A natural transdifferentiation event involving mitosis is empowered by integrating signaling inputs with conserved plasticity factors

Summary Transdifferentiation, or direct cell reprogramming, is the conversion of one fully differentiated cell type into another. Whether core mechanisms are shared between natural transdifferentiation events when occurring with or without cell division is unclear. We have previously characterized the Y-to-PDA natural transdifferentiation in Caenorhabditis elegans, which occurs without cell division and requires orthologs of vertebrate reprogramming factors. Here, we identify a rectal-to-GABAergic transdifferentiation and show that cell division is required but not sufficient for conversion. We find shared mechanisms, including erasure of the initial identity, which requires the conserved reprogramming factors SEM-4/SALL, SOX-2, CEH-6/OCT, and EGL-5/HOX. We also find three additional and parallel roles of the Wnt signaling pathway: selection of a specific daughter, removal of the initial identity, and imposition of the precise final subtype identity. Our results support a model in which levels and antagonistic activities of SOX-2 and Wnt signaling provide a timer for the acquisition of final identity.

(B) Histogram showing the percentage of animals with a "NO DVB" defect, and without (Non transgenics) or with (transgenics) overexpression of gfp::cki-1 in the rectal cells. Note that for the transgenics, the F2 generation of the transgenic lines was first scored and displayed more penetrant defects than the following generations, due to transgene silencing.

(C)
Histogram showing the percentage animals displaying an absence (No K.p) or presence (K.p present) of the K.p cell among the gfp::cki-1 overexpressing animals without DVB. Note that for each category, the GFP expression was assessed in K, K.a and K.p, when present. Solid bar: GFP expression was observed; white bar, No GFP expression.
Error bars, SD of the GFP-expressing animals.

(D)
Histogram showing the percentage of worms without DVB in mutants for the Gα and gpr-1/LGN genes involved in spindle orientation in C. elegans zygote and for the Par gene par-1. The low penetrance of DVB absence is due to an impairment in K cytokinesis.
For all the histograms, ns, not significant; n, total animal scored.   [let-413::gfp::pest], fpIs110  and oxIs12  in K.p over time, in L1 and L2 grown at 20°C. n, total animal scored. (C) lin-26 smFISH staining (white spots on the merge) on 26h post hatching L1 larvae expressing a gfp::sox-2 (CRISPR KI syb737, shown in cyan) and the rectal nuclear reporter gaIs245, shown in magenta. Intestinal cells and Pn.p cells in the same area were identified with the DAPI staining, in yellow. Scale bar for all in bottom right picture. (D) Quantifications of the lin-26 smFISH spots were performed on K.p and F rectal cells as well as on intestinal cells (Int 8/9) and Pn.p cells (P9/10.p). We considered only the spots in contact with the nuclei to reflect the most recently transcribed mRNAs. Statistical test compares the number of spots in the K.p cell to the others. Very few lin-26 mRNA molecules are detected in close proximity to the nucleus in epithelial cells generally (from 3 spots to 5 spots on average; P9/10.p where lin-26 has been shown to be expressed (Labouesse et al., 1996), 3 spots; rectal F cell, 5 spots; K.a, which is on the left side, was very difficult to image because of photobleaching (n=9) and exhibits 4 spots on average      Quantification of DVB defective L4 animals (as observed by unc-47 expression) using RNAi in a sensitized rrf-3 mutant background to target sox-2 paralogs (dpy-8 and gfp RNAi represent controls). Mutants were used for paralogs of ceh-6 (unc-86(n846) and ceh-18(mg57)) and egl-27 (lin-40(ku285)). No obvious defects were observed, although RNAi was found to work poorly in the rectal cells. n, total animal scored.     Table summarizing the binding sites on which we have focused our efforts in this study. Binding sites predicted by more than one approach, or because of the presence of two consecutive binding sites for SOX-2 (site 4), were selected. The mutations introduced into the lim-6 transcriptional reporter to abolish POP-1 binding are presented on the right. Note that these mutations most probably abolish also SOX-2 binding due to the very close similarity of their predicted binding sites.
(D) This binding site similarity can be explained by the sequence similarity of the HMG domains present in SOX-2 and POP-1.
(E) Sequence of the probes used in this study for the gel shift experiments. Note that probe 3 displayed poor annealing due to its AT rich sequence and therefore was not further used.  TACGATTTGCAAACAACAAACATTCCAGACAGAT ATGCTAAACGTTTGTTGTTTGTAAGGTCTGTCTA

3'
GTGCTACATAACTACCAAATGGTTAACCGTGTGCAGCCTTGGTG CACGATGTATTGATGGTTTACCAATTGGCACACGTCGGAACCAC   Increasing concentration (5nm, 50nM and 500nM) of purified HMG-POP-1 and SOX-2 were incubated with wild type or mutated probes 1, 2 and 4 bound to the Cy5 fluorophore. Note that the probe 4 which does not bear canonical SOX-2 binding site is able to bind SOX-2. As, in addition, the mutation of the POP-1 binding site does not seem to affect this binding, it is likely that a non-predicted SOX-2 binding site is present. Probe 3 was also able to bind both SOX-2 and HMG-POP-1, although results for are not presented because this probe annealed poorly, most probably due to its AT-rich sequence. Blue arrowhead, POP-1 bound to the probe; orange arrowhead, SOX-2 bound to the probe; light orange arrowhead, a second SOX-2 shifted band appears at high SOX-2 concentrations; open arrowhead, unbound probe.

POP-1
Free Probe

POP-1
Free Probe

POP-1
Free Probe
*, expression is seen in K.p after its birth, and disappears as lim-6 expression appears (see Fig. 5A).