression.

(i) Expression of Bsh and Ap in medulla neurons. All Bsh neurons express Ap. Anterior is to the right and posterior to the left in this and subsequent panels.

(ii) Expression of Run and Ap in medulla neurons at L3 stage. None of the Run positive neurons express Ap.

(ii’) Expression of Ap and Svp in medulla neurons at L3 stage. None of the Svp positive neurons express Ap.

(ii’’) Expression of Ap and Fd59A in medulla neurons at L3 stage. None of the Fd59A positive neurons express Ap.

(ii’’’) Expression of Ap and Hbn in medulla neurons at L3 stage. None of the Hbn positive neurons express Ap.

(iii) Expression of Ap and Vvl in medulla neurons at L3 stage. Vvl neurons that are earlier born, such as Tm9, express Ap (white arrowhead), while later born Vvl neurons, such as Dm8 and Dm11, do not (magenta arrowhead).

(iii’) Expression of Ap and Toy in medulla neurons at L3 stage. Earlier born Toy neurons, such as Dm10, do not express Ap (magenta arrowhead), while later born Toy neurons, such as Tm20, express Ap (white arrowhead).

(iii’’) Expression of Ap and TfAP-2 in medulla neurons at L3 stage. First born TfAP-2 neurons, such as Pm1, Pm2 and Pm3, do not express Ap (magenta arrowheads), while later born TfAP-2-expressing neurons, such as Tm1, express Ap (white arrowhead).

(iii’’’) Expression of Ap and D in medulla neurons at L3 stage. Earlier born D neurons do not express Ap (magenta arrowhead) and express D in the absence of this tTF in their NBs of origin, while later born D neurons express Ap (white arrowhead) and are generated in the D temporal window.

(iii’’’’) Expression of Ap and Ets65A:GFP in medulla neurons at L3 stage. Earlier born Ets65A neurons do not express Ap (magenta arrowhead), while later born Ets65A neurons express Ap (white arrowhead).

Scale bars: 10 μm

(d) Medulla clusters. Hierarchical tree relating the average variable gene expression from each cluster of the L3-P15 dataset. Low quality (LQ) clusters or clusters that have a different origin from the medulla side of the OPC neuroepithelium, such as PRs, L neurons, Lawf1-2, LCNs and LPLCs neurons are shown in red.