Ascl1 Converts Dorsal Midbrain Astrocytes into Functional Neurons In Vivo.

In vivo induction of non-neuronal cells into neurons by transcription factors offers potential therapeutic approaches for neural regeneration. Although generation of induced neuronal (iN) cells in vitro and in vivo has been reported, whether iN cells can be fully integrated into existing circuits remains unclear. Here we show that expression of achaete-scute complex homolog-like 1 (Ascl1) alone is sufficient to convert dorsal midbrain astrocytes of mice into functional iN cells in vitro and in vivo. Specific expression of Ascl1 in astrocytes by infection with GFAP-adeno-associated virus (AAV) vector converts astrocytes in dorsal midbrain, striatum, and somatosensory cortex of postnatal and adult mice into functional neurons in vivo. These iN cells mature progressively, exhibiting neuronal morphology and markers, action potentials, and synaptic inputs from and output to existing neurons. Thus, a single transcription factor, Ascl1, is sufficient to convert brain astrocytes into functional neurons, and GFAP-AAV is an efficient vector for generating iN cells from astrocytes in vivo.

Tlx3 controls cholinergic transmitter and Peptide phenotypes in a subset of prenatal sympathetic neurons.

The embryonic sympathetic nervous system consists of predominantly noradrenergic neurons and a very small population of cholinergic neurons. Postnatal development further allows target-dependent switch of a subset of noradrenergic neurons into cholinergic phenotype. How embryonic cholinergic neurons are specified at the prenatal stages remains largely unknown. In this study, we found that the expression of transcription factor Tlx3 was progressively restricted to a small population of embryonic sympathetic neurons in mice. Immunostaining for vesicular acetylcholine transporter (VAChT) showed that Tlx3 was highly expressed in cholinergic neurons at the late embryonic stage E18.5. Deletion of Tlx3 resulted in the loss of Vacht expression at E18.5 but not E12.5. By contrast, Tlx3 was required for expression of the cholinergic peptide vasoactive intestinal polypeptide (VIP), and somatostatin (SOM) at both E12.5 and E18.5. Furthermore, we found that, at E18.5 these putative cholinergic neurons expressed glial cell line-derived neurotrophic factor family coreceptor Ret but not tyrosine hydroxylase (Ret(+)/TH(-)). Deletion of Tlx3 also resulted in disappearance of high-level Ret expression. Last, unlike Tlx3, Ret was required for the expression of VIP and SOM at E18.5 but not E12.5. Together, these results indicate that transcription factor Tlx3 is required for the acquisition of cholinergic phenotype at the late embryonic stage as well as the expression and maintenance of cholinergic peptides VIP and SOM throughout prenatal development of mouse sympathetic neurons.

Ontogeny of excitatory spinal neurons processing distinct somatic sensory modalities.

Spatial and temporal cues govern the genesis of a diverse array of neurons located in the dorsal spinal cord, including dI1-dI6, dIL(A), and dIL(B) subtypes, but their physiological functions are poorly understood. Here we generated a new line of conditional knock-out (CKO) mice, in which the homeobox gene Tlx3 was removed in dI5 and dIL(B) cells. In these CKO mice, development of a subset of excitatory neurons located in laminae I and II was impaired, including itch-related GRPR-expressing neurons, PKCγ-expressing neurons, and neurons expressing three neuropeptide genes: somatostatin, preprotachykinin 1, and the gastrin-releasing peptide. These CKO mice displayed marked deficits in generating nocifensive motor behaviors evoked by a range of pain-related or itch-related stimuli. The mutants also failed to exhibit escape response evoked by dynamic mechanical stimuli but retained the ability to sense innocuous cooling and/or warm. Thus, our studies provide new insight into the ontogeny of spinal neurons processing distinct sensory modalities.

Forskolin-driven conversion of human somatic cells into induced neurons through regulation of the cAMP-CREB1-JNK signaling.

Human somatic cells can be reprogrammed into neuron cell fate through regulation of a single transcription factor or application of small molecule cocktails. Methods: Here, we report that forskolin efficiently induces the conversion of human somatic cells into induced neurons (FiNs). Results: A large population of neuron-like phenotype cells was observed as early as 24-36 h post-induction. There were >90% TUJ1-, >80% MAP2-, and >80% NEUN-positive neurons at 5 days post-induction. Multiple subtypes of neurons were present among TUJ1-positive cells, including >60% cholinergic, >20% glutamatergic, >10% GABAergic, and >5% dopaminergic neurons. FiNs exhibited typical neural electrophysiological activity in vitro and the ability to survive in vitro and in vivo more than 2 months. Mechanistically, forskolin functions in FiN reprogramming by regulating the cAMP-CREB1-JNK signals, which upregulates cAMP-CREB1 expression and downregulates JNK expression. Conclusion: Overall, our studies identify a safer and efficient single-small-molecule-driven reprogramming approach for induced neuron generation and reveal a novel regulatory mechanism of neuronal cell fate acquisition.

c-Maf is required for the development of dorsal horn laminae III/IV neurons and mechanoreceptive DRG axon projections.

Establishment of proper connectivity between peripheral sensory neurons and their central targets is required for an animal to sense and respond to various external stimuli. Dorsal root ganglion (DRG) neurons convey sensory signals of different modalities via their axon projections to distinct laminae in the dorsal horn of the spinal cord. In this study, we found that c-Maf was expressed predominantly in the interneurons of laminae III/IV, which primarily receive inputs from mechanoreceptive DRG neurons. In the DRG, c-Maf⁺ neurons also coexpressed neurofilament-200, a marker for the medium- and large-diameter myelinated afferents that transmit non-noxious information. Furthermore, mouse embryos deficient in c-Maf displayed abnormal development of dorsal horn laminae III/IV neurons, as revealed by the marked reduction in the expression of several marker genes for these neurons, including those for transcription factors MafA and Rora, GABA(A) receptor subunit α5, and neuropeptide cholecystokinin. In addition, among the four major subpopulations of DRG neurons marked by expression of TrkA, TrkB, TrkC, and MafA/GFRα2/Ret, c-Maf was required selectively for the proper differentiation of MafA⁺/Ret⁺/GFRα2⁺ low-threshold mechanoreceptors (LTMs). Last, we found that the central and peripheral projections of mechanoreceptive DRG neurons were compromised in c-Maf deletion mice. Together, our results indicate that c-Maf is required for the proper development of MafA⁺/Ret⁺/GFRα2⁺ LTMs in the DRG, their afferent projections in the dorsal horn and Pacinian corpuscles, as well as neurons in laminae III/IV of the spinal cord.

Tlx1/3 and Ptf1a control the expression of distinct sets of transmitter and peptide receptor genes in the developing dorsal spinal cord.

Establishing the pattern of expression of transmitters and peptides as well as their receptors in different neuronal types is crucial for understanding the circuitry in various regions of the brain. Previous studies have demonstrated that the transmitter and peptide phenotypes in mouse dorsal spinal cord neurons are determined by the transcription factors Tlx1/3 and Ptf1a. Here we show that these transcription factors also determine the expression of two distinct sets of transmitter and peptide receptor genes in this region. We have screened the expression of 78 receptor genes in the spinal dorsal horn by in situ hybridization. We found that receptor genes Gabra1, Gabra5, Gabrb2, Gria3, Grin3a, Grin3b, Galr1, and Npy1r were preferentially expressed in Tlx3-expressing glutamatergic neurons and their derivatives, and deletion of Tlx1 and Tlx3 resulted in the loss of expression of these receptor genes. Furthermore, we obtained genetic evidence that Tlx3 uses distinct pathways to control the expression of receptor genes. We also found that receptor genes Grm3, Grm4, Grm5, Grik1, Grik2, Grik3, and Sstr2 were mainly expressed in Pax2-expressing GABAergic neurons in the spinal dorsal horn, and their expression in this region was abolished or markedly reduced in Ptf1a and Pax2 deletion mutant mice. Together, our studies indicate that Tlx1/3 and Ptf1a, the key transcription factors for fate determination of glutamatergic and GABAergic neurons in the dorsal spinal cord, are also responsible for controlling the expression of two distinct sets of transmitter and peptide receptor genes.

Lmx1b controls peptide phenotypes in serotonergic and dopaminergic neurons.

Serotonin (5-HT) neurons synthesize a variety of peptides. How these peptides are controlled during development remains unclear. It has been reported that the co-localization of peptides and 5-HT varies by species. In contrast to the situations in the rostral 5-HT neurons of human and rat brains, several peptides do not coexist with 5-HT in the rostral 5-HT neurons of mouse brain. In this study, we found that the peptide substance P and peptide genes, including those encoding peptides thyrotropin-releasing hormone, enkephalin, and calcitonin gene-related peptide, were expressed in the caudal 5-HT neurons of mouse brain; these findings are in line with observations in rat and monkey 5-HT neurons. We also revealed that these peptides/peptide genes partially overlapped with the transcription factor Lmx1b that specifies the 5-HT cell fate. Furthermore, we found that the peptide cholecystokinin was expressed in developing dopaminergic neurons and greatly overlapped with Lmx1b that specifies the dopaminergic cell fate. By examining the phenotype of Lmx1b deletion mice, we found that Lmx1b was required for the expression of above peptides expressed in 5-HT or dopaminergic neurons. Together, our results indicate that Lmx1b, a key transcription factor for the specification of 5-HT and dopaminergic transmitter phenotypes during embryogenesis, determines some peptide phenotypes in these neurons as well.

MicroRNAs modulate the Wnt signaling pathway through targeting its inhibitors.

Increasing evidence indicates that microRNAs (miRNAs) play important roles in mouse brain development. We and several other reports recently have demonstrated that Wnt1-cre-mediated loss of Dicer, the key enzyme for miRNA biosynthesis, results in malformation of the midbrain and cerebellum and failure of neural crest and dopaminergic differentiation. The underlying mechanisms, however, remain poorly understood. The resemblance of some of the phenotypes in the Wnt1-cre Dicer conditional knockout embryos and Wnt1(-/-), Wnt1(-/-);Wnt3(-/-) and Wnt1-cre;ß-catenin(flox/flox) knockout embryos reminds us that loss of miRNA may disrupt the Wnt-ß-catenin signaling. Here we provide evidence that miRNAs modulate the Wnt signaling pathway through targeting its inhibitors. First, we predicted miRNA binding sites in the 3' UTRs of candidate inhibitors of the Wnt signaling pathway and luciferase assays revealed that several inhibitors of Wnt signaling pathway were targeted by miRNAs. Second, we demonstrated that several miRNAs could modulate the expression of Gsk3b, an inhibitor of Wnt signaling, post-transcriptional in 293T cells. Third, we found that several miRNAs were able to regulate the Wnt-ß-catenin signaling activity in 293T cells. More interestingly, the expression of ß-catenin protein was dramatically reduced in the Wnt1-cre-meidiated Dicer knockout brain tissue compared with control. Our studies therefore suggest that miRNAs might exert their functions, at least in part, by modulating the Wnt signaling pathway through targeting its inhibitors.

Reprogramming Glial Cells into Functional Neurons for Neuro-regeneration: Challenges and Promise.

The capacity for neurogenesis in the adult mammalian brain is extremely limited and highly restricted to a few regions, which greatly hampers neuronal regeneration and functional restoration after neuronal loss caused by injury or disease. Meanwhile, transplantation of exogenous neuronal stem cells into the brain encounters several serious issues including immune rejection and the risk of tumorigenesis. Recent discoveries of direct reprogramming of endogenous glial cells into functional neurons have provided new opportunities for adult neuro-regeneration. Here, we extensively review the experimental findings of the direct conversion of glial cells to neurons in vitro and in vivo and discuss the remaining issues and challenges related to the glial subtypes and the specificity and efficiency of direct cell-reprograming, as well as the influence of the microenvironment. Although in situ glial cell reprogramming offers great potential for neuronal repair in the injured or diseased brain, it still needs a large amount of research to pave the way to therapeutic application.

Molecular Mechanisms Underlying Ascl1-Mediated Astrocyte-to-Neuron Conversion.

Direct neuronal reprogramming potentially provides valuable sources for cell-based therapies. Proneural gene Ascl1 converts astrocytes into induced neuronal (iN) cells efficiently both in vitro and in vivo. However, the underlying mechanisms are largely unknown. By combining RNA sequencing and chromatin immunoprecipitation followed by high-throughput sequencing, we found that the expression of 1,501 genes was markedly changed during the early stages of Ascl1-induced astrocyte-to-neuron conversion and that the regulatory regions of 107 differentially expressed genes were directly bound by ASCL1. Among Ascl1's direct targets, Klf10 regulates the neuritogenesis of iN cells at the early stage, Myt1 and Myt1l are critical for the electrophysiological maturation of iN cells, and Neurod4 and Chd7 are required for the efficient conversion of astrocytes into neurons. Together, this study provides more insights into understanding the molecular mechanisms underlying Ascl1-mediated astrocyte-to-neuron conversion and will be of value for the application of direct neuronal reprogramming.

Regulation of apoptosis of rbf mutant cells during Drosophila development.

Inactivation of the retinoblastoma gene Rb leads to defects in cell proliferation, differentiation, or apoptosis, depending on specific cell or tissue types. To gain insights into the genes that can modulate the consequences of Rb inactivation, we carried out a genetic screen in Drosophila to identify mutations that affected apoptosis induced by inactivation of the Retinoblastoma-family protein (rbf) and identified a mutation that blocked apoptosis induced by rbf. We found this mutation to be a new allele of head involution defective (hid) and showed that hid expression is deregulated in rbf mutant cells in larval imaginal discs. We identified an enhancer that regulates hid expression in response to developmental cues as well as to radiation and demonstrated that this hid enhancer is directly repressed by RBF through an E2F binding site. These observations indicate that apoptosis of rbf mutant cells is mediated by an upregulation of hid. Finally, we showed that bantam, a miRNA that regulates hid translation, is expressed in the interommatidial cells in the larval eye discs and modulates the survival of rbf mutant cells.

Imbalance of Excitatory/Inhibitory Neuron Differentiation in Neurodevelopmental Disorders with an NR2F1 Point Mutation.

Recent studies have revealed an essential role for embryonic cortical development in the pathophysiology of neurodevelopmental disorders, including autism spectrum disorder (ASD). However, the genetic basis and underlying mechanisms remain unclear. Here, we generate mutant human embryonic stem cell lines (Mut hESCs) carrying an NR2F1-R112K mutation that has been identified in a patient with ASD features and investigate their neurodevelopmental alterations. Mut hESCs overproduce ventral telencephalic neuron progenitors (ventral NPCs) and underproduce dorsal NPCs, causing the imbalance of excitatory/inhibitory neurons. These alterations can be mainly attributed to the aberrantly activated Hedgehog signaling pathway. Moreover, the corresponding Nr2f1 point-mutant mice display a similar excitatory/inhibitory neuron imbalance and abnormal behaviors. Antagonizing the increased inhibitory synaptic transmission partially alleviates their behavioral deficits. Together, our results suggest that the NR2F1-dependent imbalance of excitatory/inhibitory neuron differentiation caused by the activated Hedgehog pathway is one precursor of neurodevelopmental disorders and may enlighten the therapeutic approaches.

Tlx1 and Tlx3 coordinate specification of dorsal horn pain-modulatory peptidergic neurons.

The dorsal spinal cord synthesizes a variety of neuropeptides that modulate the transmission of nociceptive sensory information. Here, we used genetic fate mapping to show that Tlx3(+) spinal cord neurons and their derivatives represent a heterogeneous population of neurons, marked by partially overlapping expression of a set of neuropeptide genes, including those encoding the anti-opioid peptide cholecystokinin, pronociceptive Substance P (SP), Neurokinin B, and a late wave of somatostatin. Mutations of Tlx3 and Tlx1 result in a loss of expression of these peptide genes. Brn3a, a homeobox transcription factor, the expression of which is partly dependent on Tlx3, is required specifically for the early wave of SP expression. These studies suggest that Tlx1 and Tlx3 operate high in the regulatory hierarchy that coordinates specification of dorsal horn pain-modulatory peptidergic neurons.

Ptf1a, Lbx1 and Pax2 coordinate glycinergic and peptidergic transmitter phenotypes in dorsal spinal inhibitory neurons.

Inhibitory neurons in the dorsal horn synthesize a variety of neurotransmitters, including GABA, glycine and a set of peptides. Here we show that three transcription factors, Ptf1a, Pax2, and Lbx1, which have been reported to promote a GABAergic cell fate, also specify glycinergic and peptidergic transmitter phenotypes. First, Ptf1a appears to be a master regulator, as indicated by a requirement of Ptf1a for the expression of glycinergic marker GlyT2 and a set of peptides, including neuropeptide Y (NPY), nociceptin/orphanin FQ (N/OFQ), somatostatin (SOM), enkephalin (ENK), dynorphin (DYN) and galanin (GAL). Second, Pax2 is a downstream target of Ptf1a and controls subsets of transmitter phenotypes, including the expression of GlyT2, NPY, N/OFQ, DYN, and GAL, but is dispensable for SOM or ENK expression. Third, for Lbx1, due to neuronal cell loss at late stages, our analyses focused on early embryonic stages, and we found that Lbx1 is required for the expression of GlyT2, NPY, N/OFQ and is partially responsible for SOM expression. Our studies therefore suggest a coordinated and hierarchical specification of a variety of neurotransmitters in dorsal spinal inhibitory neurons.

Lbx1 and Tlx3 are opposing switches in determining GABAergic versus glutamatergic transmitter phenotypes.

Most neurons in vertebrates make a developmental choice between two principal neurotransmitter phenotypes (glutamatergic versus GABAergic). Here we show that the homeobox gene Lbx1 determines a GABAergic cell fate in the dorsal spinal cord at early embryonic stages. In Lbx1-/- mice, the presumptive GABAergic neurons are transformed into glutamatergic cells. Furthermore, overexpression of Lbx1 in the chick spinal cord is sufficient to induce GABAergic differentiation. Paradoxically, Lbx1 is also expressed in glutamatergic neurons. We previously reported that the homeobox genes Tlx1 and Tlx3 determine glutamatergic cell fate. Here we show that impaired glutamatergic differentiation, observed in Tlx3-/- mice, is restored in Tlx3-/-Lbx1-/- mice. These genetic studies suggest that Lbx1 expression defines a basal GABAergic differentiation state, and Tlx3 acts to antagonize Lbx1 to promote glutamatergic differentiation.

Conversion of Astrocytes and Fibroblasts into Functional Noradrenergic Neurons.

Dysfunction of noradrenergic (NA) neurons is associated with a number of neuronal disorders. Diverse neuronal subtypes can be generated by direct reprogramming. However, it is still unknown how to convert non-neuronal cells into NA neurons. Here, we show that seven transcription factors (TFs) (Ascl1, Phox2b, AP-2α, Gata3, Hand2, Nurr1, and Phox2a) are able to convert astrocytes and fibroblasts into induced NA (iNA) neurons. These iNA neurons express the genes required for the biosynthesis, release, and re-uptake of noradrenaline. Moreover, iNA neurons fire action potentials, receive synaptic inputs, and control the beating rate of co-cultured ventricular myocytes. Furthermore, iNA neurons survive and integrate into neural circuits after transplantation. Last, human fibroblasts can be converted into functional iNA neurons as well. Together, iNA neurons are generated by direct reprogramming, and they could be potentially useful for disease modeling and cell-based therapies.

Tlx3 and Tlx1 are post-mitotic selector genes determining glutamatergic over GABAergic cell fates.

Glutamatergic and GABAergic neurons mediate much of the excitatory and inhibitory neurotransmission, respectively, in the vertebrate nervous system. The process by which developing neurons select between these two cell fates is poorly understood. Here we show that the homeobox genes Tlx3 and Tlx1 determine excitatory over inhibitory cell fates in the mouse dorsal spinal cord. First, we found that Tlx3 was required for specification of, and expressed in, glutamatergic neurons. Both generic and region-specific glutamatergic markers, including VGLUT2 and the AMPA receptor Gria2, were absent in Tlx mutant dorsal horn. Second, spinal GABAergic markers were derepressed in Tlx mutants, including Pax2 that is necessary for GABAergic differentiation, Gad1/2 and Viaat that regulate GABA synthesis and transport, and the kainate receptors Grik2/3. Third, ectopic expression of Tlx3 was sufficient to suppress GABAergic differentiation and induce formation of glutamatergic neurons. Finally, excess GABA-mediated inhibition caused dysfunction of central respiratory circuits in Tlx3 mutant mice.

In vivo simultaneous transcriptional activation of multiple genes in the brain using CRISPR-dCas9-activator transgenic mice.

Despite rapid progresses in the genome-editing field, in vivo simultaneous overexpression of multiple genes remains challenging. We generated a transgenic mouse using an improved dCas9 system that enables simultaneous and precise in vivo transcriptional activation of multiple genes and long noncoding RNAs in the nervous system. As proof of concept, we were able to use targeted activation of endogenous neurogenic genes in these transgenic mice to directly and efficiently convert astrocytes into functional neurons in vivo. This system provides a flexible and rapid screening platform for studying complex gene networks and gain-of-function phenotypes in the mammalian brain.

Homology-mediated end joining-based targeted integration using CRISPR/Cas9.

Targeted integration of transgenes can be achieved by strategies based on homologous recombination (HR), microhomology-mediated end joining (MMEJ) or non-homologous end joining (NHEJ). The more generally used HR is inefficient for achieving gene integration in animal embryos and tissues, because it occurs only during cell division, although MMEJ and NHEJ can elevate the efficiency in some systems. Here we devise a homology-mediated end joining (HMEJ)-based strategy, using CRISPR/Cas9-mediated cleavage of both transgene donor vector that contains guide RNA target sites and ∼800 bp of homology arms, and the targeted genome. We found no significant improvement of the targeting efficiency by the HMEJ-based method in either mouse embryonic stem cells or the neuroblastoma cell line, N2a, compared to the HR-based method. However, the HMEJ-based method yielded a higher knock-in efficiency in HEK293T cells, primary astrocytes and neurons. More importantly, this approach achieved transgene integration in mouse and monkey embryos, as well as in hepatocytes and neurons in vivo, with an efficiency much greater than HR-, NHEJ- and MMEJ-based strategies. Thus, the HMEJ-based strategy may be useful for a variety of applications, including gene editing to generate animal models and for targeted gene therapies.

Lmx1b, Pet-1, and Nkx2.2 coordinately specify serotonergic neurotransmitter phenotype.

Serotonergic (5-HT) neurons in the brainstem modulate a wide range of physiological processes and behaviors. Two transcription factor genes, Pet-1 and Nkx2.2, are necessary but not sufficient to specify the 5-HT transmitter phenotype. Here we show that the Lim class homeobox gene Lmx1b is required for proper formation of the entire 5-HT system in the hindbrain, as indicated by the loss of expression of genes necessary for serotonin synthesis and transport in Lmx1b null mice. Lmx1b and Pet1 act downstream of Nkx2.2, and their expression is independently regulated at the time when 5-HT transmitter phenotype is specified. Ectopic expression of Lmx1b plus Pet-1 is able to induce formation of 5-HT cells in the most ventral spinal cord, where Nkx2.2 is normally expressed. Combined expression of all three genes, Lmx1b, Pet-1, and Nkx2.2, drives 5-HT differentiation in the dorsal spinal cord. Our studies therefore define a molecular pathway necessary and sufficient to specify the serotonergic neurotransmitter phenotype.