Selective Roles of Normal and Mutant Huntingtin in Neural Induction and Early Neurogenesis

<div><p>Huntington's disease (HD) is a neurodegenerative disorder caused by abnormal polyglutamine expansion in the amino-terminal end of the huntingtin protein (Htt) and characterized by progressive striatal and cortical pathology. Previous reports have shown that Htt is essential for embryogenesis, and a recent study by our group revealed that the pathogenic form of Htt (mHtt) causes impairments in multiple stages of striatal development. In this study, we have examined whether HD-associated striatal developmental deficits are reflective of earlier maturational alterations occurring at the time of neurulation by assessing differential roles of Htt and mHtt during neural induction and early neurogenesis using an <i>in vitro</i> mouse embryonic stem cell (ESC) clonal assay system. We demonstrated that the loss of Htt in ESCs (KO ESCs) severely disrupts the specification of primitive and definitive neural stem cells (pNSCs, dNSCs, respectively) during the process of neural induction. In addition, clonally derived KO pNSCs and dNSCs displayed impaired proliferative potential, enhanced cell death and altered multi-lineage potential. Conversely, as observed in HD knock-in ESCs (Q111 ESCs), mHtt enhanced the number and size of pNSC clones, which exhibited enhanced proliferative potential and precocious neuronal differentiation. The transition from Q111 pNSCs to fibroblast growth factor 2 (FGF2)-responsive dNSCs was marked by potentiation in the number of dNSCs and altered proliferative potential. The multi-lineage potential of Q111 dNSCs was also enhanced with precocious neurogenesis and oligodendrocyte progenitor elaboration. The generation of Q111 epidermal growth factor (EGF)-responsive dNSCs was also compromised, whereas their multi-lineage potential was unaltered. These abnormalities in neural induction were associated with differential alterations in the expression profiles of <i>Notch</i>, <i>Hes1</i> and <i>Hes5</i>. These cumulative observations indicate that Htt is required for multiple stages of neural induction, whereas mHtt enhances this process and promotes precocious neurogenesis and oligodendrocyte progenitor cell elaboration.</p></div

Htt is required for the elaboration of LIF-responsive pNSs, whereas mHtt differentially deregulates this process.

<p>(A, B) Quantification of the size and number of KO and Q111 pNSs as compared to CTL and Q18 pNSs, respectively. Error bars represent ± SEM; unless otherwise stated, *p-value<0.05. (C, D) Quantification of the percentage of positive cells for the proliferation markers, KI67 and pHisH3, and for the cell death marker, TUNEL, in KO pNSs as compared to CTL pNSs, and in Q111 pNSs as compared to Q18 pNSs, respectively. (E) Immunofluorescence micrographs of KI67 and pHisH3 immunoreactive cells in CTL, KO, Q18 and Q111 pNSs. (F, G) Quantification of the percentage of positive cells for the ESC marker, SSEA1, and the NSC marker, Nestin, in KO pNSs as compared to CTL pNSs, and in Q111 pNSs as compared to Q18 pNSs, respectively. (H) Immunofluorescence micrographs of SSEA1 and Nestin immunoreactive cells in CTL, KO, Q18 and Q111 pNSs. (I) Quantification of pNSs expressing the early neuronal marker, β-TubIII, from CTL, KO, Q18 and Q111 ESCs. (J) Immunofluorescence micrographs of β-TubIII immunoreactive in CTL, KO, Q18 and Q111 pNSs. Error bars represent ±95% CI; unless otherwise stated, *p-value<0.05. All scale bars = 25 μm.</p

Htt is associated with Notch signaling pathways, whereas mHtt differentially deregulates this signaling cascade.

<p>(A, B) QPCR expression analysis of <i>Notch</i>, <i>Hes1</i> and <i>Hes5</i> in KO as compared to CTL pNSs and dNSs. (C, D) QPCR expression analysis of <i>Notch</i>, <i>Hes1</i> and <i>Hes5</i> in Q111 as compared to Q18 pNSs and dNSs. Error bars represent ±95% CI; unless otherwise stated, *p-value<0.05.</p

Functions of Huntingtin in Germ Layer Specification and Organogenesis

<div><p>Huntington’s disease (HD) is a neurodegenerative disease caused by abnormal polyglutamine expansion in the huntingtin protein (Htt). Although both Htt and the HD pathogenic mutation (mHtt) are implicated in early developmental events, their individual involvement has not been adequately explored. In order to better define the developmental functions and pathological consequences of the normal and mutant proteins, respectively, we employed embryonic stem cell (ESC) expansion, differentiation and induction experiments using huntingtin knock-out (KO) and mutant huntingtin knock-in (Q111) mouse ESC lines. In KO ESCs, we observed impairments in the spontaneous specification and survival of ectodermal and mesodermal lineages during embryoid body formation and under inductive conditions using retinoic acid and Wnt3A, respectively. Ablation of BAX improves cell survival, but failed to correct defects in germ layer specification. In addition, we observed ensuing impairments in the specification and maturation of neural, hepatic, pancreatic and cardiomyocyte lineages. These developmental deficits occurred in concert with alterations in Notch, Hes1 and STAT3 signaling pathways. Moreover, in Q111 ESCs, we observed differential developmental stage-specific alterations in lineage specification and maturation. We also observed changes in Notch/STAT3 expression and activation. Our observations underscore essential roles of Htt in the specification of ectoderm, endoderm and mesoderm, in the specification of neural and non-neural organ-specific lineages, as well as cell survival during early embryogenesis. Remarkably, these developmental events are differentially deregulated by mHtt, raising the possibility that HD-associated early developmental impairments may contribute not only to region-specific neurodegeneration, but also to non-neural co-morbidities.</p> </div

Specification and survival of three germ layers requires Htt whereas mHtt differentially impairs these processes.

<p>(A) Representative images of different sizes of CTL and KO EBs at 10 DIV. (B, C) Quantification of the size and number of CTL and KO EBs at 4DIV, 6DIV, 8DIV and 10DIV. Error bars represent ±SEM; *p<0.0001. (D) Representative images of TUNEL assay in CTL and KO EB at 4 DIV. (E) Representative images of different sizes of Q18 and Q111 EBs at 10 DIV. (F, G) Quantification of the size and number of Q18 and Q111 EBs at 4DIV, 6DIV, 8DIV and 10DIV. Error bars represent ±SEM; *p<0.0001. (H) Representative images of TUNEL assay in Q18 and Q111 EBs at 4 DIV. (I, J) QPCR analysis of developmental markers representing the ectodermal (FGF5), endodermal (NODAL) and mesodermal (BRACHYURY) germ layers. Error bars represent ±95% CI; *p<0.0001. (K) Quantification of the size of CTL and KO EBs formed after knocking down BAX using lentiviral transfection with a double short hairpin RNA (shRNA). (L) QPCR analysis of germ layer developmental markers in CTL-shSCR, CTL-shBAX, KO-shSCR and KO-shBAX specimens. Error bars represent ±95% CI; *p<0.0001, #p<0.0001 unless otherwise noted. Scale bar = 20 µm (A, E); 200 µm (D, H).</p

mHtt differentially alters the specification and maturation of organ-specific lineage species.

<p>(A–B) QPCR expression analysis of markers representing pancreatic progenitors and mature pancreatic species, respectively, during pancreatic differentiation of Q18 and Q111 ESCs. (C–D) QPCR expression analysis of markers representing early hepatoblasts and mature hepatocytes, respectively, during hepatic differentiation of Q18 and Q111 ESCs. (E–G) QPCR expression analysis of markers representing early cardiomyocyte progenitors and mature contractile cardiomyocytes, respectively, during cardiomyocyte differentiation of Q18 and Q111 ESCs. (H–I) Quantification of EBs containing contractile cardiomyocytes and immunofluorescence analysis of MF20 in CTL and KO ESCs in response to cardiomyocyte differentiation. All error bars represent ±95% CI; *p<0.0001 unless otherwise noted. Scale bar = 20 µm.</p

mHtt impairs the spontaneous differentiation of ESCs in ways analogous to Htt ablation.

<p>(A, D) Immunofluorescence analysis of the ESC marker (SSEA1) and the pluripotency factors (Nanog, Oct4, Sox2) in undifferentiated ESC maintained with LIF. (B, E) Quantification of Nanog+, Oct4+, Sox2+ (n=1203, 995, 1138 and 608 for CTL, KO, Q18 and Q111, respectively) and Klf4+ (n=1228, 592, 1113 and 1112 for CTL, KO, Q18 and Q111, respectively) cells present in ESCs after 4 DIV following removal of LIF. (C, F) ESC cultures pulsed with BrdU for 4 hrs in media without LIF. The ESCs were then fixed at 1DIV, 2DIV, and 4DIV and quantification of BrdU+ cells was assessed at these three time points (n=1426, 1162, 1571 and 2033 for CTL, KO, Q18 and Q111, respectively). All error bars represent ±95% CI; *p-values < 0.0001 unless otherwise noted. Scale bar = 20 µm.</p

Htt is required for the maintenance of lineage potential of pNSCs, whereas mHtt promotes neurogenesis.

<p>(A, B) QPCR expression analysis of proneural genes in KO pNSs as compared to CTL pNSs, and in Q111 pNSs as compared to Q18 pNSs, respectively. (C, D) QPCR expression analysis of the endodermal gene, <i>GATA4</i>, and the mesodermal genes, <i>Brachyury</i> and <i>Hnf-4A</i>, in KO pNSs as compared to CTL pNSs, and in Q111 pNSs as compared to Q18 pNSs, respectively. (E, F) pNSs were cultured under differentiating conditions for 7DIV and analyzed by immunofluorescence microscopy for SSEA1, Nestin and Doublecortin (DCX), which are markers for ESCs, NSCs and neuronal precursor species respectively. Error bars represent ±95% CI; unless otherwise stated, *p-value<0.05. All scale bars = 25 μm.</p

Htt is required for the maintenance of lineage potential in dNSCs, whereas mHtt selectively deregulates this process.

<p>(A, C) QPCR expression analysis of proneural genes in KO FGF2-responsive dNSs as compared to CTL FGF2-responsive dNSs, and in Q111 FGF2-responsive dNSs as compared to Q18 FGF2-responsive dNSs, respectively. (B, D) QPCR expression analysis of the endodermal gene, <i>GATA4</i>, and the mesodermal genes, <i>Brachyury</i> and <i>Hnf-4A</i>, in KO FGF2-responsive dNSs as compared to CTL FGF2-responsive dNSs, and in Q111 FGF2-responsive dNSs as compared to Q18 FGF2-responsive dNSs, respectively. (E, F) FGF2-responsive dNSs were cultured under differentiating conditions for 7DIV and analyzed by immunofluorescence microscopy for assessment of the expression profiles of Nestin, β-TubIII, GFAP, NG2 and O4, which are markers for NSCs, neurons, astrocytes, oligodendrocyte precursors and oligodendrocyte progenitors, respectively. Error bars represent ±95% CI; unless otherwise stated, *p-value<0.05. All scale bars = 25 μm.</p

The regulation of Notch/Hes1/STAT3 signaling pathways requires Htt whereas mHtt differentially alters this process.

<p>(A) QPCR expression analysis of Notch, Hes1 and Hes5 in CTL and KO EBs at 4DIV. (B) QPCR expression analysis of STAT3 in CTL and KO EBs at 4DIV. (C) Quantification of protein levels of non-phosphorylated and phosphorylated STAT3 measured by Western blot analysis in CTL and KO EBs at 4DIV. Error bars represent ± SEM; *p<0.05. (D) QPCR expression analysis of Notch, Hes1 and Hes5 in Q18 and Q111 EBs at 4DIV. (E) QPCR expression analysis of STAT3 in Q18 and Q111 EBs at 4DIV. (F) Quantification of protein levels of non-phosphorylated and phosphorylated STAT3 measured by Western blot analysis in Q18 and Q111 EBs. Error bars represent ± SEM; *p<0.05. (G–I) QPCR expression analysis of the germ layer markers, FGF5, NODAL and BRACHYURY in CTL and KO Hes1-overexpression EBs. Dotted lines refer to expression levels of control ESCs expressing the scrambled construct, CTL-SCR. Error bars represent ±95% CI. *p<0.0001 unless otherwise noted, as compared to CTL-SCR; ^# p<0.0001 unless otherwise noted, as compared to KO–SCR (G, H) and CTL-HES1 (I).</p