Functions of Huntingtin in Germ Layer Specification and Organogenesis

Abstract

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.

Faulty neuronal determination and cell polarization are reverted by modulating HD early phenotypes

“…So far, the main evidence suggesting a neurodevelopmental component in HD is threefold. First, HTT interacts with a number of developmental factors (9,10) and is implicated in several aspects of neural development, such as epiblast formation (11,12), neural rosette and neural tube formation (13,14), as well as neuronal survival and cortical maturation and migration (12,15,16). Second, there are predictions that the patho-logical process develops linearly from birth (17) and, accordingly, measurement of the intracranial volume as a representation of the maximal brain growth obtained during development has shown consistent changes in HD subjects before symptoms arise (18)(19)(20).…”

Postnatal and adult consequences of loss of huntingtin during development: Implications for Huntington's disease

“…Whether HD developmental impairments associated with late life disease hallmarks are a consequence of gain or loss of function mechanisms or a compendium of both pathological processes is unknown. Interestingly, our previous in vitro studies have shown that developmental deficits associated with wild-type Huntingtin (Htt) ablation parallel many of those observed in specimens carrying mHtt (Nguyen et al, 2013a; Nguyen et al, 2013b), which supports a loss of function mechanism. In addition, other studies have shown that loss of huntingtin, via impairments of mitotic spindle orientation, affects neural stem cell (NSC) cycle progression and corresponding cell fate specification potential, further supporting an underlining loss-of-function mechanism (Godin et al, 2010).…”

“…The fact that lethality of mutant hypomorph mice can be overcome by the presence of a single non-pathogenic allele, even at reduced expression levels (Hdh neoQ20 ), strongly indicates that the polyQ expansion is interfering with an important developmental function(s) of Htt. This possible dominant-negative mechanism is also supported by the following facts: (1) the array of developmental and postnatal neurological deficits exhibited by Hdh d•hyp , Hdh neoQ20/null , Hdh neoQ111/neoQ20 and Hdh neoQ50/neoQ20 are analogous; (2) the phenotype and course of neurological disorders in Hdh neoQ50/neoQ20 and Hdh neoQ111/neoQ20 mice are more aggressive than in Hdh neoQ20/null , possibly reflecting a more severe loss of Htt developmental functions (Auerbach et al, 2001; White et al, 1997); (3) developmental abnormalities in Hdh d•hyp , Hdh neoQ20/null and Hdh neoQ111/neoQ20 parallel those described in a HD knock-in model (Auerbach et al, 2001; Molero et al, 2009; White et al, 1997); and (4) many stem cell-mediated developmental alterations associated with mHtt are recapitulated by wild-type Htt ablation (Nguyen et al, 2013a; Nguyen et al, 2013b). This putative dominant-negative mechanism may be secondary to polyQ-dependent aberrant splicing of Htt or aberrant cleavage of the protein resulting in a short amino-terminal fragment (Sathasivam et al, 2013; Zheng and Diamond, 2012).…”

Loss-of-Huntingtin in Medial and Lateral Ganglionic Lineages Differentially Disrupts Regional Interneuron and Projection Neuron Subtypes and Promotes Huntington's Disease-Associated Behavioral, Cellular, and Pathological Hallmarks

“…Htt is a pleiotropic protein that encompasses a wide spectrum of seminal cellular roles during both the embryonic and postdevelopmental periods (Cattaneo et al, 2005;Marques Sousa and Humbert, 2013). We and others have demonstrated a compendium of neural developmental abnormalities associated with mutant Htt ranging from defects in neuroectodermal specification (Nguyen et al, 2013a;Conforti et al, 2018), abnormalities in neurogenesis within pallial and subpallial neuronal subtypes (Nguyen et al, 2013b;Molina-Calavita et al, 2014;Siebzehnrübl et al, 2018), changes in the timing of striatal medium spiny neuron (MSN) specification and migration within striatal germinative zones, and neuronal differentiation as well as maturational abnormalities of the chemo-architecture of the striatum (Molero et al, 2009;HD iPSC Consortium, 2017). Using an inducible Cre ERT2 system to drive the excisional recombination of mutant Htt in a temporally defined manner, we demonstrated that restricted expression of this pathogenic protein during development elicits the key hallmarks of HD during later life .…”