A non-canonical role for the proneural gene Neurog1 as a negative regulator of neocortical neurogenesis.

Neural progenitors undergo temporal identity transitions to sequentially generate the neuronal and glial cells that make up the mature brain. Proneural genes have well-characterised roles in promoting neural cell differentiation and subtype specification, but they also regulate the timing of identity transitions through poorly understood mechanisms. Here, we investigated how the highly related proneural genes Neurog1 and Neurog2 interact to control the timing of neocortical neurogenesis. We found that Neurog1 acts in an atypical fashion as it is required to suppress rather than promote neuronal differentiation in early corticogenesis. In Neurog1-/- neocortices, early born neurons differentiate in excess, whereas, in vitro, Neurog1-/- progenitors have a decreased propensity to proliferate and form neurospheres. Instead, Neurog1-/- progenitors preferentially generate neurons, a phenotype restricted to the Neurog2+ progenitor pool. Mechanistically, Neurog1 and Neurog2 heterodimerise, and while Neurog1 and Neurog2 individually promote neurogenesis, misexpression together blocks this effect. Finally, Neurog1 is also required to induce the expression of neurogenic factors (Dll1 and Hes5) and to repress the expression of neuronal differentiation genes (Fezf2 and Neurod6). Neurog1 thus employs different mechanisms to temper the pace of early neocortical neurogenesis.

Neurog2 and Ascl1 together regulate a postmitotic derepression circuit to govern laminar fate specification in the murine neocortex.

A derepression mode of cell-fate specification involving the transcriptional repressors Tbr1, Fezf2, Satb2, and Ctip2 operates in neocortical projection neurons to specify six layer identities in sequence. Less well understood is how laminar fate transitions are regulated in cortical progenitors. The proneural genes Neurog2 and Ascl1 cooperate in progenitors to control the temporal switch from neurogenesis to gliogenesis. Here we asked whether these proneural genes also regulate laminar fate transitions. Several defects were observed in the derepression circuit in Neurog2-/-;Ascl1-/- mutants: an inability to repress expression of Tbr1 (a deep layer VI marker) during upper-layer neurogenesis, a loss of Fezf2+/Ctip2+ layer V neurons, and precocious differentiation of normally late-born, Satb2+ layer II-IV neurons. Conversely, in stable gain-of-function transgenics, Neurog2 promoted differentiative divisions and extended the period of Tbr1+/Ctip2+ deep-layer neurogenesis while reducing Satb2+ upper-layer neurogenesis. Similarly, acute misexpression of Neurog2 in early cortical progenitors promoted Tbr1 expression, whereas both Neurog2 and Ascl1 induced Ctip2. However, Neurog2 was unable to influence the derepression circuit when misexpressed in late cortical progenitors, and Ascl1 repressed only Satb2. Nevertheless, neurons derived from late misexpression of Neurog2 and, to a lesser extent, Ascl1, extended aberrant subcortical axon projections characteristic of early-born neurons. Finally, Neurog2 and Ascl1 altered the expression of Ikaros and Foxg1, known temporal regulators. Proneural genes thus act in a context-dependent fashion as early determinants, promoting deep-layer neurogenesis in early cortical progenitors via input into the derepression circuit while also influencing other temporal regulators.

Neurog1 and Neurog2 control two waves of neuronal differentiation in the piriform cortex.

The three-layered piriform cortex, an integral part of the olfactory system, processes odor information relayed by olfactory bulb mitral cells. Specifically, mitral cell axons form the lateral olfactory tract (LOT) by targeting lateral olfactory tract (lot) guidepost cells in the piriform cortex. While lot cells and other piriform cortical neurons share a pallial origin, the factors that specify their precise phenotypes are poorly understood. Here we show that in mouse, the proneural genes Neurog1 and Neurog2 are coexpressed in the ventral pallium, a progenitor pool that first gives rise to Cajal-Retzius (CR) cells, which populate layer I of all cortical domains, and later to layer II/III neurons of the piriform cortex. Using loss-of-function and gain-of-function approaches, we find that Neurog1 has a unique early role in reducing CR cell neurogenesis by tempering Neurog2's proneural activity. In addition, Neurog1 and Neurog2 have redundant functions in the ventral pallium, acting in two phases to first specify a CR cell fate and later to specify layer II/III piriform cortex neuronal identities. In the early phase, Neurog1 and Neurog2 are also required for lot cell differentiation, which we reveal are a subset of CR neurons, the loss of which prevents mitral cell axon innervation and LOT formation. Consequently, mutation of Trp73, a CR-specific cortical gene, results in lot cell and LOT axon displacement. Neurog1 and Neurog2 thus have unique and redundant functions in the piriform cortex, controlling the timing of differentiation of early-born CR/lot cells and specifying the identities of later-born layer II/III neurons.

Single-cell approaches define two groups of mammalian oligodendrocyte precursor cells and their evolution over developmental time.

Here, we used single-cell RNA sequencing (scRNA-seq), single-cell ATAC sequencing (scATAC-seq), and single-cell spatial transcriptomics to characterize murine cortical OPCs throughout postnatal life. During development, we identified two groups of differentially localized PDGFRα+ OPCs that are transcriptionally and epigenetically distinct. One group (active, or actOPCs) is metabolically active and enriched in white matter. The second (homeostatic, or hOPCs) is less active, enriched in gray matter, and predicted to derive from actOPCs. In adulthood, these two groups are transcriptionally but not epigenetically distinct, and relative to developing OPCs are less active metabolically and have less open chromatin. When adult oligodendrogenesis is enhanced during experimentally induced remyelination, adult OPCs do not reacquire a developmental open chromatin state, and the oligodendrogenesis trajectory is distinct from that seen neonatally. These data suggest that there are two OPC groups subserving distinct postnatal functions and that neonatal and adult OPC-mediated oligodendrogenesis are fundamentally different.

bHLH transcription factors in neural development, disease, and reprogramming.

The formation of functional neural circuits in the vertebrate central nervous system (CNS) requires that appropriate numbers of the correct types of neuronal and glial cells are generated in their proper places and times during development. In the embryonic CNS, multipotent progenitor cells first acquire regional identities, and then undergo precisely choreographed temporal identity transitions (i.e. time-dependent changes in their identity) that determine how many neuronal and glial cells of each type they will generate. Transcription factors of the basic-helix-loop-helix (bHLH) family have emerged as key determinants of neural cell fate specification and differentiation, ensuring that appropriate numbers of specific neuronal and glial cell types are produced. Recent studies have further revealed that the functions of these bHLH factors are strictly regulated. Given their essential developmental roles, it is not surprising that bHLH mutations and de-regulated expression are associated with various neurological diseases and cancers. Moreover, the powerful ability of bHLH factors to direct neuronal and glial cell fate specification and differentiation has been exploited in the relatively new field of cellular reprogramming, in which pluripotent stem cells or somatic stem cells are converted to neural lineages, often with a transcription factor-based lineage conversion strategy that includes one or more of the bHLH genes. These concepts are reviewed herein.

Normally lethal amino acid substitutions suppress an ultramutator DNA Polymerase δ variant.

In yeast, the pol3-01,L612M double mutant allele, which causes defects in DNA polymerase delta (Pol δ) proofreading (pol3-01) and nucleotide selectivity (pol3-L612M), confers an "ultramutator" phenotype that rapidly drives extinction of haploid and diploid MMR-proficient cells. Here, we investigate antimutator mutations that encode amino acid substitutions in Pol δ that suppress this lethal phenotype. We find that most of the antimutator mutations individually suppress the pol3-01 and pol3-L612M mutator phenotypes. The locations of many of the amino acid substitutions in Pol δ resemble those of previously identified antimutator substitutions; however, two novel mutations encode substitutions (R674G and Q697R) of amino acids in the fingers domain that coordinate the incoming dNTP. These mutations are lethal without pol3-L612M and markedly change the mutation spectra produced by the pol3-01,L612M mutator allele, suggesting that they alter nucleotide selection to offset the pol3-L612M mutator phenotype. Consistent with this hypothesis, mutations and drug treatments that perturb dNTP pool levels disproportionately influence the viability of pol3-L612M,R674G and pol3-L612M,Q697R cells. Taken together, our findings suggest that mutation rate can evolve through genetic changes that alter the balance of dNTP binding and dissociation from DNA polymerases.

Forebrain neurogenesis: From embryo to adult.

A satellite symposium to the Canadian Developmental Biology Conference 2016 was held on March 16-17, 2016 in Banff, Alberta, Canada, entitled Forebrain Neurogenesis: From embryo to adult. The Forebrain Neurogenesis symposium was a focused, high-intensity meeting, bringing together the top Canadian and international researchers in the field. This symposium reported the latest breaking news, along with 'state of the art' techniques to answer fundamental questions in developmental neurobiology. Topics covered ranged from stem cell regulation to neurocircuitry development, culminating with a session focused on neuropsychiatric disorders. Understanding the underlying causes of neurodevelopmental disorders such as autism spectrum disorder (ASD) and attention deficit/hyperactivity disorder (ADHD) is of great interest as diagnoses of these conditions are climbing at alarming rates. For instance, in 2012, the Centers for Disease Control reported that the prevalence rate of ASD in the U.S. was 1 in 88; while more recent data indicate that the number is as high as 1 in 68 (Centers for Disease Control and Prevention MMWR Surveillance Summaries. Vol. 63. No. 2). Similarly, the incidence of ASD is on the rise in Canada, increasing from 1 in 150 in 2000 to 1 in 63 in 2012 in southeastern Ontario (Centers for Disease Control and Prevention). Currently very little is known regarding the deficits underlying these neurodevelopmental conditions. Moreover, the development of effective therapies is further limited by major gaps in our understanding of the fundamental processes that regulate forebrain development and adult neurogenesis. The Forebrain Neurogenesis satellite symposium was thus timely, and it played a key role in advancing research in this important field, while also fostering collaborations between international leaders, and inspiring young researchers.

Central neuropathic itch from spinal-cord cavernous hemangioma: a human case, a possible animal model, and hypotheses about pathogenesis.

Cavernous hemangiomas (cavernomas) of the spinal cord are rare congenital malformations that comprise less than 5% of all intramedullary lesions. Despite this rarity, we describe the third case of central neuropathic itch associated with intramedullary cavernoma. Since fewer than 10 cases of central spinal itch from all causes have been published, this concurrence suggests the possibility of a specific association. A middle-aged man developed chronic disabling neuropathic itch and pain affecting his left shoulder and arm after frank hemorrhage of a midcervical cavernoma. We hypothesize that the relatively rostro-dorsal location of his lesion increased its likelihood of causing itch as well as pain. The microscopic pathology of cavernomas, specifically their gliotic rim containing hemosiderin-laden phagocytes, fosters ectopic firing of nearby neurons and makes cranial cavernomas highly epileptogenic. We hypothesize that these pathological features predispose cavernomas to cause central itch if they are located near, but spare, the central itch projection neurons in lamina I of the dorsal horn. Quisqualate injections into the deeper layers (neck) of the dorsal horns of rats produce pathologically similar lesions. Such rats develop unilateral dermatomal hyperalgesia and self-injurious scratching and biting (autotomy). Although this pathological grooming is currently interpreted as a response to chronic pain, we propose that it more likely models scratching provoked by central neuropathic itch, as seen in our patient and others. Study of quisqualate-injected rats may provide leads towards new treatments for neuropathic itch.

Neurog1 and Neurog2 coordinately regulate development of the olfactory system.

BACKGROUND: Proneural genes encode basic helix-loop-helix transcription factors that specify distinct neuronal identities in different regions of the nervous system. In the embryonic telencephalon, the proneural genes Neurog1 and Neurog2 specify a dorsal regional identity and glutamatergic projection neuron phenotype in the presumptive neocortex, but their roles in cell fate specification in the olfactory bulb, which is also partly derived from dorsal telencephalic progenitors, have yet to be assessed. Given that olfactory bulb development is guided by interactions with the olfactory epithelium in the periphery, where proneural genes are also expressed, we investigated the roles of Neurog1 and Neurog2 in the coordinated development of these two olfactory structures. RESULTS: Neurog1/2 are co-expressed in olfactory bulb progenitors, while only Neurog1 is widely expressed in progenitors for olfactory sensory neurons in the olfactory epithelium. Strikingly, only a remnant of an olfactory bulb forms in Neurog1-/-;Neurog2-/- double mutants, while this structure is smaller but distinguishable in Neurog1-/- single mutants and morphologically normal in Neurog2-/- single mutants. At the cellular level, fewer glutamatergic mitral and juxtaglomerular cells differentiate in Neurog1-/-;Neurog2-/- double-mutant olfactory bulbs. Instead, ectopic olfactory bulb interneurons are derived from dorsal telencephalic lineages in Neurog1-/-;Neurog2-/- double mutants and to a lesser extent in Neurog2-/- single mutants. Conversely, cell fate specification is normal in Neurog1-/- olfactory bulbs, but aberrant patterns of cell proliferation and neuronal migration are observed in Neurog1-/- single and Neurog1-/-;Neurog2-/- double mutants, probably contributing to their altered morphologies. Finally, in Neurog1-/- and Neurog1-/-;Neurog2-/- embryos, olfactory sensory neurons in the epithelium, which normally project to the olfactory bulb to guide its morphogenesis, fail to innervate the olfactory bulb. CONCLUSIONS: We have identified a cell autonomous role for Neurog1/2 in specifying the glutamatergic identity of olfactory bulb neurons. Furthermore, Neurog1 (and not Neurog2) is required to guide olfactory sensory neuron innervation of the olfactory bulb, the loss of which results in defects in olfactory bulb proliferation and tissue morphogenesis. We thus conclude that Neurog1/2 together coordinate development of the olfactory system, which depends on tissue interactions between the olfactory bulb and epithelium.