Keqin Yan

Snf2h-mediated chromatin organization and histone H1 dynamics govern cerebellar morphogenesis and neural maturation

Chromatin compaction mediates progenitor to post-mitotic cell transitions and modulates gene expression programs, yet the mechanisms are poorly defined. Snf2h and Snf2l are ATP-dependent chromatin remodelling proteins that assemble, reposition and space nucleosomes, and are robustly expressed in the brain. Here we show that mice conditionally inactivated for Snf2h in neural progenitors have reduced levels of histone H1 and H2A variants that compromise chromatin fluidity and transcriptional programs within the developing cerebellum. Disorganized chromatin limits Purkinje and granule neuron progenitor expansion, resulting in abnormal post-natal foliation, while deregulated transcriptional programs contribute to altered neural maturation, motor dysfunction and death. However, mice survive to young adulthood, in part from Snf2l compensation that restores Engrailed-1 expression. Similarly, Purkinje-specific Snf2h ablation affects chromatin ultrastructure and dendritic arborization, but alters cognitive skills rather than motor control. Our studies reveal that Snf2h controls chromatin organization and histone H1 dynamics for the establishment of gene expression programs underlying cerebellar morphogenesis and neural maturation.

Establishment of a cone photoreceptor transplantation platform based on a novel cone-GFP reporter mouse line.

We report successful retinal cone enrichment and transplantation using a novel cone-GFP reporter mouse line. Using the putative cone photoreceptor-enriched transcript Coiled-Coil Domain Containing 136 (Ccdc136) GFP-trapped allele, we monitored developmental reporter expression, facilitated the enrichment of cones, and evaluated transplanted GFP-labeled cones in wildtype and retinal degeneration mutant retinas. GFP reporter and endogenous Ccdc136 transcripts exhibit overlapping temporal and spatial expression patterns, both initiated in cone precursors of the embryonic retina and persisting to the adult stage in S and S/M opsin+ cones as well as rod bipolar cells. The trapped allele does not affect cone function or survival in the adult mutant retina. When comparing the integration of GFP+ embryonic cones and postnatal Nrl−/− ‘cods’ into retinas of adult wildtype and blind mice, both cell types integrated and exhibited a degree of morphological maturation that was dependent on donor age. These results demonstrate the amenability of the adult retina to cone transplantation using a novel transgenic resource that can advance therapeutic cone transplantation in models of age-related macular degeneration.

Combinatorial Hedgehog and Mitogen Signaling Promotes the In Vitro Expansion but Not Retinal Differentiation Potential of Retinal Progenitor Cells

PURPOSE The in vitro expansion of multilineage competent primary neural progenitor cells is typically limited. Hedgehog (Hh) signaling is required in vivo for the maintenance of stem cell (SC) and progenitor populations in the central nervous system, including the retina. Here we investigated the impact of Hh signaling on in vitro expansion of perinatal mouse retinal progenitor cells (RPCs). METHODS Perinatal mouse retinal cells were treated with combinations of Hh agonist (Hh-Ag), epidermal growth factor (EGF)/fibroblast growth factor 2 (FGF2) and the cultures were assayed for long-term growth, gene expression, and dependence on Gli2. Differentiation was assessed in monolayer cultures, following in vivo transplantation and in cellular reaggregates. RESULTS Using a combination of Hh-Ag, EGF, and FGF2, we were able to establish long-term RPC cultures (termed Hh-RPCs). The ability of this combinatorial signaling approach to block quiescence of these was not associated with altered TP53/MDM2 levels or Hh-EGF cooperativity gene expression. Efficient Hh-RPC expansion and monolayer culture establishment requires Gli2, as Hh-RPCs derived from Gli2 knockout retinal tissue fail to generate cultures that can be passaged long-term in vitro. Hedgehog RPCs retain competence for neurogenic and gliogenic differentiation in vitro; however, they fail to engraft and differentiate into retinal cell types following in vivo transplantation to the eye or in vitro when mixed with acutely dissociated perinatal retinal cells. CONCLUSIONS Our data show that combining Hh and mitogen signaling is sufficient to promote the expansion of RPCs in vitro, but it is insufficient to maintain competence of these cells for retinal differentiation.

Erratum: Corrigendum: Establishment of a cone photoreceptor transplantation platform based on a novel cone-GFP reporter mouse line

Scientific Reports 6: Article number: 22867 ; published online: 11 March 2016; updated: 22 April 2016 The original version of this Article contained a typographical error in the spelling of the author Yves De Repentigny, which was incorrectly given as Yves De Repentingy. This has now been corrected in the PDF and HTML versions of the Article.

Recovery from impaired muscle growth arises from prolonged postnatal accretion of myonuclei in Atrx mutant mice

Reduced muscle mass due to pathological development can occur through several mechanisms, including the loss or reduced proliferation of muscle stem cells. Muscle-specific ablation of the α-thalassemia mental retardation syndrome mutant protein, Atrx, in transgenic mice results in animals with a severely reduced muscle mass at three weeks of age; yet this muscle mass reduction resolves by adult age. Here, we explore the cellular mechanism underlying this effect. Analysis of Atrx mutant mice included testing for grip strength and rotorod performance. Muscle fiber length, fiber volume and numbers of myofiber-associated nuclei were determined from individual EDL or soleus myofibers isolated at three, five, or eight weeks. Myofibers from three week old Atrx mutant mice are smaller with fewer myofiber-associated nuclei and reduced volume compared to control animals, despite similar fiber numbers. Nonetheless, the grip strength of Atrx mutant mice was comparable to control mice when adjusted for body weight. Myofiber volume remained smaller at five weeks, becoming comparable to controls by 8 weeks of age. Concomitantly, increased numbers of myofiber-associated nuclei and Ki67+ myoblasts indicated that the recovery of muscle mass likely arises from the prolonged accretion of new myonuclei. This suggests that under disease conditions the muscle satellite stem cell niche can remain in a prolonged active state, allowing for the addition of a minimum number of myonuclei required to achieve a normal muscle size.

Retinal interneuron survival requires non-cell-autonomous Atrx activity

ATRX is a chromatin remodeling protein that is mutated in several intellectual disability disorders including alpha-thalassemia/mental retardation, X-linked (ATR-X) syndrome. We previously reported the prevalence of ophthalmological defects in ATR-X syndrome patients, and accordingly we find morphological and functional visual abnormalities in a mouse model harboring a mutation occurring in ATR-X patients. The visual system abnormalities observed in these mice parallels the Atrx-null retinal phenotype characterized by interneuron defects and selective loss of amacrine and horizontal cells. The mechanisms that underlie selective neuronal vulnerability and neurodegeneration in the central nervous system upon Atrx mutation or deletion are unknown. To interrogate the cellular specificity of Atrx for its retinal neuroprotective functions, we employed a combination of temporal and lineage-restricted conditional ablation strategies to generate five different conditional knockout mouse models, and subsequently identified a non-cell-autonomous requirement for Atrx in bipolar cells for inhibitory interneuron survival in the retina. Atrx-deficient retinal bipolar cells exhibit functional, structural and molecular alterations consistent with impairments in neuronal activity and connectivity. Gene expression changes in the Atrx-null retina indicate defective synaptic structure and neuronal circuitry, suggest excitotoxic mechanisms of neurodegeneration, and demonstrate that common targets of ATRX in the forebrain and retina may contribute to similar neuropathological processes underlying cognitive impairment and visual dysfunction in ATR-X syndrome.

Coordinated epigenetic regulation of Engrailed-1 by the chromatin remodelers Smarca1 and Smarca5 mediates cerebellar morphogenesis

Background Morphological patterning of the cerebellum requires precise changes in Engrailed homeotic gene expression yet the mechanisms controlling this process remain elusive. Here, we show that the Iswi chromatin remodeling proteins, Smarca5 and Smarca1, are required for the dynamic regulation of Engrailed-1 (En1). Conditional Smarca5-null mice display abnormal cerebellar foliation, ataxia-like symptoms and young mortality. Postnatal granule neuron progenitor expansion and Purkinje cell (PC) development are compromised and attributed to loss of En1 expression. Mutants survive to early adulthood via upregulation of Smarca1 and restoration of En1 expression in PCs, while ablation of both Iswi genes results in lethality at birth. During late cerebellar development, we observe co-binding of the Iswi proteins at the En1 locus and an altered H2AZ/ H3.3 chromatin profile that accompanies changes in En1 expression.

Myoblast expansion defects leads to muscle growth delay and subsequent compensatory adaptation in adult Atrx cKO skeletal muscle

Background The growth of muscle tissue and its regeneration from injury is crucially dependent on a self-renewing population of muscle progenitor cells called satellite cells. Activated satellite cells give rise to a rapidly expanding population of myoblasts that increase muscle mass by differentiating into new or adding into pre-existing muscle fibers. Patients with mutations in the chromatin remodeling gene ATRX are clinically characterised by severe cognitive disabilities and muscular hypotonia, thus dramatically compromising their independent locomotory ability. Materials and methods We generated a skeletal muscle specific knockout mouse model by interbreeding Myf5-Cre mice with mice harbouring the Atrx floxed conditional knockout allele (herein referred to as Atrx cKO). Body mass of Atrx cKO and control littermates were measured along with general morphometric analysis of select hindlimb muscles at 3weeks, 5-weeks, and greater than 8-weeks of age. Methods of analysis included single muscle fiber preparations from the extensor digitorum longus (EDL) and soleus muscles as well as RNA expression analysis from the EDL and soleus muscles. Primary myoblasts were also cultured from dissociated muscle tissue from the hindlimbs and analyzed via immuno-fluorescent microscopy. Neuromuscular acuity and endurance was assessed in Atrx cKO and control littermates by the roto-rod apparatus. Muscle strength was assessed by utilizing a digital grip strength measuring apparatus. Results Atrx cKO mice presented with telltale characteristics of weaker musculature, exemplified by spinal kyphosis and reduced body mass at 3-weeks of age. Satellite cell derived myoblasts from Atrx cKO mice were incapable of rapid expansion in culture but were fully capable of terminally differentiating. Atrx cKO myoblasts displayed delayed cell cycle progression through mid-late S-phase and rampant signs of genomic instability characterised by fragmented nuclei, g-H2AX foci, and telomeric aberrations. Despite inefficient myoblast proliferative capacity, Atrx cKO animals were able to re-establish normal body mass by adulthood. Muscular fitness and function in Atrx cKO mice was also age dependent, as younger 3-week old Atrx cKO mice had a reduced capacity to stay on the roto-rod and poorer gripping strength. However, differences in grip strength of adult Atrx cKO mice were almost indistinguishable from their control littermates. Data regarding pathways mediating the hypertrophic compensatory adaptation in Atrx cKO mice will also be presented. Conclusions Inefficient expansion of activated satellite cells in Atrx cKO mice results in delayed muscle development up to 3-weeks of age, when myonuclear accretion reaches its plateau. Reduced muscle mass at 3-weeks of age also correlated with poorer performance in physical tasks that require muscular force, endurance, and coordination. Compensatory mechanisms are triggered after 3-weeks of age in Atrx cKO mice that allow for the eventual recovery of body mass and muscle functionality in adults.