Fate mapping reveals mixed embryonic origin and unique developmental codes of mouse forebrain septal neurons.

The septum is a key structure at the core of the forebrain that integrates inputs and relays information to other brain areas to support cognition and behaviours such as feeding and locomotion. Underlying these functions is a rich diversity of neuronal types and an intricate complexity of wiring across and within the septal region. We currently have very little understanding of how septal neuronal diversity emerges during development. Using transgenic mice expressing Cre in different subsets of telencephalic precursors we explored the origins of the three main neuronal types of the septal complex: GABAergic, cholinergic and glutamatergic neurons. We find that septal neurons originate from distinct neuroepithelial domains of the developing septum and are born at different embryonic time points. An exception to this is the GABAergic medial septal Parvalbumin-expressing population which is generated outside the septum from surrounding germinal zones. We identify the transcription factor BSX as being expressed in the developing glutamatergic neuron population. Embryonic elimination of BSX in the septum results in a reduction of septal glutamatergic cell numbers and a consequent deficit in locomotion. Further refinement of septal neuron diversity is needed to understand the multiple roles of septal neurons and their contribution to distinct behaviours.

MTG8 interacts with LHX6 to specify cortical interneuron subtype identity.

Cortical interneurons originating in the embryonic medial ganglionic eminence (MGE) diverge into a range of different subtypes found in the adult mouse cerebral cortex. The mechanisms underlying this divergence and the timing when subtype identity is set up remain unclear. We identify the highly conserved transcriptional co-factor MTG8 as being pivotal in the development of a large subset of MGE cortical interneurons that co-expresses Somatostatin (SST) and Neuropeptide Y (NPY). MTG8 interacts with the pan-MGE transcription factor LHX6 and together the two factors are sufficient to promote expression of critical cortical interneuron subtype identity genes. The SST-NPY cortical interneuron fate is initiated early, well before interneurons migrate into the cortex, demonstrating an early onset specification program. Our findings suggest that transcriptional co-factors and modifiers of generic lineage specification programs may hold the key to the emergence of cortical interneuron heterogeneity from the embryonic telencephalic germinal zones.

Single cell enhancer activity distinguishes GABAergic and cholinergic lineages in embryonic mouse basal ganglia.

Enhancers integrate transcription factor signaling pathways that drive cell fate specification in the developing brain. We paired enhancer labeling and single-cell RNA-sequencing (scRNA-seq) to delineate and distinguish specification of neuronal lineages in mouse medial, lateral, and caudal ganglionic eminences (MGE, LGE, and CGE) at embryonic day (E)11.5. We show that scRNA-seq clustering using transcription factors improves resolution of regional and developmental populations, and that enhancer activities identify specific and overlapping GE-derived neuronal populations. First, we mapped the activities of seven evolutionarily conserved brain enhancers at single-cell resolution in vivo, finding that the selected enhancers had diverse activities in specific progenitor and neuronal populations across the GEs. We then applied enhancer-based labeling, scRNA-seq, and analysis of in situ hybridization data to distinguish transcriptionally distinct and spatially defined subtypes of MGE-derived GABAergic and cholinergic projection neurons and interneurons. Our results map developmental origins and specification paths underlying neurogenesis in the embryonic basal ganglia and showcase the power of scRNA-seq combined with enhancer-based labeling to resolve the complex paths of neuronal specification underlying mouse brain development.

Regulatory Elements Inserted into AAVs Confer Preferential Activity in Cortical Interneurons.

Cortical interneuron (CIN) dysfunction is thought to play a major role in neuropsychiatric conditions like epilepsy, schizophrenia and autism. It is therefore essential to understand how the development, physiology, and functions of CINs influence cortical circuit activity and behavior in model organisms such as mice and primates. While transgenic driver lines are powerful tools for studying CINs in mice, this technology is limited in other species. An alternative approach is to use viral vectors such as AAV, which can be used in multiple species including primates and also have potential for therapeutic use in humans. Thus, we sought to discover gene regulatory enhancer elements (REs) that can be used in viral vectors to drive expression in specific cell types. The present study describes the systematic genome-wide identification of putative REs (pREs) that are preferentially active in immature CINs by histone modification chromatin immunoprecipitation and sequencing (ChIP-seq). We evaluated two novel pREs in AAV vectors, alongside the well-established Dlx I12b enhancer, and found that they drove CIN-specific reporter expression in adult mice. We also showed that the identified Arl4d pRE could drive sufficient expression of channelrhodopsin for optogenetic rescue of behavioral deficits in the Dlx5/6+/- mouse model of fast-spiking CIN dysfunction.

Tsc1 represses parvalbumin expression and fast-spiking properties in somatostatin lineage cortical interneurons.

Medial ganglionic eminence (MGE)-derived somatostatin (SST)+ and parvalbumin (PV)+ cortical interneurons (CINs), have characteristic molecular, anatomical and physiological properties. However, mechanisms regulating their diversity remain poorly understood. Here, we show that conditional loss of the Tuberous Sclerosis Complex (TSC) gene, Tsc1, which inhibits the mammalian target of rapamycin (MTOR), causes a subset of SST+ CINs, to express PV and adopt fast-spiking (FS) properties, characteristic of PV+ CINs. Milder intermediate phenotypes also occur when only one allele of Tsc1 is deleted. Notably, treatment of adult mice with rapamycin, which inhibits MTOR, reverses the phenotypes. These data reveal novel functions of MTOR signaling in regulating PV expression and FS properties, which may contribute to TSC neuropsychiatric symptoms. Moreover, they suggest that CINs can exhibit properties intermediate between those classically associated with PV+ or SST+ CINs, which may be dynamically regulated by the MTOR signaling.

Neonatal Tbr1 Dosage Controls Cortical Layer 6 Connectivity.

An understanding of how heterozygous loss-of-function mutations in autism spectrum disorder (ASD) risk genes, such as TBR1, contribute to ASD remains elusive. Conditional Tbr1 deletion during late mouse gestation in cortical layer 6 neurons (Tbr1layer6 mutants) provides novel insights into its function, including dendritic patterning, synaptogenesis, and cell-intrinsic physiology. These phenotypes occur in heterozygotes, providing insights into mechanisms that may underlie ASD pathophysiology. Restoring expression of Wnt7b largely rescues the synaptic deficit in Tbr1layer6 mutant neurons. Furthermore, Tbr1layer6 heterozygotes have increased anxiety-like behavior, a phenotype seen ASD. Integrating TBR1 chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) data from layer 6 neurons and activity of TBR1-bound candidate enhancers provides evidence for how TBR1 regulates layer 6 properties. Moreover, several putative TBR1 targets are ASD risk genes, placing TBR1 in a central position both for ASD risk and for regulating transcriptional circuits that control multiple steps in layer 6 development essential for the assembly of neural circuits.

Tangential migration of corridor guidepost neurons contributes to anxiety circuits.

In mammals, thalamic axons are guided internally toward their neocortical target by corridor (Co) neurons that act as axonal guideposts. The existence of Co-like neurons in non-mammalian species, in which thalamic axons do not grow internally, raised the possibility that Co cells might have an ancestral role. Here, we investigated the contribution of corridor (Co) cells to mature brain circuits using a combination of genetic fate-mapping and assays in mice. We unexpectedly found that Co neurons contribute to striatal-like projection neurons in the central extended amygdala. In particular, Co-like neurons participate in specific nuclei of the bed nucleus of the stria terminalis, which plays essential roles in anxiety circuits. Our study shows that Co neurons possess an evolutionary conserved role in anxiety circuits independently from an acquired guidepost function. It furthermore highlights that neurons can have multiple sequential functions during brain wiring and supports a general role of tangential migration in the building of subpallial circuits.

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.

Genetic programs controlling cortical interneuron fate.

The origins of cortical interneurons in rodents have been localized to the embryonic subcortical telencephalon where distinct neuroepithelial precursors generate defined interneuron subsets. A swathe of research activity aimed at identifying molecular determinants of subtype identity has uncovered a number of transcription factors that function at different stages of interneuron development. Pathways that lead to the acquisition of mature interneuron traits are therefore beginning to emerge. As genetic programs are influenced by external factors the search continues not only into genetic determinants but also extrinsic influences and the interplay between the two in cell fate specification.

PROX1: a lineage tracer for cortical interneurons originating in the lateral/caudal ganglionic eminence and preoptic area.

The homeobox-encoding gene Prox1 and its Drosophila homologue prospero are key regulators of cell fate-specification. In the developing rodent cortex a sparse population of cells thought to correspond to late-generated cortical pyramidal neuron precursors expresses PROX1. Using a series of transgenic mice that mark cell lineages in the subcortical telencephalon and, more specifically, different populations of cortical interneurons, we demonstrate that neurons expressing PROX1 do not represent pyramidal neurons or their precursors but are instead subsets of cortical interneurons. These correspond to interneurons originating in the lateral/caudal ganglionic eminence (LGE/CGE) and a small number of preoptic area (POA)-derived neurons. Expression within the cortex can be detected from late embryonic stages onwards when cortical interneurons are still migrating. There is persistent expression in postmitotic cells in the mature brain mainly in the outer cortical layers. PROX1(+ve) interneurons express neurochemical markers such as calretinin, neuropeptide Y, reelin and vasoactive intestinal peptide, all of which are enriched in LGE/CGE- and some POA-derived cells. Unlike in the cortex, in the striatum PROX1 marks nearly all interneurons regardless of their origin. Weak expression of PROX1 can also be detected in oligodendrocyte lineage cells throughout the forebrain. Our data show that PROX1 can be used as a genetic lineage tracer of nearly all LGE/CGE- and subsets POA-derived cortical interneurons at all developmental and postnatal stages in vivo.

Multiple embryonic origins of nitric oxide synthase-expressing GABAergic neurons of the neocortex.

CORTICAL GABAERGIC INTERNEURONS IN RODENTS ORIGINATE IN THREE SUBCORTICAL REGIONS: the medial ganglionic eminence (MGE), the lateral/caudal ganglionic eminence (LGE/CGE), and the preoptic area (POA). Each of these neuroepithelial precursor domains contributes different interneuron subtypes to the cortex. Neuronal NOS (nNOS)-expressing neurons represent a heterogenous population of cortical interneurons. We examined the development of these cells in the mouse embryonic cortex and their abundance and distribution in adult animals. Using genetic lineage tracing in transgenic mice we find that nNOS type I cells originate only in the MGE whereas type II cells have a triple origin in the MGE, LGE/CGE, and POA. The two populations are born at different times during development, occupy different layers in the adult cortex and have distinct neurochemical profiles. nNOS neurons are more numerous in the adult cortex than previously reported and constitute a significant proportion of the cortical interneuron population. Our data suggest that the heterogeneity of nNOS neurons in the cortex can be attributed to their multiple embryonic origins which likely impose distinct genetic specification programs.

Altered regulation of tau phosphorylation in a mouse model of down syndrome aging.

Down syndrome (DS) results from trisomy of human chromosome 21 (Hsa21) and is associated with an increased risk of Alzheimer's disease (AD). Here, using the unique transchromosomic Tc1 mouse model of DS we investigate the influence of trisomy of Hsa21 on the protein tau, which is hyperphosphorylated in Alzheimer's disease. We show that in old, but not young, Tc1 mice increased phosphorylation of tau occurs at a site suggested to be targeted by the Hsa21 encoded kinase, dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A). We show that DYRK1A is upregulated in young and old Tc1 mice, but that young trisomic mice may be protected from accumulating aberrantly phosphorylated tau. We observe that the key tau kinase, glycogen synthase kinase3-ß (GSK-3ß) is aberrantly phosphorylated at an inhibitory site in the aged Tc1 brain which may reduce total glycogen synthase kinase3-ß activity. It is possible that a similar mechanism may also occur in people with DS.

The germinal zones of the basal ganglia but not the septum generate GABAergic interneurons for the cortex.

Cortical interneurons originate from subpallial precursors and migrate into the cortex during development. Using genetic lineage tracing in transgenic mice we examine the contribution of two germinal zones, the septum and the lateral ganglionic eminence/caudal ganglionic eminence (LGE/CGE) to interneurons of the cortex. We find that the septal neuroepithelium does not generate interneurons for the neocortex. There is, however, clear migration of cells from the LGE/CGE to the cortex. Comparison of the dynamics of cortical colonization by the two major cohorts of interneurons originating in the medial ganglionic eminence (MGE) and the LGE/CGE has shown differences in the timing of migration and initial route of entry into the cortex. LGE/CGE-derived interneurons enter the cortex later than the MGE-derived ones. They invade the cortex through the subventricular/intermediate zone route and only later disperse within the cortical plate and the marginal zone. During the first postnatal week MGE interneurons move extensively to acquire their laminar position within the cortical plate whereas LGE/CGE-derived cells remain largely within the upper layers of the cortex. The two populations intermingle in the adult cortex but have distinct neurochemical properties and different overall distributions. LGE/CGE-derived interneurons account for one third of the total GABAergic interneuron population in the adult cortex.

Decreased expression of gastrokine 1 and the trefoil factor interacting protein TFIZ1/GKN2 in gastric cancer: influence of tumor histology and relationship to prognosis.

PURPOSE: Transcriptional profiling showed decreased expression of gastrokine 1 (GKN1) and the related trefoil factor interacting protein (TFIZ1/GKN2) in Helicobacter pylori infection. Decreased GKN1 and GKN2 mRNA expression has been reported in gastric adenocarcinoma. We have examined GKN1 and GKN2 protein expression in a large gastric cancer series, correlated expression with tumor subtype, and evaluated their utility as prognostic biomarkers. EXPERIMENTAL DESIGN: GKN1, GKN2, and the trefoil factors TFF1 and TFF3 were examined in tissue microarrays from 155 distal gastric adenocarcinomas. Immunohistochemical expression was correlated with clinical outcome. GKN1 and GKN2 expression was measured by real-time PCR and Western analysis in samples of gastric cancer and adjacent nonneoplastic mucosa. RESULTS: GKN1 was lost in 78% of diffuse and 42% of intestinal cancers (P < 0.0001, diffuse versus intestinal). GKN2 expression was lost in 85% of diffuse and 54% of intestinal type cancers (P < 0.002). GKN1 and GKN2 down-regulation were confirmed by Western and real-time PCR analysis. Loss of either protein was associated with significantly worse outcome in intestinal-type tumors by univariate analysis; and GKN2 loss remained a predictor of poor outcome in multivariate analysis (P < 0.033). TFF1 was lost in >70%, and TFF3 was expressed in approximately 50% of gastric cancers. CONCLUSIONS: Loss of GKN1 and GKN2 expression occurs frequently in gastric adenocarcinomas, especially in the diffuse subtype. GKN1 and GKN2 loss are associated with shorter overall survival in the intestinal subtype.

Clinical significance of colonoscopic findings associated with colonic thickening on computed tomography: is colonoscopy warranted when thickening is detected?

GOAL: To determine the utility of colonoscopy in the management of patients with abdominal pain found to have colonic thickening on computed tomography (CT). BACKGROUND: CT is often used in the investigation of abdominal pain. Clinical guidelines regarding colonoscopy when colonic wall thickening is reported at CT are lacking. STUDY: From July 2000 to April 2004, the abdominal CT reports of all patients at a major teaching hospital who were investigated for abdominal pain were reviewed. Cases were selected if any colonic wall thickening was reported. Patients were excluded if they had a previously diagnosed gastrointestinal condition, or if they had not undergone colonoscopy within 30 days of the abnormal CT. Clinical, endoscopic, and pathologic data were extracted from the medical records of all eligible patients. RESULTS: One hundred seven cases were identified. Of these, 8 (7.4%) had colorectal adenocarcinoma. In 10 patients (9.3%), a new diagnosis of inflammatory bowel disease (IBD) was made. Sixteen (15.0%) had findings consistent with infectious colitis, 39 (36.4%) ischemic colitis, and 6 patients (5.6%) had miscellaneous findings possibly responsible for the colonic thickening (diverticulitis, appendicitis, proctitis, and melanosis coli). In 28 patients (26.1%), no abnormality was found that could explain the CT finding. Of those diagnosed with colorectal carcinoma or IBD, only 4 of the 18 patients (28%) presented with evidence of gastrointestinal bleeding or anemia. CONCLUSIONS: On the basis of the rate of new diagnoses of colorectal carcinoma and IBD, we recommend colonoscopy be performed after clinical evaluation in patients with abdominal pain and colonic thickening on CT.