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Clonal analysis of neur.

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posted on 08.01.2015 by Tobias Troost, Markus Schneider, Thomas Klein

Clones are labelled by the loss of GFP (green). (A–D) A neur mutant clone that includes the complete aSA cluster (arrow in A). The region is shown at higher magnification in (B–D). Comparison of sca and Hnt expression reveals that a subgroup of 7 cells has adopted the SOP fate in the absence of neur function. (E–H) A clone where a wildtype SOP is surrounded by neur mutant cells (arrow in E). (F–H) Magnification of the region highlighted by the arrow in (E). Only the wildtype cell has adopted the SOP fate indicated by Hnt expression (arrowhead in F, G, H). Hence, the wildtype Neur positive SOP can prevent all neur mutant neighbours from becoming SOPs. (I) A clone that includes the DC cluster (arrow). This region is shown at higher magnification in (J–L). At the aDC position all cells are mutant and three cells have adopted the SOP fate as indicated by expression of Hnt (arrowhead). At the pDC position only the GFP positive wildtype cell expresses Hnt (arrow). Note, while this SOP inhibits its neighbours, it is unable to prevent the SOP fate in the mutant cells only two diameters away (arrowhead). This indicates that the Neur dependent signal emitted by the pDC SOP reaches only the adjacent cells. (M–O) In cases where the clones include a wildtype SOP, a halo of Gbe+Su(H) expression is observed. Arrowheads in (M) point to the regions shown in (N, O) at higher magnification. Arrowheads in (N, O) point to the wildtype SOPs surrounded by the neur mutant cells. (P, Q) The halos were lost in cases where the whole cluster is mutant (arrowhead). (R–X) Expression of Hnt and sca-lacZ neur mutant PNCs at 12 h and 6 h bpf. At the position highlighted by the arrowhead, four cells express Hnt, but one stronger than its neighbours. The cell is also larger. Only one cell expresses Hnt at the other position of the mutant cluster (arrow). (T–W) A neur clone that includes the DC cluster at 12 h bpf. The arrowhead highlights the position of the pDC in the early arising DC cluster, the yellow arrows that of later arising PNCs. The white arrow highlights a single wildtypic SOP surrounded by mutant cells. (S, T) Magnification of the DC region highlighted by the arrowhead in (R). At this early time the pDC arises. Although all cell of the cluster are neur mutant, only one cell has elevated levels of sca expression (S) and weakly expresses Hnt (T) at this stage. This indicates that this cell has elevated proneural activity and is already determined as SOP. Thus, this cell is advanced in its development towards the SOP fate. In the older disc shown in (U–X) four Hnt expressing SOPs can be seen at the pDC position. This indicates that the advanced cell was not able to inhibit its neighbours in the absence of Neur function. The yellow arrows point to the late arising cluster mutant for neur. In these cluster only one Hnt expressing cells is recognised, indicating the these cells are also advanced in their development. Note, that the expression of sca-lacZ is not elevated between the PNCs in the mutant territory as observed upon complete loss of Notch function. White scale bar 50µm; cyan scale bar: 10µm.