Cytochrome p450 cyp26a1 alters spinal motor neuron subtype identity in differentiating embryonic stem cells

Abstract

The ability to differentiate embryonic stem cells (ESCs) into specific cell types is critical for improved regenerative medicine strategies, cancer chemotherapeutic approaches, and regimens to combat chronic diseases associated with aging. Subclasses of motor neurons (MNs) are generated at different positions along the rostrocaudal axis of the spinal cord, and the signals that specify MN subtype fates remain poorly defined. We show here that the cytochrome P450 enzyme Cyp26a1, which metabolizes all-trans-retinoic acid (RA) and thereby reduces RA levels, plays a crucial role in specifying MN columnar subtypes. Lack of Cyp26a1 in ESCs during differentiation to spinal MNs increases Aldh1a2 (RALDH2) and Hoxc6, markers of the Hox-dependent, lateral motor column (LMC) subtype identity. In contrast, Lhx3, a marker for median motor column identity, showed lower expression in Cyp26a1(-/-)-derived MNs compared with WT. Without Cyp26a1, an increase in intracellular RA concentration plus sonic hedgehog agonist treatment confer an LMC fate on differentiating MNs. Our data suggest a strategy for increasing LMC-type MNs from ESCs by blocking Cyp26a1 in cell replacement/ESC differentiation therapy to treat neurodegenerative diseases, such as amyotrophic lateral sclerosis.

Keywords: Cytochrome P450; Differentiation; Neurological Diseases; Retinoid; Stem Cells.

FIGURE 1.

Cyp26a1^−/− murine ESCs differentiate to pMN despite an altered class I HD profile. A, time line of ESC differentiation, showing expression of key developmental markers. ES, embryonic stem cells; PE, primitive ectoderm; NP, neural plate; pMN, progenitor motor neuron. B, qPCR analysis of oct4 mRNA to confirm ESC status. C, qPCR analysis of Pax6, a class I HD transcription factor, transcripts. Cyp26a1^−/− knockout (KO) cells at day 5 exhibit a 2.6 ± 0.3-fold (p = 0.0146) higher pax6 mRNA level as compared with WT. D, qPCR analysis of nkx6.1, a class II HD transcription factor, transcripts. Day 5 WT neural progenitors express 1.9 ± 0.2-fold (p = 0.026) higher nkx6.1 mRNA levels than Cyp26a1^−/− neural progenitors. E, qPCR analysis of olig2, a pMN marker. No statistically significant differences in olig2 expression were detected at any time point in the WT or Cyp26a1^−/− cells. F, qPCR analysis of shh transcript levels. These qPCR data are from at least three independent biological repeats (n ≥ 3), run in triplicate in each experiment, and all qPCR data are normalized to 36b4 mRNA as a control. KO, mCyp26a1^−/− ESCs; SHH, treatment with sonic hedgehog agonist, Hh Ag-1.3). y axes, arbitrary units; note differences in y axes in different panels. Error bars, S.E.

FIGURE 2.

Cyp26a1^−/−-derived MNs have an altered retinoid profile compared with WT derived MNs. A and B, qPCR analysis of Hb9, a motor neuron marker, and VAChT, vesicular acetylcholine transporter, indicative of their cholinergic transmitter status. No significant differences in Hb9 or VAChT mRNA levels were detected between the RA plus SHH-treated WT and Cyp26a1^−/− cells at day 7, the peak of MN differentiation. C, aldh1a2 mRNA levels in WT versus KO MNs. There was an 8.6 ± 2.5-fold (p = 0.0123) higher aldh1a2 mRNA level in the Cyp26a1^−/− MNs as compared with WT MNs at day 7. D, cyp26a1 mRNA levels at various time points during MN differentiation. E, HPLC analysis of retinoids from both cells and media of WT and Cyp26a1^−/− MNs (day 7). A 92.4 ± 25.1-fold (p = 0.0219) higher amount of total RA was detected in the Cyp26a1^−/− MNs as compared with WT MNs at day 7. R. palmitate, retinyl palmitate. F, cyp26b1 mRNA levels at day 7 during MN differentiation. No differences were seen between WT and Cyp26a1^−/− MNs. These data are from at least three biological repeats (n > 3), and all qPCR data, in triplicate for each experiment, are normalized to 36b4 mRNA. y axes, arbitrary units; note differences in y axes in different panels.

FIGURE 3.

Immunocytochemistry of key protein markers identifies Cyp26a1^−/− MNs as LMC MNs. A, HB9 protein (all panels, red fluorescence), ALDH1a2 (top panels, green fluorescence), Hoxc6 (middle panels, green fluorescence), VAChT (bottom panels, green fluorescence), and DAPI (all panels, blue fluorescence). The “merge” in each set of panels shows overlap of HB9, DAPI, and ALDH1a2 (top panels), Hoxc6 (middle panels), and VAChT (bottom panels). WT (left) and cytochrome P450 Cyp26a1^−/− ESCs were treated with RA and SHH, and representative pictures were taken at day 7. B, panels are as described in A except that WT (left) and Cyp26a1^−/− ESCs (right) were treated only with RA (negative control), and representative pictures were taken at day 7. These data are representative of data from three independent, biological repeats. Magnification is ×200 in all pictures.

FIGURE 4.

Cyp26a1^−/− MNs have an altered Hoxc expression profile. A, rostrocaudal domains of Hoxc expression in MNs, with respect to molecularly defined MN columns (modified from Ref. 45). HMC, hypaxial motor column; R, rostral; C, caudal. B, Cyp26a1^−/− MNs exhibit 7 ± 1.4-fold (p = 0.0016) higher levels of hoxc6 mRNA as compared with WT MNs upon RA plus SHH treatment. The Cyp26a1^−/− MNs also exhibited 3 ± 0.8-fold (p = 0.0301) higher hoxc6 mRNA levels compared with WT MNs at day 7 with RA treatment alone. C, hoxc8 mRNA expression was 16.4 ± 2.4-fold (p < 0.0001) higher in the Cyp26a1^−/− MNs as compared with WT MNs at day 7. The Cyp26a1^−/− MNs also exhibited 4.5 ± 1.2-fold (p = 0.0242) higher hoxc8 mRNA levels compared with WT MNs at day 7 with RA treatment alone. D, Cyp26a1^−/− MNs exhibit 9.5 ± 2.7-fold (p = 0.0154) higher levels of hoxc9 mRNA compared with WT MNs at day 7. The Cyp26a1^−/− MNs also exhibited 5.3 ± 0.5-fold (p = 0.0005) higher hoxc9 mRNA levels compared with WT MNs at day 7 with RA treatment alone. E, bmp5 mRNA expression levels were 3.7 ± 0.8-fold (p = 0.0390) higher in Cyp26a1^−/− MNs at day 7 compared with WT MNs. The Cyp26a1^−/− MNs also exhibited 3.3 ± 0.2-fold (p = 0.0257) higher bmp5 mRNA levels compared with WT MNs at day 7 with RA treatment alone. F, foxP1 mRNA was 2.4 ± 0.4-fold (p = 0.0211) higher in the Cyp26a1^−/− MNs compared with WT MNs at day 7. These data are from at least three biological repeats, and all qPCR data, from triplicate samples, are normalized to 36b4 mRNA. y axes, arbitrary units; note differences in y axes in different panels. Error bars, S.E.

FIGURE 5.

The LIM HD code is altered in the Cyp26a1^−/− MNs. A, LIM HD code of motor columns. LMC(m), medial subtype of lateral motor column; LMC(l), lateral subtype of lateral motor column; MMC(m), medial subtype of median motor column. B, isl1 mRNA expression was 2 ± 0.4-fold (p = 0.021) higher in the Cyp26a1^−/− MNs compared with WT MNs at day 7. In contrast, WT MNs exhibited 2.93 ± 0.2-fold (p = 0.0001) higher isl1 mRNA levels compared with Cyp26a1^−/− MNs at day 7 with SHH treatment alone. C, isl2 mRNA is expressed in both WT and Cyp26a1^−/− MNs at day 7. D, lhx3 mRNA, a marker for the MMC, is 2.4 ± 0.4-fold (p = 0.0298) higher in the WT MNs compared with Cyp26a1^−/− MNs at day 7. E, lim1 mRNA is 2.4 ± 0.4-fold (p = 0.012) higher in the Cyp26a1^−/− MNs compared with the WT MNs at day 7. The Cyp26a1^−/− MNs also exhibited 2.45 ± 0.5-fold (p = 0.0396) higher Lim1 mRNA levels compared with WT MNs at day 7 with RA treatment alone. These data are from at least three biological repeats, and all qPCR data are normalized to 36b4 mRNA. y axes, arbitrary units; note differences in y axes in different panels. Error bars, S.E. F, summary diagram of WT and Cyp26a1^−/− ESC differentiation into various columns of motor neurons (modified from Jessell et al. (56)). The LMC MNs are generated only at forelimb and hindlimb levels and send axons into limb mesenchyme. The MMCs innervate the dorsal axial musculature. WT ESCs express Cyp26a1 so that they have lower RA levels (Fig. 2E) and higher RA polar metabolite levels. The Cyp26a1^−/− ESCs have sustained, higher intracellular RA levels (Fig. 2E) and very low levels of polar metabolites of RA. These differences lead to differences in the types of MNs. The thicker arrow indicates sustained, higher [RA].