The ability of control (REST-100) and Rest mutant ES cells (REST/KD-50 and REST-null) to generate the different neural cell populations was assessed by real time-PCR using the following markers: NSCs Pax6, Msi1 and Nestin (A–C); NPCs Mash1 and Ngn1 (D–E); early neurons Tubb3 (F) and mature neurons, Scn2a and L1cam (G–H). Data are represented as mean±SEM. *P<0.05 and **P<0.01, significantly different from REST-100 and REST/KD-50.

The control REST-100 and 4 Rest mutant ES cells were either plated on gelatinised glass coverslips (A–J) or on glass coverslips coated with laminins (K–T) and subjected to standard neural differentiation. Cultures were fixed after 4 days of differentiation (A–E and K–O), the peak of NSC generation or fixed after 14 days of differentiation (F–J and P–T), when neurons were prevalent, and cell death in the cultures was assessed using TUNEL staining (green). In all cases cell nuclei were identified by DAPI staining (blue). As expected, cell death was observed when NSCs were generated in all groups (A–E). However, a marked reduction in cell death was observed in all cases when ES cells were plated and differentiated on laminin substrates (compare A–E with K–O). After 14 days of differentiation on gelatinised glass coverslips, markedly more cell death was observed in REST-null than in REST-100 and REST/KD-50 (compare H with F and G). A similar level of death was observed in REST-null+MTV (I). REST-null+REST exhibited less cell apoptosis (J). Laminin-treatment reduced apoptosis in all Rest mutants (compare H–J with R–T). Scale bar: 20 µm.

The expression patterns of Lama1 (A), Lamb1 (B), Lamc1 (C) and Lama2 (D) were analysed throughout ES cell-derived neural differentiation by real time-PCR. Data are represented as mean±SEM. *P<0.05 and **P<0.01, significantly different from REST-100 and REST/KD-50. ^#P<0.05 and ^¥P<0.01, significantly different from REST-null and REST-null+MTV.

(A) Summary of neural stage-specific markers used in this study. NSC: neural stem cells; NPC: neural progenitor cells. (B) Down-regulation of Oct4 and Rest was observed as neural differentiation proceeded. (C) From day 2–8, the expression of NSC markers, Nestin and Pax6 is observed. (D) NPC markers, Mash1 and Ngn1, appeared in an overlapping but slightly later wave than NSC markers. (E) After 10 days of differentiation, markers of early (Tubb3) and mature neurons (Syn1 and L1cam) appeared.

(A–E) NSCs were derived from the control (REST-100) and 4 Rest mutant ES cells, and identified by Sox1 (green) and Nestin (red) after 4 days of differentiation. Nuclei were stained with DAPI (blue). Few, if any, NSCs generated from REST-null ES cells (C and D) expressed Nestin, but showed positive immunostaining with Sox1. NSCs generated from control (REST-100) and REST/KD-50 ES cells showed much higher levels of Nestin immunostaining in Sox1⁺ cells (A–B). Constitutive expression of Rest (REST-null+REST) rescues, at least partially, this phenotype with many Sox1⁺ cells now expressing Nestin (E). (F–O) Neurons derived from the control and 4 Rest mutant ES cells were identified using NeuN (red) and Map2 (green) after 14 days of differentiation. Neurons generated from REST-100 and REST/KD-50 ES cells showed elaborate neurite outgrowth, fasciculation between aggregates/colonies (F, G and in higher power K, L) and migration of NeuN+ cells (see arrows in L). Conversely, neurons derived from REST-null and REST-null+MTV exhibited fragmented neuronal colonies that lacked elaborate processes and migration (H, I and in higher power M, N). Constitutively expressing Rest (REST-null+REST) attenuated the phenotypic effects of Rest ablation: neurite outgrowth, neurite fasciculation (J, O) and neuronal migration was observed (arrows in O). Scale bars: 20 µm (A to E and K to O) and 100 µm (F to J).

Control (REST-100) and 4 Rest mutants ES cells were grown under neural-differentiating conditions on laminin coated glass coverslips. After 4 days of differentiation some cultures were fixed and labelled with the NSC markers Nestin (red) and Sox1 (green) (A–E). The results show that laminins restored the ability to generate Nestin⁺ cells in Rest mutants (C–E, compared with Fig. 3C–3E). Sister cultures were allowed to differentiate for 14 days to analyse neuronal differentiation using NeuN (red) and Map2 (green) (F–J and in higher power K–O). Laminins rescued the neuronal phenotypes derived from REST-null ES cells, which included elaboration processes, neurite outgrowth and migration (see arrows in M and N) similar to that seen in neurons derived from control ES cells. Nuclei were stained with DAPI (blue) in all. Scale bars: 20 µm (A to E and K to O) and 100 µm (F to J).