Thuret R , Auger H , Papalopulu N .

BB/E003044/1 Biotechnology and Biological Sciences Research Council , BB_BB/E003044/1 Biotechnology and Biological Sciences Research Council

	Fig. 1. Sox3+ progenitors are maintained across Xenopus development. (A) Representation of Xenopus laevis stages used for the study. For each NF stage, the age in hours or days post-fertilisation (hpf or dpf) and the size of the animal (in mm) are provided. (B-J) Fluorescent immunodetection of Sox3 (red) and pH3 (green) at representative stages of Xenopus development. NF stages are indicated on each panel. Nuclei are counterstained with DAPI. Scale bars=50 µm. Note that during metamorphic stages, we identify a population of cells negative for Sox3 outside the ventricular zone but positive for PH3 in the posterior hindbrain/anterior spinal cord. These cells could represent a population of non-neurogenic glia such as astrocytes or oligodendrocytes (Maier and Miller, 1995; Yoshida et al., 1999). (K) Mean absolute number of Sox3-positive cells per section during development. After the restriction of the expression of Sox3 to the neural plate, the number of positive cells decreases slightly until NF25 to augment and then reach a plateau by NF54. (L) Quantification of mitotic index (green curve) and labelling index (grey bars) of Sox3+ progenitors. The number of pH3-positive nuclei amongst Sox3-positive ones has been counted. Mitotic index is found to be high during neurulation (NF 14 to 25) and prometamorphosis (NF54-56). In between NF25 and NF54, the mitotic index decrease gradually to become nearly negligible at NF50. The labelling index represent the percentage of BrdU-positive Sox3-positive cells from animals injected with BrdU and fixed 3 h later. (M) Cumulative BrdU incorporation in neural progenitors at NF14 and NF35. More than 95% of the Sox3+ progenitors are positive for BrdU after 6 h at NF14 and after 10 to 12 h at NF35. Data represented as mean±s.e.m. ***P<0.001.
	Fig. 2. Neuronal birth during development. (A) Rationale of the birth dating experiment. Animals are injected with BrdU and kept for 24 h to allow BrdU incorporation and cell division to occur. (B) Schematic representation of spinal cord with identified cell populations. In a section of spinal cord, we can identify two categories of cells. (C) Fluorescent immunostaining against xMyT1 (green) and BrdU (red). Nuclei were counterstained with DAPI. Each developmental stage is indicated on the images. Scale bars=50 µm.
	Fig. 3. The behaviour of neural progenitors changes during development. The quantification of the birth dating results enables the estimation of two parameters according to the populations considered. Since mitotic index was negligible at NF66, no birth dating data have been collected for this stage and values have been extrapolated to 0 (dashed lines). (A) Rate of neuronal birth. This graph represents the percentage of BrdU-positive cells among the xMyT1-positive cells and corresponds to the proportion of newly born neurons appeared during the last 24 h. Two main phases of neuronal birth can be identified at NF14 and NF54. In between, the rate of newly born neurons is very low. After NF54, the rate of newly born neurons decreases again. (B) Progenitors fate. This diagram represents the percentage of xMyT1-positive cells among the BrdU-positive population and is an indication of the choice made by progenitors to stay in the cell cycle by proliferating or to exit the cell cycle by differentiating into neurons. Data represented as mean±s.e.m. ***P<0.001, *P<0.05.
	Fig. 4. Estimation of cell cycle length during Xenopus development. (A) Protocols used for estimation of cell cycle length by dual pulse S-phase labelling. Different strategies have been used for stages corresponding to primary neurogenesis (NF14, n=13), progenitor amplification phase (NF35, n=9), prometamorphosis (NF50, n=5), and secondary neurogenesis during metamorphosis (NF54, n=5). (B) Total cell cycle length (Tc) and (C) S-phase length (Ts) at corresponding stages. Both Ts and Tc are increasing during development in a manner that seems independent of the neural progenitors behaviour. (D) Cell cycle length of the dorsal part versus the ventral part of the spinal cord at NF54. The overall cell cycle at this stage is 35 h represent a composite of a heterogeneous population of Sox3 progenitors where dorsal progenitors cycle faster (25 h) than the ventral ones (80 h). Data represented as mean±s.e.m. ***P<0.001, **P<0.01, *P<0.05.
	Supplementary Figure 1: A. Estimation of uridine analogue bioavailablity in Xenopus. An initial shot of BrdU was provided to NF35 embryos. Then, an EdU dose was injected after different incubation times ranging from 1 to 3.5 hours. Red: BrdU, green: EdU, scale bar = 50μm. A similar representative section located in the hindbrain of NF35 embryos is presented shown for each time point. For clarity, DAPI staining is not shown. We determine the bioavailability of uridine analogues by the time needed for the appearance of EdU only nuclei (arrowheads). B. Double immunostaining for Sox3 (green) and xMyT1 (red) in spinal cord section of NF40 embryo. Nuclei were counterstained with DAPI. Scale bar = 50μm. C. X-Delta-1 in situ hybridisation combined with MyT1 antibody staining at NF20. White arrowheads indicate the areas of expression of X-Delta-1. Green arrowheads indicate X-MyT1+ nuclei. MyT1 positive nuclei are close to the domain of expression of X-Delta-1 but do not overlap. D. Table compiling the various data collected.

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