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

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 in vitro 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 Notch, Hes1 and Hes5. 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.


Introduction
HD is a neurodegenerative disorder caused by abnormal polyglutamine expansion in the amino-terminal end of huntingtin protein (Htt) and characterized by preferential striatal and cortical cellular dysfunction and death associated with late-onset neuropsychiatric and motor disabilities [1]. The molecular basis underlying the selective cellular vulnerability in HD and HD pathogenesis in general remains largely elusive. Htt is a large cytosolic protein of ,340 kDa with ubiquitous cellular localization and adult functional pleiotropism, involving cellular processes ranging from transcriptional regulation to cell survival, whereas mutant Htt (mHtt) causes selective and progressive striatal and cortical neuronal dysfunction and subsequent cell death [2][3][4][5][6]. These observations suggest that Htt may mediate a distinct, selective and previously uncharacterized set of developmental functions, and the pathogenic mutation may therefore have the potential to deregulate these maturational processes and predispose to neurodegeneration. Identifying and characterizing this potential developmental diathesis may have important implications for defining an earlier HD pathogenic window, for explaining the occurrence of a protracted prodromal phase of the disease and for developing novel disease modifying therapies.
An increasing number of reports have begun to implicate Htt in seminal early neural developmental processes. For example, conditional deletion of the huntingtin gene (htt) in the whole brain (Hdh flox/2 ;Camk2a Cre/+ ) from as early as embryonic day 14.5 (E14.5) causes widespread neurodegeneration that mimics several HD phenotypes [7]. Hypomorphic expression of Htt (Hdh neoQ50 homozygotes) leads to severe malformation of fore-and mid-brain regions, whereas the complete ablation of Htt results in embryonic lethality as early as E6.5 with a range of severe neural developmental defects, including impairments in the formation of the neural plate and absence of head-folds [8][9][10][11][12]. KO embryos also display a shortened primitive streak and absence of the embryonic organizer, which are essential for neural development [11]. These cumulative observations suggest that Htt has additional roles in earlier stages of neural induction and early neurogenesis. In line with these observations, a spectrum of neural developmental deficits have recently been demonstrated by our group in a HD knock-in mouse model (Q111) as early as E13.5, including impairments in striatal neural stem cell (NSC) maintenance, and NSC-mediated medium spiny neuron (MSN) specification and maturation [13]. Other reports of aberrant profiles of adult neurogenesis, including enhanced NSC proliferation, have also been documented in HD models and human pathological specimens [14][15][16]. Another recent study reported significantly reduced neuronal differentiation in HD knock-in embryonic stem cell (ESC)-derived NSCs with enhanced cell death [17]. In concert with these overall findings, there is increasing evidence of abnormalities of brain morphology, alterations in synaptic and neural plasticity, the presence of subtle neuropsychological impairments and other HD-associated manifestations occurring long before the advent of overt clinical deficits in HD patients and mouse models [18][19][20][21][22][23][24][25][26][27].
In the present study, we examined the roles of Htt during neural induction and early neurogenesis, and also assessed whether the HD pathogenic mutation (mHtt) may affect the integrity of these essential developmental processes. To accomplish this goal, we utilized an established ESC neural induction culture model that recapitulates the progressive stages of neural induction and early neurogenesis occurring in vivo.

Results
Htt is required for the specification, self-renewal and proliferation of LIF-responsive pNSCs, whereas mHtt enhances these processes and promotes precocious neurogenesis Utilizing the ESC paradigm for neural induction, pNSCs can be identified with a colony-forming assay in the presence of leukemia inhibitory factor (LIF) in which they form clonally derived primitive neurospheres (pNSs) [28]. The pNSs express nestin and proneural genes while suppressing expression of the ESC marker, SSEA1 and alternate endodermal/mesodermal lineage genes [29]. To determine whether neural induction can normally occur in the absence of Htt, we compared ESC-derived pNSCs generated from Hdh ex4/5 /Hdh ex4/5 ESCs, hereafter referred to as KO ESCs, and control ESCs, hereafter referred to as CTL ESCs [10]. Both the size and number of the KO pNSs were significantly smaller than the CTL pNSs (size: 1.5610 4 vs 3.0610 4 mm 2 respectively, p = 0.0002; number: 9 vs 44 respectively, pvalue,0.0001; Fig. 1A). In addition, KO pNSs were composed of significantly fewer KI67+ and phosphorylated histone H3 (pHisH3)+ cells, markers for dividing cells and G2/M-phase cells, respectively, as compared to CTL pNSs ( Fig. 1F and H).
However, when we compared Q111 ESCs, which has an expansion of 111 CAG repeats [30], with control Q18 ESCs, which express 18 CAG repeats, we observed an aberrant enhancement in the specification of pNSCs. Both the size and the number of Q111 pNSs were significantly increased as compared to Q18 pNSs (size: 2.7610 4 vs 2.1610 4 mm 2 ; number: 62 vs 28, respectively, all p-values,0.0001, respectively; Fig. 1B). In addition, there was an increase in the percentage of Figure 1. Htt is required for the elaboration of LIF-responsive pNSs, whereas mHtt differentially deregulates this process. (A, B) Quantification of the size and number of KO and Q111 pNSs as compared to CTL and Q18 pNSs, respectively. Error bars represent 6 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 proliferating cells that were KI67 and pHisH3 positive, while no differences were observed in the percentage of TUNEL positive dying cells in Q111 ESCs as compared to Q18 ESCs (Ki67: 54.7 vs 48.2%, p-value,0.0001; pHisH3: 24.8 vs 16.5%, p-value = 0.0127; TUNEL: 13.7 vs 11.4%, p-values = 0.0912, respectively; Fig. 1D and E; Fig. S1A). Moreover, compared to Q18 pNSs, there was a significant increase in the percentage of nestin+ cells and a concomitant reduction in SSEA1+ cells in Q111 pNSs (SSEA1: 33.7 vs 43.7%; nestin: 53.9 vs 38.1%, respectively, all pvalues,0.0001; Fig. 1G and H). It has previously been shown that only about 1% of all pNSCs express b-TubIII; however, about 20% of all Q111 pNS displayed expression of b-TubIII, which indicates the presence of precocious neurogenesis in the presence of mHtt ( Fig. 1I and J). These overall observations suggest that Htt is required for the incipient program of neural induction as well as for the self-renewal, proliferation and survival capacity of LIFresponsive pNSCs. Furthermore, the mutation in Htt enhances ESC-derived neural induction and leads to precocious neuronal lineage specification.
Htt is required for the specification, self-renewal and proliferation of FGF2-and EGF-responsive dNSCs, whereas mHtt differentially alters these processes and further promotes precocious neurogenesis in FGF2responsive dNSCs We next examined the role of Htt in the program of neural specification of dNSCs and assessed whether the presence of mHtt alters this developmental program. Individual LIF-responsive pNSCs were dissociated and re-propagated in the presence of FGF2 to generate FGF2-responsive dNSCs [29,31]. These dNSCs are equivalent to their in vivo counterparts that exist from E8.5 through adulthood. Analogous to the developmental profiles observed with KO pNSs, both the size and number of the KO FGF2-responsive dNSs were significantly decreased as compared to CTL FGF2-responsive dNSs (size: 3. In contrast to the findings with the KO ECSs, the presence of mHtt resulted in significantly higher numbers of Q111 FGF2responsive dNSs than those of Q18 FGF2-responsive dNSs even though there was no difference in their respective sizes (number: 27.9 vs 11.9, respectively, p-value,0.0001; size: 3.7610 4 vs 3.9610 4 mm 2 , respectively, p-value = 0.0521; Fig. 2B). In addition, as compared to Q18 FGF2-responsive dNSs, there was an increase in the percentage of KI67+ and pHisH3+cells in Q111 FGF2responsive dNSs, whereas the percentage of TUNEL+ cells was unchanged (Ki67: 45.7 vs 33.3%, p-value,0.0001; pHisH3: 16.  Fig. 2G-J). The increase in the percentage of b-TubIII+ cells suggests an enhanced specification of committed neuronal progenitors. These cumulative observations suggest that Htt is required for the transition of LIF-responsive pNSCs to FGF2responsive dNSCs and for the promotion of self-renewal, proliferation and neuronal lineage fate of FGF2-responsive dNSCs. Conversely, mHtt enhanced the transition from LIF-responsive pNSCs to FGF2-responsive dNSCs with alterations in proliferative potential and precocious neurogenesis.
FGF2-responsive dNSCs are the direct precursors of EGFresponsive dNSCs [31]. To further investigate the role of Htt and the effects of mHtt in the specification of EGF-responsive dNSCS from FGF2-responsive dNSCs, we dissociated and re-propagated FGF2-responsive dNSs in EGF to generate EGF-responsive dNSs. Both the number and size of the KO EGF-responsive dNSs were significantly decreased as compared with CTL EGF-responsive dNSs (size: 0.1610 4 vs 3.5610 4 mm 2 ; number: 0 vs 9, respectively, all p-values,0.0001; Fig. S1C). Upon differentiation after 7 days in vitro (DIV), few irregularly shaped EGF-responsive dNSs were formed from KO ESCs and these clones failed to differentiate into neurons and glia (Fig. S1E). In contrast, the size of Q111 EGFresponsive dNSs was comparable to Q18 EGF-responsive dNSs, whereas the number of Q111 EGF-responsive dNSs was significantly decreased as compared to those of Q18 EGFresponsive dNSs (2.0610 4 vs 1.8610 4 mm 2 , p-values = 0.5550; 1 vs 2; p-values,0.0001, respectively, Fig. S1D). Nonetheless, the elaboration of b-TubIII+ neuronal species and GFAP+ astrocytes in Q111 EGF-responsive dNSs were comparable to those in the Q18 EGF-responsive dNSs under differentiating conditions. (Fig. S1E). These observations indicate that Htt is required for the developmental transition of FGF2-responsive dNSCs to EGFresponsive dNSCs and the subsequent differentiation into neurons and glia, whereas mHtt selectively impairs the specification of EGF-responsive dNSCs but does not alter their neural lineage potential.
Htt is required for the expression of ectodermal and neural genes, and the repression of genes specifying alternate endodermal cell fate in LIF-responsive pNSCs, whereas mHtt selectively enhances ectodermal and neuronal gene expression To investigate the roles of Htt and mHtt in mediating lineage potential during the process of early neural induction, we assessed expression levels of genes involved in promoting neural and nonneural lineage decisions from pNSCs, and further examined NSC maintenance and lineage potential under differentiating conditions. KO pNSCs exhibited significant downregulation in the expression level of the primitive ectoderm gene, FGF5 (0.333, pvalue,0.001), the proneural genes, Ngn2 and Mash1 (0.31 and 0.055, respectively; p,0.001), the neurogenic gene, NeuroD1 (0.207, p-value,0.001) and the early patterning and gliogenic gene, Nkx2.2 (0.329, p-value = 0.012), as compared to CTL pNSCs (Fig. 3A). Additionally, contrary to previous studies reporting that wild type pNSCs do not express any endodermal or mesodermal genes [28], the expression of the endodermal gene, GATA4, was significantly increased in KO pNSs as compared to CTL pNSs (7.387, p-value,0.001), thereby suggesting Htt modulates the repression of endodermal lineages during neural induction in ESCs (Fig. 3C). Furthermore, immunofluorescence lineage analysis after 7DIV under differentiating conditions revealed that in contrast to CTL pNSCs, KO pNSCs retained SSEA1 expression and failed to express nestin and the neuronal precursor marker, doublecortin (DCX) (Fig. 3E). Conversely, gene expression analysis in Q111 pNSs revealed a significant upregulation in the expression levels of the primitive ectodermal gene, FGF5 (5.888, pvalue,0.001) and the neurogenic gene, NeuroD1 (2.989, pvalue,0.001), as compared to Q18 pNSs (Fig. 3B). Additionally, there were no discernible differences in the expression levels of genes involved in specifying the alternate endodermal lineage (GATA4, 0.875, p-value = 0.071) when comparing Q111 and Q18 pNSs (Fig. 3D). Further, after 7DIV under differentiating conditions, the expression of SSEA1 was completely repressed in Q111 pNSs, whereas there were no significant differences in the specification of nestin+ NSCs and DCX+ neuronal precursor species between Q111 and Q18 pNSs (Fig. 3F). These experimental findings suggest that Htt is required for promoting ectodermal, and later neural lineage fates and for preventing the elaboration of selective alternate lineage (e.g., endodermal) fates, whereas mHtt enhances the expression of ectodermal and neurogenic genes in pNSCs that may have resulted in precocious neurogenesis. However, increases in the proportion of b-TubIIIexpressing cells between Q18 and Q111 ESCs without corresponding differences in the profiles of DCX-expressing cells suggest that mHtt is promoting precocious neuronal specification but not progressive neuronal maturation.

Discussion
In this study, we employed a specialized ESC clonal culture paradigm to characterize the entire program of neural induction and early neurogenesis [28,29], and demonstrated the essential roles of Htt in the program of neural induction, progressive specification of neural progenitor cell types and the subsequent elaboration of neural lineage species. Our study also revealed that the HD pathogenic mutation aberrantly enhanced ESC-derived neural fate specification, resulting in precocious neurogenesis of pNSCs, and enhanced the elaboration of neuronal and glial lineages from dNSCs.
The development of the central nervous system (CNS) begins with the early program of neural induction within the anterior region of the epiblast. It has been shown that neural fate specification in the pre-gastrula epiblast exists as a 'default' state and FGF signaling from the organizer antagonizes the inhibitory effects of bone morphogenetic proteins (BMPs) on anterior neural fate [34]. However, following gastrulation, the organizer further 'induces' the elaboration and patterning of neural tissue by antagonizing other neural inhibitory signals, such as Nodal and Wnt [35]. Although Htt has previously been shown to be essential for neural development at the time of gastrulation, the roles of Htt for early neural induction have not been adequately explored, in part, due to the early lethality of the KO embryo [11]. Furthermore, the presence of severe mesodermal impairments in KO embryos prevents definitive assessment of the direct roles of Htt in neural development, as these mesodermal structures play critical inductive roles for neural development [35]. The in vitro clonal ESC neural induction model recapitulates the in vivo program of neural induction that follows the ''default'' pathway in the absence of confounding extrinsic factors [28,29]. Thus this experimental paradigm provides an important alternative approach that circumvents many of the aforementioned experimental limitations. However, control conditions for the KO and Q111 ESC lines differed in several lineage parameters. These observations are likely due to the fact that the appropriate controls differed by the presence of mouse (R1 ESC) and human (Q18) 59 sequences within the huntingtin gene, which did not exhibit complete homology. The utilization of separate controls for the KO and Q111 ES cell conditions was necessary because the mutant huntingtin ESC line was constructed with the humanized expansion repeat sequence.
The early stage of LIF-responsive pNSC induction in vitro, however, is a particularly vulnerable developmental phase due to the enhanced sensitivity of apoptosis signaling pathways to caspase-mediated cell death [28,29]. Htt has been shown to display primary anti-apoptotic functions that are mediated, in part, through direct inhibition of activation of caspase 3 and 9, and therefore the absence of Htt may enhance the cellular vulnerability of the KO pNSCs [5]. The presence of LIF has also been demonstrated to have important pro-survival roles in pNSCs, and Htt is known to interact with Grb2 and RasGAP, two adapter molecules of the LIF receptor [36]. Thus, the absence of Htt may disrupt the LIF receptor-mediated pro-survival pathway and further impair the survival of the KO pNSCs. Interestingly, these adapter molecules are also part of the FGF receptor-signaling pathway that is essential for the specification and proliferation of pNSCs and their transition to dNSCs [28,37].
The Notch pathway is an active signaling cascade regulating the early program of neural induction. Our study has shown that the absence of Htt disrupted expression of Notch as well as Hes5, an essential Notch effector, in KO pNSCs. By contrast, the ablation of Notch (Notch 2/2 ) in mouse embryos has been demonstrated to only reduce Hes5 expression, but does not disrupt the generation of pNSCs [33]. This strongly indicates that the requirement of Htt for the specification of pNSCs is independent from its putative role in modulating Notch signaling pathways. Alternatively, high Hes1 in KO pNSCs can suppress proliferation as it has been shown that high Hes1 levels in neural progenitors can repress cyclin D1 and result in G1 phase retardation [38]. Additionally, and consistent with our observations, high Hes1 expression levels have also been shown to promote preferential mesodermal differentiation over neural differentiation, possibly via repression of Notch signaling [39,40].
Interestingly, Notch 2/2 embryonic brains as well as Notch 2/2 ESCs are severely impaired in the generation of FGF2-and EGFresponsive dNSCs [33]. Similarly, knockdown of RBP-Jk, a downstream mediator of Notch signaling, has also been shown to deplete early dNSCs [32]. Thus, the reduced Notch/Hes5 expression we observed in KO pNSCs may have resulted in impairment in their transition to dNSCs. However, some KO pNSCs were capable of undergoing early neural developmental transition to form FGF2-responsive dNSs, which also displayed comparable levels of expression of Notch and Hes5 as the controls. As appropriate Notch expression levels are important to direct ESC differentiation to neural lineages [41], the apparent normal levels of expression of Notch and Hes5 in KO FGF2-responsive dNSs suggest that Htt may not play a role in the regulation of Notch signaling in dNSCs, and that alterations in neuronal and glial lineage elaboration may be due, in part, to additional nonredundant developmental signaling pathways. Thus far, Htt has not been shown to have any direct interaction with components of the Notch signaling cascade, with the exception of a single study reporting an indirect functional association between Huntingtin interacting protein 1 (HIP-1) and deltex-dependent Notch signaling in Drosophila that plays a role in neurogenesis [42]. Additional studies are required to elucidate the mechanisms underlying this regulatory function.
Htt may also play an important role at the intersection of neural and non-neural fate decisions during the incipient program of neural induction as both KO pNSCs and dNSCs displayed preferential increases in mesodermal and endodermal gene expression over pro-neural gene expression. These specialized roles of Htt in cell fate decisions may be orchestrated by modulating the functions of the neuron-restrictive silencing factor/RE1-silencing transcription factor (NRSF/REST) by normally sequestering it within the cytoplasm [43]. REST is a transcriptional and epigenetic regulator of both neural and nonneural cell fate specification programs [43]. It has previously been demonstrated that overexpression of REST in ESCs can promote early differentiation of ESC-derived embryoid bodies to primitive endoderm and also disrupt specification of the epiblast [44]. Further studies are required to show whether the loss of Htt may enhance aberrant accumulation of REST in the nucleus and contribute to the preferential acquisition of endodermal over ectodermal fates during the program of neural induction.
On the other hand, in Q111 pNSCs the presence of mHtt enhanced Notch and Hes5 expression levels. Interestingly, enhanced FGF receptor signaling in dNSC can also potentiate Notch signaling and enhance neurogenesis [45,46]. Constitutive Notch activation (NotchIC) has been shown to not only upregulate Hes5 expression levels but also more importantly to enhance the generation of dNSCs, which is consistent with our observations in Q111 dNSCs [33]. These dNSCs then progressively become Notch/Hes5-dependent and undergo asymmetric cell division to modulate the balance between the maintenance of NSC populations and neural lineage commitment [47,48]. Thus, the sustained increase in the expression of Notch in Q111 dNSCs may differentially enhance asymmetric cell divisions resulting in premature specification of committed neural progenitors, which is consistent with our observation of enhanced generation of neuronal and glial lineages [47,[49][50][51]. Furthermore, this may also lead to the premature depletion of Q111 FGF2-responsive dNSCs, and thus to deficits in the generation of Q111 EGF-responsive dNSCs. Conversely, Hes1 expression levels were significantly downregulated in Q111 pNSCs and dNSCs and may have contributed to the preferential expression of both neuroectodermal and neurogenic genes, to enhanced proliferative capacity and to precocious neurogenesis. Indeed, low Hes1 expression in ESCs has been shown to preferentially enhance neural differentiation, whereas the complete ablation of Hes1 further promoted premature neurogenesis [40,49]. High Hes1 expression has also been reported to have suppressive effects on the maturation of NG2+/O4-OL precursors, which is consistent with the enhanced elaboration of O4+ OL progenitors in the low Hes1-expressing Q111 dNS culture condition [51]. The latter observation may have important implications for defining the mechanistic underpinnings of previous findings of increased oligodendrocyte density reported in the caudate nucleus in HD patients [52]. Remarkably, Notch signaling, particularly with respect to Notch1/3, has been shown to play pivotal roles in the developmental stage-specific regulation of neural progenitors in the ventricular zone that contribute to striatal development [53]. These findings suggest that mHtt alters Notch signaling cascades during neural induction, and these and related molecular pathways may have important implications for explaining the regional striatal developmental deficits previously reported by our group in the HD knock-in Q111 mouse model [13].
Our findings of significant alterations in proliferative potential, self-renewal as well as neural and non-neural lineage potential in Q111 pNSCs and dNSCs have important implications for HD. First, these cellular alterations may result in impairments in neural lineage specification in neurogenic zones that, in part, is consistent with several reports of enhanced self-renewal and precocious neurogenesis in the subventricular zone (SVZ) of R6/2 and Hdh-Q150 KI HD mouse models [14,54]. Second, highly proliferative Q111 pNSCs undergo enhanced DNA replication and are therefore at increased risk for accelerated DNA damage and repair responses, which have been shown to promote mutational instability of CAG repeats and potentially contribute to the pathogenesis of HD [55,56]. Putative DNA instability may persist in mutant pNSCs and subsequently in their progeny, thereby promoting the propagation of developmental mutation lengthmediated cellular and functional impairments into adult life. These pathogenic possibilities are consistent with several reports of increased DNA instability and CAG expansion mosaicism in the brains of HD patients and mouse models [55,57].
A recent study by Conforti and colleagues reported that the loss of Htt and the presence of mHtt (NS-Hdh ex4/5 and NS-Hdh Q111/7 , respectively) did not disrupt the in vitro derivation of ESC-derived NSCs or impair their self-renewal and proliferative properties. In addition, the Hdh Q111/7 NSCs were shown to display reduced neurogenesis and increased cell death [17]. The differences observed between these findings and those of the present study may stem from the use of alternate experimental protocols. Importantly, the current study extends our previous published observations in Q111 mice that mHtt deregulates cell cycle parameters of NSCs and results in aberrant expansion of intermediate progenitors in the absence of increased cell death [13]. Furthermore, the previous work from our group [13] and the current findings strongly suggest that HD-associated abnormalities in adult life (reviewed in [58]) may stem from early and cumulative neurodevelopmental impairments, and may therefore support the notion that HD represents a primary neurodevelopmental disorder in addition to a neurodegenerative disease [59]. Equally important is the concept that seminal impairments occurring during early stages of the neural developmental program can potentially lead to multiple foci of regional cellular vulnerabilities along the entire neuraxis, observations increasingly shown to be associated with HD and other neurodegenerative disease phenotypes [26,27,[60][61][62][63].
It is imperative to corroborate the observations in this study with other in vivo HD models to better refine our understanding of the potential contributions of the HD pathogenic mutation and of differing numbers of pathogenic expansion repeats during incipient stages of embryonic and neural development. Moreover, it is also important to define key molecular impairments occurring along the continuum of developmental and adult stages in affected individuals and in robust HD animal models by identifying potentially unique developmental protein partners of Htt, such as regulators of transcriptional, epigenetic and additional diverse cellular processes. These essential initiatives will open up new possibilities for innovative and efficacious diagnostic, therapeutic and preventative strategies for HD.

Immunofluorescence Analysis
NSs were collected by centrifugation at 300 rpm for 5 minutes, washed once in PBS and fixed in 4% PFA for 20 minutes at room temperature. NSs were then collected in 20% sucrose until they became totally submerged and then frozen in M-1 Embedding Matrix (Thermo) for cryo-sectioning. Immunofluorescence analysis was carried out as previously described [13,64] (See Table S1 for the list of utilized antibodies). TUNEL analysis was performed according to the manufacturer's protocols (Roche, 11684795910). BrdU analysis was carried out as previously described [13].

Quantitative Real-Time PCR (QPCR)
Harvesting of RNA from samples was carried out using TRI reagentH (Molecular Research Center Inc, Cincinnati, OH, USA) according to manufacturer's protocol. The quantification of total RNA concentration was determined using the QubitH RNA assay kit and QubitH 2.0 Fluorometer (Invitrogen). Single strand cDNA synthesis was performed using the High Capacity RNA Reverse Transcription KitH (Applied Biosystems, 4368814) following the manufacturer's recommendations. TaqMan primers were purchased from PE Applied Biosystems and SYBR Green probes were generated using the Invitrogen service (See Table S1). We utilized either TaqMan Universal PCR Master MixH or SYBR Green Master Mix and ran samples in triplicate in the Model 7000 Real Time PCR systemH (Applied Biosystems, CA, USA). The housekeeping gene employed was hypoxanthine guanine phosphoribosyl transferase 1 (HPRT1). Data collection and quality assessment were performed utilizing the 7000 SDS 1.1 RQ Software (Applied Biosystems, CA, USA). The analysis was accomplished with the 2(-Delta-Delta C(T)) relative quantification method with the Relative Expression Software Tool (REST) developed by Corbett Research [65][66]. Gene expression levels were reported using the relative RQ values with 695% Confidence Interval (CI).

Statistical Analysis
Statistical comparisons were evaluated according to the type of data analyzed: proportions were compared with Chi-square test or Fisher's Test. The means of samples were analyzed with either Mann-Whitney U test or t-test. Statistically significant differences between samples were considered using a probability of at least ,0.05. Table S1 List of antibodies, TaqMan probes and SYBR Green probes utilized in the study. All antibodies are listed with manufacturers' names, catalogue numbers, as well as concentration used. All TaqMan probes are listed with catalogue numbers from Applied Biosystems. All SYBR Green probes are listed with forward and reverse sequences. (DOCX)