Huntingtin Is Required for Neural But Not Cardiac/Pancreatic Progenitor Differentiation of Mouse Embryonic Stem Cells In vitro

Abstract

Mutation in the huntingtin (HTT) gene causes Huntington's disease (HD). It is an autosomal dominant trinucleotide-repeat expansion disease in which CAG repeat sequence expands to >35. This results in the production of mutant HTT protein with an increased stretch of glutamines near the N-terminus. The wild type HTT gene encodes a 350 kD protein whose function remains elusive. Mutant HTT protein has been implicated in transcription, axonal transport, cytoskeletal structure/function, signal transduction, and autophagy. HD is characterized by the appearance of nuclear inclusions and degeneration of the striatum. Although HTT protein is expressed early in embryos, most patients develop symptoms in mid-life. It is also unclear why the ubiquitously expressed mutant HTT specifically causes striatal atrophy. Wild type Htt is essential for development as Htt knockout mice die at day E7.5. Increasing evidence suggests mutant Htt may alter neurogenesis and development of striatal neurons resulting in neuronal loss. Using a mouse embryonic stem cell model, we examined the role of Htt in neural differentiation. We found cells lacking Htt inefficient in generating neural stem cells. In contrast differentiation into progenitors of mesoderm and endoderm lineages was not affected. The data suggests Htt is essential for neural but not cardiac/pancreatic progenitor differentiation of embryonic stem cells in vitro.

Keywords: Huntington’s disease; embryoid bodies; embryonic stem cells; huntingtin gene; neural differentiation; neural stem cells.

FIGURE 1

Neural differentiation of mESC lines carrying different forms of Htt. Using the 5-stage neural differentiation method, four mESC lines were differentiated into neurons/glia. Morphology of cells at different stages is shown. (A) Stage 1: mESCs. The pluripotency of mESCs was examined by the AP assay. (B) Stage 2: floating EBs at Day 3. (C) Stage 3: Selection of NSCs in ITSFn medium. EBs were plated onto cell culture plates for attachment and differentiation. Morphology of NSCs on Day 1 and Day 8 are shown. The average number of cells collected from one 100 mm dish after Stage 3 (ITSFn, Day 8) for each cell line is shown in the table. (D) Stage 4: Expansion of nestin-positive NSCs in N2 medium at Day 6. Anti-nestin (green) and TO-PRO-3 dye (blue, nuclei). (E) Stage 5: Differentiation of neurons/glia at Day 6 (R1, HN, 7Q) and Day 4 (140Q). Shown are β-III tubulin in red, GFAP in green, and TO-PRO-3 in blue. Scale bar: (A–C) 200 μm, (D,E) 25 μm.

FIGURE 2

Neural progenitor cell differentiation by the hanging-drop EB method (ectodermal lineage). (A) Diagram showing the timeline of neural progenitor differentiation. (B) Morphology of floating EBs at Day 2. Graph at right shows efficiency of EB formation, calculated as the percentage of EBs formed over the total number of EBs seeded. R1: 95.62 ± 0.75, HN: 91.04 ± 7.41, 7Q: 94.37 ± 1.71, 140Q: 95.88 ± 2.88. No statistical significance. (C) Morphology and average diameter of Day 5 EBs cultured in ultra low attachment 96-well plate. ^∗∗∗∗P < 0.0001, compared to R1, 7Q, and 140Q, by one-way ANOVA test. n = 3 independent experiments. (D) Generation of NSCs. Morphology of NSCs cultured in ITSFn medium at Day 1 and Day 8. Differentiation potential of neural EBs was calculated as the percentage of EBs attached and differentiated over the total number of EBs plated. R1: 97.09 ± 4.47, HN: 7.60 ± 12.04, 7Q: 97.08 ± 4.76, 140Q: 95.31 ± 5.86. ^∗∗∗∗P < 0.0001, compared to R1, 7Q, and 140Q, by one-way ANOVA test. n = 3 independent experiments. Scale bar (B–D): 200 μm. (E) Expression of different markers during neural progenitor cell differentiation. Quantitative RT-qPCR was performed for Nestin, Pax6, Oct4, Htt exon 16-17, and GAPDH using RNA collected from cells at different neural differentiation stages including mESC, EB (Day 5), and ITSFn (Day 8). Gene expression was calculated as fold change (over R1 mESC) after normalization to GAPDH expression. ^∗∗∗P < 0.001, compared to mESCs, by Dunnett’s multiple comparisons test (one-way ANOVA). n.s., non-significant (P > 0.05). Separate graphs for Oct4 and Htt exon 16-17 are shown in Supplementary Figure 3A.

FIGURE 3

Cardiac progenitor cell differentiation by the hanging-drop EB method (mesodermal lineage). (A) Diagram showing the timeline of cardiac progenitor differentiation. (B) Morphology of R1 and HN cells during cardiac progenitor differentiation. Scale bar: 200 μm. (C) mRNA expression of cardiac progenitor cell-specific markers αMHC and Nkx2.5 at Day 12 and 19 was determined by quantitative RT-qPCR. Gene expression was calculated as fold change (over R1 mESC) after normalization to GAPDH expression. ^∗∗∗P < 0.001, ^∗∗P < 0.01, and ^∗P < 0.05, compared to the mESC group by Dunnett’s multiple comparisons test (one-way ANOVA). Separate graphs for Oct4 and Htt exon 16-17 are shown in Supplementary Figure 3B.

FIGURE 4

Pancreatic progenitor cell differentiation by the hanging-drop EB method (endodermal lineage). (A) Diagram showing the timeline of pancreatic progenitor differentiation. (B) Morphology of R1 and HN cells during pancreatic progenitor differentiation. Scale bar: 200 μm. (C) mRNA expression of pancreatic progenitor cell-specific marker Isl1 and Gcg at Day 15, 20, and 35 was determined by quantitative RT-qPCR. Gene expression was calculated as fold change (over R1 mESC) after normalization to GAPDH expression. ^∗∗∗∗P < 0.0001, ^∗∗∗P < 0.001, ^∗∗P < 0.01, and ^∗P < 0.05, compared to the mESC group by Dunnett’s multiple comparisons test (one-way ANOVA). Separate graphs for Oct4 and Htt exon 16-17 are shown in Supplementary Figure 3C.

FIGURE 5

Differentially expressed genes in R1 and Htt-null mESCs. mRNA levels of genes (A) up-regulated or (B) down-regulated in HN mESCs compared with R1 cells were confirmed by quantitative RT-qPCR. Gene expression was calculated as relative expression after normalization to18S rRNA. All experiments were carried out using RNA samples extracted from four different batches of R1 and HN mESCs (N = 4). ^∗∗∗∗P < 0.0001, ^∗∗P < 0.01, and ^∗P < 0.05, compared to R1, by unpaired t-test. a.u., arbitrary units. (C) Protein levels of Olig2 and Kdm6a were examined by immunoblotting. Vinculin and YY1 served as markers of cytoplasmic and nuclear fractions, respectively. ^∗Kdm6a (170 kD), ^∗∗Olig2 (32 kD).