DLX1 and the NuRD complex cooperate in enhancer decommissioning and transcriptional repression.

In the developing subpallium, the fate decision between neurons and glia is driven by expression of Dlx1/2 or Olig1/2, respectively, two sets of transcription factors with a mutually repressive relationship. The mechanism by which Dlx1/2 repress progenitor and oligodendrocyte fate, while promoting transcription of genes needed for differentiation, is not fully understood. We identified a motif within DLX1 that binds RBBP4, a NuRD complex subunit. ChIP-seq studies of genomic occupancy of DLX1 and six different members of the NuRD complex show that DLX1 and NuRD colocalize to putative regulatory elements enriched near other transcription factor genes. Loss of Dlx1/2 leads to dysregulation of genome accessibility at putative regulatory elements near genes repressed by Dlx1/2, including Olig2. Consequently, heterozygosity of Dlx1/2 and Rbbp4 leads to an increase in the production of OLIG2+ cells. These findings highlight the importance of the interplay between transcription factors and chromatin remodelers in regulating cell-fate decisions.

Genomic Resolution of DLX-Orchestrated Transcriptional Circuits Driving Development of Forebrain GABAergic Neurons.

DLX transcription factors (TFs) are master regulators of the developing vertebrate brain, driving forebrain GABAergic neuronal differentiation. Ablation of Dlx1&2 alters expression of genes that are critical for forebrain GABAergic development. We integrated epigenomic and transcriptomic analyses, complemented with in situ hybridization (ISH), and in vivo and in vitro studies of regulatory element (RE) function. This revealed the DLX-organized gene regulatory network at genomic, cellular, and spatial levels in mouse embryonic basal ganglia. DLX TFs perform dual activating and repressing functions; the consequences of their binding were determined by the sequence and genomic context of target loci. Our results reveal and, in part, explain the paradox of widespread DLX binding contrasted with a limited subset of target loci that are sensitive at the epigenomic and transcriptomic level to Dlx1&2 ablation. The regulatory properties identified here for DLX TFs suggest general mechanisms by which TFs orchestrate dynamic expression programs underlying neurodevelopment.

Dlx1 and Dlx2 Promote Interneuron GABA Synthesis, Synaptogenesis, and Dendritogenesis.

The postnatal functions of the Dlx1&2 transcription factors in cortical interneurons (CINs) are unknown. Here, using conditional Dlx1, Dlx2, and Dlx1&2 knockouts (CKOs), we defined their roles in specific CINs. The CKOs had dendritic, synaptic, and survival defects, affecting even PV+ CINs. We provide evidence that DLX2 directly drives Gad1, Gad2, and Vgat expression, and show that mutants had reduced mIPSC amplitude. In addition, the mutants formed fewer GABAergic synapses on excitatory neurons and had reduced mIPSC frequency. Furthermore, Dlx1/2 CKO had hypoplastic dendrites, fewer excitatory synapses, and reduced excitatory input. We provide evidence that some of these phenotypes were due to reduced expression of GRIN2B (a subunit of the NMDA receptor), a high confidence Autism gene. Thus, Dlx1&2 coordinate key components of CIN postnatal development by promoting their excitability, inhibitory output, and survival.

Transcriptional Networks Controlled by NKX2-1 in the Development of Forebrain GABAergic Neurons.

The embryonic basal ganglia generates multiple projection neurons and interneuron subtypes from distinct progenitor domains. Combinatorial interactions of transcription factors and chromatin are thought to regulate gene expression. In the medial ganglionic eminence, the NKX2-1 transcription factor controls regional identity and, with LHX6, is necessary to specify pallidal projection neurons and forebrain interneurons. Here, we dissected the molecular functions of NKX2-1 by defining its chromosomal binding, regulation of gene expression, and epigenetic state. NKX2-1 binding at distal regulatory elements led to a repressed epigenetic state and transcriptional repression in the ventricular zone. Conversely, NKX2-1 is required to establish a permissive chromatin state and transcriptional activation in the sub-ventricular and mantle zones. Moreover, combinatorial binding of NKX2-1 and LHX6 promotes transcriptionally permissive chromatin and activates genes expressed in cortical migrating interneurons. Our integrated approach provides a foundation for elucidating transcriptional networks guiding the development of the MGE and its descendants.

OTX2 Transcription Factor Controls Regional Patterning within the Medial Ganglionic Eminence and Regional Identity of the Septum.

The Otx2 homeodomain transcription factor is essential for gastrulation and early neural development. We generated Otx2 conditional knockout (cKO) mice to investigate its roles in telencephalon development after neurulation (approximately embryonic day 9.0). We conducted transcriptional profiling and in situ hybridization to identify genes de-regulated in Otx2 cKO ventral forebrain. In parallel, we used chromatin immunoprecipitation sequencing to identify enhancer elements, the OTX2 binding motif, and de-regulated genes that are likely direct targets of OTX2 transcriptional regulation. We found that Otx2 was essential in septum specification, regulation of Fgf signaling in the rostral telencephalon, and medial ganglionic eminence (MGE) patterning, neurogenesis, and oligodendrogenesis. Within the MGE, Otx2 was required for ventral, but not dorsal, identity, thus controlling the production of specific MGE derivatives.

The Ink4a/Arf locus is a barrier to direct neuronal transdifferentiation.

Non-neurogenic cell types, such as cortical astroglia and fibroblasts, can be directly converted into neurons by the overexpression of defined transcription factors. Normally, the cellular phenotype of such differentiated cells is remarkably stable and resists direct cell transdifferentiation. Here we show that the Ink4a/Arf (also known as Cdkn2a) locus is a developmental barrier to direct neuronal transdifferentiation induced by transcription factor overexpression. With serial passage in vitro, wild-type postnatal cortical astroglia become progressively resistant to Dlx2-induced neuronal transdifferentiation. In contrast, the neurogenic competence of Ink4a/Arf-deficient astroglia is both greatly increased and does not diminish through serial cell culture passage. Electrophysiological analysis further demonstrates the neuronal identity of cells induced from Ink4a/Arf-null astroglia, and short hairpin RNA-mediated acute knockdown of p16Ink4a and p19Arf p16(Ink4a) and p19(Arf) indicates that these gene products function postnatally as a barrier to cellular transdifferentiation. Finally, we found that mouse fibroblasts deficient for Ink4a/Arf also exhibit greatly enhanced transcription factor-induced neuronal induction. These data indicate that Ink4a/Arf is a potent barrier to direct neuronal transdifferentiation and further suggest that this locus functions normally in the progressive developmental restriction of postnatal astrocytes.

Analysis of Mll1 deficiency identifies neurogenic transcriptional modules and Brn4 as a factor for direct astrocyte-to-neuron reprogramming.

BACKGROUND: Mixed lineage leukemia-1 (Mll1) epigenetically regulates gene expression patterns that specify cellular identity in both embryonic development and adult stem cell populations. In the adult mouse brain, multipotent neural stem cells (NSCs) in the subventricular zone generate new neurons throughout life, and Mll1 is required for this postnatal neurogenesis but not for glial cell differentiation. Analysis of Mll1-dependent transcription may identify neurogenic genes useful for the direct reprogramming of astrocytes into neurons. OBJECTIVE: To identify Mll1-dependent transcriptional modules and to determine whether genes in the neurogenic modules can be used to directly reprogram astrocytes into neurons. METHODS: We performed gene coexpression module analysis on microarray data from differentiating wild-type and Mll1-deleted subventricular zone NSCs. Key developmental regulators belonging to the neurogenic modules were overexpressed in Mll1-deleted cells and cultured cortical astrocytes, and cell phenotypes were analyzed by immunocytochemistry and electrophysiology. RESULTS: Transcriptional modules that correspond to neurogenesis were identified in wild-type NSCs. Modules related to astrocytes and oligodendrocytes were enriched in Mll1-deleted NSCs, consistent with their gliogenic potential. Overexpression of genes selected from the neurogenic modules enhanced the production of neurons from Mll1-deleted cells, and overexpression of Brn4 (Pou3f4) in nonneurogenic cortical astroglia induced their transdifferentiation into electrophysiologically active neurons. CONCLUSION: Our results demonstrate that Mll1 is required for the expression of neurogenic but not gliogenic transcriptional modules in a multipotent NSC population and further indicate that specific Mll1-dependent genes may be useful for direct reprogramming strategies.

Patient selection criteria for primary total hip Arthroplasty in displaced intracapsular hip fractures: Are they appropriate?

INTRODUCTION: This study aims to determine, by outcome analysis, the appropriateness of current criteria employed to select patients for total hip arthroplasty (THA) as the primary treatment for displaced intracapsular hip fracture (DICHF). This study is intended to inform prospective randomised controlled trials investigating the efficacy of THA as a primary treatment. MATERIALS AND METHODS: Contemporary THA eligibility criteria were derived from recent publications relating to pre-fracture residence, mobility and independence. Outcome data were analysed for 96 patients (19% of 506 consecutive patients with DICHF between March 2003 and February 2005) who fulfilled those criteria. The variables analysed included age, gender, co-existing injuries, co-morbidities, social circumstances, mobility, independence, delay to surgery, readmission and death. The 96 patients included in the study were followed for 3 years. The primary outcome was the combined achievement of home or warden-assisted accommodation at 3 months, no patient readmission within 6 weeks of discharge and survival to 1 year. Secondary outcome was survival to 3 years. RESULTS: At 3 months, 86 patients (90%) had returned home, three (3.1%) required nursing or residential home placement, four (4.2%) were still resident in a community hospital and three (3.1%) had died. A total of ten patients failed to return to their own home. Eight patients (8.3%) were readmitted within 6 weeks. At 1 year, eight patients (8.3%) had died; by 3 years, the mortality rate was 25%. Patients not achieving return to home were older (84.8 years vs. 79.7 years, p = 0.19), prior to fracture weremore likely to use a walking aid (odds ratio [OR] 2.35) or required home support (OR 1.74). The number of co-morbidities was not an association. Backward selection applied to the above data produced an OR of 1.12 (confidence interval [CI] 1.01-1.21) for age as a variable in patients successfully discharged home, so that for each increase in age by year, the odds of home not being the final destination increased by 12%. These factors were also reflected in the patients requiring readmission within 6 weeks from discharge. CONCLUSIONS: If maintaining a high level of activity and independence is the expectation in patients considered for THA for DICHFs, then current selection criteria appear to be appropriate in identifying those capable of returning home, remaining independent and surviving to 1 year compared to the whole DICHF population. With a 75% 3-year survival, the postulated benefit of THA will not be realised in many patients and this needs to be considered. Cost-effectiveness trials are required before broad practice change occurs.

An N-of-1 randomized controlled trial ('N-of-1 trial') of donepezil in the treatment of non-progressive amnestic syndrome.

INTRODUCTION: a professional man sustained a residual, persisting, isolated impairment of short-term memory secondary to severe carbon monoxide poisoning. Informal, open trial of the cholinesterase inhibitor donepezil resulted in uncertain benefit. PROTOCOL: an N-of-1 randomized, double-blind controlled trial of donepezil against placebo. Placebo-induced headache permitted evaluation of donepezil at only the 5 mg dose. RESULTS: there was no improvement in short-term memory. CONCLUSION: the trial excluded significant benefit of donepezil 5 mg in this patient, preventing long-term unnecessary prescription. Beneficial effects in other similar patients or at higher dosage cannot be excluded.