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. 2024 Mar 13;22(3):e3002555. doi: 10.1371/journal.pbio.3002555

Fig 6. Specification of PNs and OCs from Islet-negative cells by MAPK and Notch pathways.

Fig 6

(A) Diagram showing effect of MAPK inhibition with the pharmacological MEK inhibitor U0126, based on findings from Wagner and colleagues and Liu and Satou. Inhibition of FGF/MAPK results in expansion of Foxg and Islet from 3 discrete foci to a large “U-shaped” swath, transforming 3 papillae into a single, enlarged papilla (similar results reported with BMP inhibition by Roure and colleagues). (B) The 10 μm U0126 treatment at 7.5 hpf/20 °C (st. 16) results in loss of PNs (assayed by C4.78>Unc-76::GFP at 17 hpf/20 °C, ~st. 27, green) upon expansion of Islet+ cells (pink nuclei), relative to DMSO alone. (C) The same treatment results in loss of OCs (L141.36>Unc-76::GFP at either 17 or 20 hpf/20 °C, ~st. 27–29 green) upon expansion of Islet+ cells (pink nuclei). Both U0126 experiments were performed in duplicate, with 100 larvae per condition per duplicate. (D) Two-color, whole-mount mRNA in situ hybridization for Foxg (green in merged image) and Ascl.a (KH. L9.13, pink). (E) Larva electroporated with Ascl.a>Unc-76::GFP labeling several papilla cells including PNs. (F) Myt1>mNeonGreen labeling PNs and other neurons including CENs. (G) Two-color in situ hybridization of Foxg (green) and Pou4 (pink), the latter labeling adjacent PNs and possibly RTENs. (H) Lineage-specific CRISPR/Cas9-mediated mutagenesis of Pou4 results in loss of PN reporter expression (C4.78>Unc-76::GFP, green). Asterisk denoted background/leaky expression in mesenchyme. Larvae at 17 hpf/20 °C (~st. 27). (I) Scoring of Foxc>H2B::mCherry+ larvae represented in panel H and in Sp6/7/8 CRISPR mutagenesis condition showing Sp6/7/8 does not appear to play a major role in PN reporter expression like Pou4. Experiments repeated in duplicate with 100 larvae in each. (J) Inhibition of Delta/Notch signaling using Foxc>SUH-DBM results in reduced expression of OC reporter (L141.36>Unc-76::GFP, green) at 21 hpf/20 °C (~st. 29). Experiment was repeated in duplicate with 100 larvae in each. (K) Notch inhibition also results in concomitant expansion of supernumerary PNs at 17 hpf/20 °C (~st. 27, labeled by C4.78>Unc-76::GFP, green) relative to Foxc>lacZ control. Experiment was repeated in duplicate, with 42 to 50 larvae in each. (L) Summary diagram and model of effects of Delta/Notch inhibition on PN/OC fate choice in Islet-negative (but formerly Foxg+) papilla progenitor cells. All Islet reporters are the Islet intron 1 + bpFOG>H2B::mCherry. All error bars indicate upper and lower limits. **** p < 0.0001, *** p = 0.0003 in both duplicates as determined by Fisher’s exact test, ns = not statistically significant in at least 1 duplicate. See S4 Data for the data underlying the graphs and for statistical test details. CEN, caudal epidermal neuron; OC, outer collocyte; PN, papilla neuron; RTEN, rostral trunk epidermal neuron.