Dbx1 is a dorsal midbrain-specific determinant of GABAergic neuron fate and regulates differentiation of the dorsal midbrain into the inferior and superior colliculi.

The mechanism underlying the differentiation of the dorsal midbrain into two morphologically and functionally distinct compartments, the inferior colliculus (IC) and superior colliculus (SC), which process auditory and visual information, respectively, remains largely unexplored. By using null and conditional alleles, we uncover the roles of a homeodomain transcription factor Dbx1 in the regulation of IC and SC differentiation. We show that Dbx1 regulates GABAergic neuron development in the dorsal midbrain. In the absence of Dbx1 function, the dorsal-most m1-m2 progenitor domains in the midbrain fail to activate GABAergic neuron-specific gene expression and instead switch to a glutamatergic phenotype. These results identify Dbx1 as a dorsal midbrain-specific GABAergic determinant that regulates the selector genes, Helt, Gata2, and Tal2. Furthermore, we demonstrate that maturation of the dorsal midbrain into the IC and SC is dependent on Dbx1. Null mutation of Dbx1 impairs the identity and fate of IC and SC neurons. Surprisingly, Dbx1 is required for preventing IC into SC fate switch and thus Dbx1-deficient IC neurons undergo acquisition of SC identity. Conditional inactivation of Dbx1 at late developmental phase leads to alteration in the identity and fate of the IC, but not the SC. These results suggest that SC differentiation is dependent on the early function of Dbx1, and that the IC requires the prolonged action for its normal formation. Furthermore, we uncover that Tcf7l2 acts downstream of Dbx1 selectively to promote IC differentiation. Altogether, our study identifies a molecular mechanism underlying spatial and temporal control of dorsal midbrain development.

The embryonic patterning gene Dbx1 governs the survival of the auditory midbrain via Tcf7l2-Ap2δ transcriptional cascade.

At the top of the midbrain is the inferior colliculus (IC), which functions as the major hub for processing auditory information. Despite the functional significance of neurons in the IC, our understanding of their formation is limited. In this study, we identify the embryonic patterning gene Dbx1 as a key molecular player that governs genetic programs for IC survival. We find that Dbx1 plays a critical role in preventing apoptotic cell death in postnatal IC by transcriptionally repressing c-Jun and pro-apoptotic BH3 only factors. Furthermore, by employing combined approaches, we uncover that Tcf7l2 functions downstream of Dbx1. Loss of Tcf7l2 function causes IC phenotypes with striking similarity to those of Dbx1 mutant mice, which include defective embryonic maturation and postnatal deletion of the IC. Finally, we demonstrate that the Dbx1-Tcf7l2 cascade functions upstream of Ap-2δ, which is essential for IC development and survival. Together, these results unravel a novel molecular mechanism for IC maintenance, which is indispensable for normal brain development.

Application of Antimicrobial Peptide LL-37 as an Adjuvant for Middle East Respiratory Syndrome-Coronavirus Antigen Induces an Efficient Protective Immune Response Against Viral Infection After Intranasal Immunization.

The human antimicrobial peptide LL-37 has chemotactic and modulatory activities in various immune cells, including dendritic cells. Because of its characteristics, LL-37 can be considered an adjuvant for vaccine development. In this study, we confirmed the possible adjuvant activity of LL-37 in mucosal vaccine development against Middle East respiratory syndrome-coronavirus (MERS-CoV) by means of intranasal immunization in C57BL/6 and human dipeptidyl peptidase 4 (hDPP4)-transgenic (hDPP4-Tg) mice. Intranasal immunization using the receptor-binding domain (RBD) of MERS-CoV spike protein (S-RBD) recombined with LL-37 (S-RBD-LL-37) induced an efficient mucosal IgA and systemic IgG response with virus-neutralizing activity, compared with S-RBD. Ag-specific CTL stimulation was also efficiently induced in the lungs of mice that had been intranasally immunized with S-RBD-LL-37, compared with S-RBD. Importantly, intranasal immunization of hDPP4-Tg mice with S-RBD-LL-37 led to reduced immune cell infiltration into the lungs after infection with MERS-CoV. Finally, intranasal immunization of hDPP4-Tg mice with S-RBD-LL-37 led to enhanced protective efficacy, with increased survival and reduced body weight loss after challenge infection with MERS-CoV. Collectively, these results suggest that S-RBD-LL-37 is an effective intranasal vaccine candidate molecule against MERS-CoV infection.

Intranasal immunization with a Middle East respiratory syndrome-coronavirus antigen conjugated to the M-cell targeting ligand Co4B enhances antigen-specific mucosal and systemic immunity and protects against infection.

Middle East respiratory syndrome (MERS) is a threat to public health worldwide. A vaccine against the causative agent of MERS, MERS-coronavirus (MERS-CoV), is urgently needed. We previously identified a peptide ligand, Co4B, which can enhance antigen (Ag) delivery to the nasal mucosa and promote Ag-specific mucosal and systemic immune responses following intranasal immunization. MERS-CoV infects via the respiratory route; thus, we conjugated the Co4B ligand to the MERS-CoV spike protein receptor-binding domain (S-RBD), and used this to intranasally immunize C57BL/6 and human dipeptidyl peptidase 4-transgenic (hDPP4-Tg) mice. Ag-specific mucosal immunoglobulin (Ig) A and systemic IgG, together with virus-neutralizing activities, were highly induced in mice immunized with Co4B-conjugated S-RBD (S-RBD-Co4B) compared to those immunized with unconjugated S-RBD. Ag-specific T cell-mediated immunity was also induced in the spleen and lungs of mice intranasally immunized with S-RBD-Co4B. Intranasal immunization of hDPP4-Tg mice with S-RBD-Co4B reduced immune cell infiltration into the tissues of virus-challenged mice. Finally, S-RBD-Co4B-immunized mice exhibited were better protected against infection, more likely to survive, and exhibited less body weight loss. Collectively, our results suggest that S-RBD-Co4B could be used as an intranasal vaccine candidate against MERS-CoV infection.

Long Noncoding RNA Expression Profiling Reveals Upregulation of Uroplakin 1A and Uroplakin 1A Antisense RNA 1 under Hypoxic Conditions in Lung Cancer Cells.

Hypoxia plays important roles in cancer progression by inducing angiogenesis, metastasis, and drug resistance. However, the effects of hypoxia on long noncoding RNA (lncRNA) expression have not been clarified. Herein, we evaluated alterations in lncRNA expression in lung cancer cells under hypoxic conditions using lncRNA microarray analyses. Among 40,173 lncRNAs, 211 and 113 lncRNAs were up- and downregulated, respectively, in both A549 and NCI-H460 cells. Uroplakin 1A (UPK1A) and UPK1A-antisense RNA 1 (AS1), which showed the highest upregulation under hypoxic conditions, were selected to investigate the effects of UPK1AAS1 on the expression of UPK1A and the mechanisms of hypoxia-inducible expression. Following transfection of cells with small interfering RNA (siRNA) targeting hypoxiainducible factor 1α (HIF-1α), the hypoxia-induced expression of UPK1A and UPK1A-AS1 was significantly reduced, indicating that HIF-1α played important roles in the hypoxiainduced expression of these targets. After transfection of cells with UPK1A siRNA, UPK1A and UPK1A-AS1 levels were reduced. Moreover, transfection of cells with UPK1A-AS1 siRNA downregulated both UPK1A-AS1 and UPK1A. RNase protection assays demonstrated that UPK1A and UPK1A-AS1 formed a duplex; thus, transfection with UPK1A-AS1 siRNA decreased the RNA stability of UPK1A. Overall, these results indicated that UPK1A and UPK1A-AS1 expression increased under hypoxic conditions in a HIF-1α-dependent manner and that formation of a UPK1A/UPK1A-AS1 duplex affected RNA stability, enabling each molecule to regulate the expression of the other.

Conjugation of Human ß-Defensin 2 to Spike Protein Receptor-Binding Domain Induces Antigen-Specific Protective Immunity against Middle East Respiratory Syndrome Coronavirus Infection in Human Dipeptidyl Peptidase 4 Transgenic Mice.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory symptoms. Due to the lack of medical countermeasures, effective and safe vaccines against MERS-CoV infection are urgently required. Although different types of candidate vaccines have been developed, their immunogenicity is limited, and the dose and administration route need optimization to achieve optimal protection. We here investigated the potential use of human ß-defensin 2 (HBD 2) as an adjuvant to enhance the protection provided by MERS-CoV vaccination. We found that immunization of human dipeptidyl peptidase 4 (hDPP4)-transgenic (hDPP4-Tg) mice with spike protein receptor-binding domain (S RBD) conjugated with HBD 2 (S RBD-HBD 2) induced potent antigen (Ag)-specific adaptive immune responses and protected against MERS-CoV infection. In addition, immunization with S RBD-HBD 2 alleviated progressive pulmonary fibrosis in the lungs of MERS-CoV-infected hDPP4-Tg mice and suppressed endoplasmic reticulum stress signaling activation upon viral infection. Compared to intramuscular administration, intranasal administration of S RBD-HBD 2 induced more potent mucosal IgA responses and was more effective for protecting against intranasal MERS-CoV infection. In conclusion, our findings suggest that HBD 2 potentiates Ag-specific immune responses against viral Ag and can be used as an adjuvant enhancing the immunogenicity of subunit vaccine candidates against MERS-CoV.

Efficient production process of bioactive recombinant human leukemia inhibitory factor in Chinese hamster ovary cells.

The rhLIF is widely used as an essential factor in stem cell cultures for cell therapies. However, all the recombinant LIFs commercially available are expensive, and no commercially available rhLIF meet the standards recommended by USP for use in cell therapies. The current study reports the efficient production of N-glycosylated and bioactive rhLIF in CHO cells. The production rate of established rhLIF-expressing rCHO cells was approximately 0.85 g/l in 12-day fed-batch cultures using a 7.5 l bioreactor. The rhLIF protein was purified via a four-step purification procedure with approximately 57% recovery rate and greater than 99% purity. The purified rhLIF was N-glycosylated and biologically active with an EC50 of 0.167 ng/ml and a specific activity of 0.86 × 103 IU/mg. The purification procedure controlled the levels of process-related impurities below critical levels recommended by USP for cytokines used in cell therapies. The current work is the first production process of N-glycosylated and bioactive rhLIF, which can be applied to large-scale manufacture of GMP-grade rhLIF for use as an ancillary material in cell therapy.

Middle East Respiratory Syndrome-Coronavirus Infection into Established hDPP4-Transgenic Mice Accelerates Lung Damage Via Activation of the Pro-Inflammatory Response and Pulmonary Fibrosis.

Middle East respiratory syndrome coronavirus (MERS-CoV) infects the lower respiratory airway of humans, leading to severe acute respiratory failure. Unlike human dipeptidyl peptidase 4 (hDPP4), a receptor for MERS-CoV, mouse DPP4 (mDPP4) failed to support MERS-CoV infection. Consequently, diverse transgenic mouse models expressing hDPP4 have been developed using diverse methods, although some models show no mortality and/or only transient and mild-to-moderate clinical signs following MERS-CoV infection. Additionally, overexpressed hDPP4 is associated with neurological complications and breeding difficulties in some transgenic mice, resulting in impeding further studies. Here, we generated stable hDPP4-transgenic mice that were sufficiently susceptible to MERS-CoV infection. The transgenic mice showed weight loss, decreased pulmonary function, and increased mortality with minimal perturbation of overexpressed hDPP4 after MERS-CoV infection. In addition, we observed histopathological signs indicative of progressive pulmonary fibrosis, including thickened alveolar septa, infiltration of inflammatory monocytes, and macrophage polarization as well as elevated expression of profibrotic molecules and acute inflammatory response in the lung of MERS-CoV-infected hDPP4-transgenic mice. Collectively, we suggest that this hDPP4-transgenic mouse is useful in understanding the pathogenesis of MERS-CoV infection and for antiviral research and vaccine development against the virus.

Tcf7l2 transcription factor is required for the maintenance, but not the initial specification, of the neurotransmitter identity in the caudal thalamus.

BACKGROUND: Dysfunction of GABAergic and glutamatergic neurons in the brain, which establish inhibitory and excitatory networks, respectively, may cause diverse neurological disorders. The mechanism underlying the determination of GABAergic vs. glutamatergic neurotransmitter phenotype in the caudal diencephalon remains largely unknown. RESULTS: In this study, we investigated the consequence of Tcf7l2 (transcription factor 7-like 2) ablation on the neurotransmitter identity of GABAergic and glutamatergic neurons in the caudal diencephalon. We identified positive and negative activity in the control of glutamatergic and GABAergic neuronal gene expression by Tcf7l2. Loss of Tcf7l2 did not alter the initial acquisition of the neurotransmitter identity in thalamic neurons. However, glutamatergic thalamic neurons failed to maintain their excitatory neurotransmitter phenotype in the absence of Tcf7l2. Moreover, a subset of Tcf7l2-deficient thalamic neurons underwent a glutamatergic to GABAergic neurotransmitter identity switch. Our data indicate that Tcf7l2 may promote glutamatergic neuronal differentiation and repress GABAergic neurotransmitter identity in the caudal thalamus. CONCLUSIONS: This study provides evidence for a novel and crucial role of Tcf7l2 in the molecular mechanism by which the neurotransmitter identity of glutamatergic thalamic neurons is established. Our findings exemplify a clear case of neurotransmitter identity regulation that is partitioned into initiation and maintenance phases.

MiR-200c downregulates HIF-1α and inhibits migration of lung cancer cells.

BACKGROUND: Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor with a pivotal role in physiological and pathological responses to hypoxia. While HIF-1α is known to be involved in hypoxia-induced upregulation of microRNA (miRNA) expression, HIF-1α is also targeted by miRNAs. In this study, miRNAs targeting HIF-1α were identified and their effects on its expression and downstream target genes under hypoxic conditions were investigated. Cell migration under the same conditions was also assessed. METHODS: microRNAs that target HIF-1α were screened using 3'-untranslated region luciferase (3'-UTR-luciferase) reporter assays. The expression levels of HIF-1α and its downstream target genes after transfection with miRNA were assessed using quantitative RT-PCR and western blot analyses. The effect of the miRNAs on the transcriptional activity of HIF-1α was determined using hypoxia-responsive element luciferase (HRE-luciferase) assays. Cell migration under hypoxia was examined using the wound-healing assay. RESULTS: Several of the 19 screened miRNAs considerably decreased the luciferase activity. Transfection with miR-200c had substantial impact on the expression level and transcription activity of HIF-1α. The mRNA level of HIF-1α downstream genes decreased in response to miR-200c overexpression. MiR-200c inhibited cell migration in normoxia and, to a greater extent, in hypoxia. These effects were partly reversed by HIF-1α expression under hypoxic conditions. CONCLUSION: miR-200c negatively affects hypoxia-induced responses by downregulating HIF-1α, a key regulator of hypoxia. Therefore, overexpression of miR-200c might have therapeutic potential as an anticancer agent that inhibits tumor hypoxia.

Efficient Development of Stable Recombinant Chinese Hamster Ovary (rCHO) Cell Lines to Produce Antibodies by Using Dimethyl Sulfoxide (DMSO) in Electroporation.

Development of stable rCHO cell lines is still time consuming and labor intensive, although it is a critical step in the commercial development of recombinant antibodies. The current work demonstrates, for the first time, that electroporation of CHO cells with DMSO can enhance stable expression of recombinant antibodies in rCHO cells. Electroporation with DMSO resulted in an average 3.7-fold and 2.8-fold increases in expression levels of aflibercept and pembrolizumab, respectively, in pools of stable rCHO cells. It also resulted in an average of 2.2-fold and 2.6-fold increases in the expression of aflibercept and pembrolizumab, respectively, in single-cell derived rCHO clones. Simple batch cultures of rCHO cell clones with the highest expression produced 1.0 g/l for aflibercept and 1.4 g/l for pembrolizumab without a time-consuming gene amplification process. Electroporation with DMSO also shortened the development of rCHO cell lines to 2-3 months, allowing rapid establishment of stable rCHO cell lines with a desirable expression level antibodies.

Specification of neurotransmitter identity by Tal1 in thalamic nuclei.

BACKGROUND: The neurons contributing to thalamic nuclei are derived from at least two distinct progenitor domains: the caudal (cTH) and rostral (rTH) populations of thalamic progenitors. These neural compartments exhibit unique neurogenic patterns, and the molecular mechanisms underlying the acquisition of neurotransmitter identity remain largely unclear. RESULTS: T-cell acute lymphocytic leukemia protein 1 (Tal1) was expressed in the early postmitotic cells in the rTH domain, and its expression was maintained in mature thalamic neurons in the ventrolateral geniculate nucleus (vLG) and the intergeniculate leaflet (IGL). To investigate a role of Tal1 in thalamic development, we used a newly generated mouse line driving Cre-mediated recombination in the rTH domain. Conditional deletion of Tal1 did not alter regional patterning in the developing diencephalon. However, in the absence of Tal1, rTH-derived thalamic neurons failed to maintain their postmitotic neuronal features, including neurotransmitter profile. Tal1-deficient thalamic neurons lost their GABAergic markers such as Gad1, Npy, and Penk in IGL/vLG. These defects may be associated at least in part with down-regulation of Nkx2.2, which is known as a critical regulator of rTH-derived GABAergic neurons. CONCLUSIONS: Our results demonstrate that Tal1 plays an essential role in regulating neurotransmitter phenotype in the developing thalamic nuclei. Developmental Dynamics 246:749-758, 2017. © 2017 Wiley Periodicals, Inc.

Expression and Purification of Biologically Active Human Bone Morphogenetic Protein-4 in Recombinant Chinese Hamster Ovary Cells.

Bone morphogenetic protein-4 (BMP-4) is considered to have therapeutic potential for various diseases, including cancers; however, the high expression of biologically active recombinant human BMP-4 (rhBMP-4) needed for its manufacture for therapeutic purposes has yet to be established. In the current study, we established a recombinant Chinese hamster ovary (rCHO) cell line overexpressing rhBMP-4 as well as a production process using 7.5-l bioreactor (5 L working volume). The expression of the mature rhBMP-4 was significantly enhanced by recombinant furin expression. The combination of a chemically defined medium and a nutrient supplement solution for high expression of rhBMP-4 was selected and used for bioreactor cultures. The 11-day fed-batch cultures of the established rhBMP-4-expressing rCHO cells in the 7.5-L bioreactor produced approximately 32 mg/l of rhBMP-4. The mature rhBMP-4 was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure, resulting in a recovery rate of approximately 55% and a protein purity greater than 95%. The N-terminal amino acid sequences and N-linked glycosylation of the purified rhBMP-4 were confirmed by N-terminal sequencing and de-N-glycosylation analysis, respectively. The mature purified rhBMP-4 has been proved to be functionally active, with an effective dose concentration of EC50 of 2.93 ng/ml.

Tcf7l2 plays crucial roles in forebrain development through regulation of thalamic and habenular neuron identity and connectivity.

The thalamus acts as a central integrator for processing and relaying sensory and motor information to and from the cerebral cortex, and the habenula plays pivotal roles in emotive decision making by modulating dopaminergic and serotonergic circuits. These neural compartments are derived from a common developmental progenitor domain, called prosomere 2, in the caudal forebrain. Thalamic and habenular neurons exhibit distinct molecular profile, neurochemical identity, and axonal circuitry. However, the mechanisms of how their progenitors in prosomere 2 give rise to these two populations of neurons and contribute to the forebrain circuitry remains unclear. In this study, we discovered a previously unrecognized role for Tcf7l2, a transcription factor known as the canonical Wnt nuclear effector and diabetes risk-conferring gene, in establishing neuronal identity and circuits of the caudal forebrain. Using genetic and chemical axon tracers, we showed that efferent axons of the thalamus, known as the thalamocortical axons (TCAs), failed to elongate normally and strayed from their normal course to inappropriate locations in the absence of Tcf7l2. Further experiments with thalamic explants revealed that the pathfinding defects of Tcf7l2-deficient TCAs were associated at least in part with downregulation of guidance receptors Robo1 and Robo2 expression. Moreover, the fasciculus retroflexus, the main habenular output tract, was missing in embryos lacking Tcf7l2. These axonal defects may result from dysregulation of Nrp2 guidance receptor. Strikingly, loss of Tcf7l2 caused a post-mitotic identity switch between thalamic and habenular neurons. Despite normal acquisition of progenitor identity in prosomere 2, Tcf7l2-deficient thalamic neurons adopted a molecular profile of a neighboring forebrain derivative, the habenula. Conversely, habenular neurons failed to maintain their normal post-mitotic neuronal identity and acquired a subset of thalamic neuronal features in the absence of Tcf7l2. Our findings suggest a unique role for Tcf7l2 in generating distinct neuronal phenotypes from homogeneous progenitor population, and provide a better understanding of the mechanism underlying neuronal specification, differentiation, and connectivity of the developing caudal forebrain.

Identification of a conserved cis-regulatory element controlling mid-diencephalic expression of mouse Six3.

The sine oculis homeobox protein Six3 plays pivotal roles in the development of the brain and craniofacial structures. In humans, SIX3 haploinsufficiency results in holoprosencephaly, a defect in anterior midline formation. Although much is known about the evolutionarily conserved functions of Six3, the regulatory mechanism responsible for the expression pattern of Six3 remains relatively unexplored. To understand how the transcription of Six3 is controlled during embryogenesis, we screened ∼300 kb of genomic DNA encompassing the Six3 locus for cis-acting regulatory elements capable of directing reporter gene expression to sites of Six3 transcription in transgenic mouse embryos. We identified a novel enhancer element, whose activity recapitulates endogenous Six3 expression in the ventral midbrain, pretectum, and thalamus. Cross-species comparisons revealed that this Six3 brain enhancer is functionally conserved in other vertebrates. We also showed that normal Six3 transcription in the ventral midbrain and pretectum is dependent on Ascl1, a basic helix-loop-helix proneural factor. Moreover, loss of Ascl1 resulted in downregulation of the Six3 brain enhancer activity, emphasizing its unique role in regulating Six3 expression in the developing brain.

Cis-regulatory architecture of a brain signaling center predates the origin of chordates.

Genomic approaches have predicted hundreds of thousands of tissue-specific cis-regulatory sequences, but the determinants critical to their function and evolutionary history are mostly unknown. Here we systematically decode a set of brain enhancers active in the zona limitans intrathalamica (zli), a signaling center essential for vertebrate forebrain development via the secreted morphogen Sonic hedgehog (Shh). We apply a de novo motif analysis tool to identify six position-independent sequence motifs together with their cognate transcription factors that are essential for zli enhancer activity and Shh expression in the mouse embryo. Using knowledge of this regulatory lexicon, we discover new Shh zli enhancers in mice and a functionally equivalent element in hemichordates, indicating an ancient origin of the Shh zli regulatory network that predates the chordate phylum. These findings support a strategy for delineating functionally conserved enhancers in the absence of overt sequence homologies and over extensive evolutionary distances.

Overproduction of recombinant human bone morphogenetic protein-7 in Chinese hamster ovary cells.

Bone morphogenetic protein-7 is a multifunctional growth factor involved in various cellular processes such as osteogenesis, kidney and eye development, brown adipogenesis, and bone metastasis, and thus has been considered to have therapeutic potential for treating various diseases. In this study, we established a Chinese hamster ovary (CHO) cell line stably overexpressing recombinant human BMP-7 (rhBMP-7). Over the course of a 14-day fed-batch culture process in a 7.5-l bioreactor (5-l working volume) using chemically defined medium, the established cells could produce over 188 mg/l of rhBMP-7 protein. The rhBMP-7 was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure that resulted in a recovery rate of approximately 55%, with protein purity greater than 95%. The purified rhBMP-7 was further demonstrated to be functionally active by measuring the proliferation of MC3T3-E1 cells, revealing a half-maximal effective concentration of 28.31 ng/ml.

Functional Diversification of Motor Neuron-specific Isl1 Enhancers during Evolution.

Functional diversification of motor neurons has occurred in order to selectively control the movements of different body parts including head, trunk and limbs. Here we report that transcription of Isl1, a major gene necessary for motor neuron identity, is controlled by two enhancers, CREST1 (E1) and CREST2 (E2) that allow selective gene expression of Isl1 in motor neurons. Introduction of GFP reporters into the chick neural tube revealed that E1 is active in hindbrain motor neurons and spinal cord motor neurons, whereas E2 is active in the lateral motor column (LMC) of the spinal cord, which controls the limb muscles. Genome-wide ChIP-Seq analysis combined with reporter assays showed that Phox2 and the Isl1-Lhx3 complex bind to E1 and drive hindbrain and spinal cord-specific expression of Isl1, respectively. Interestingly, Lhx3 alone was sufficient to activate E1, and this may contribute to the initiation of Isl1 expression when progenitors have just developed into motor neurons. E2 was induced by onecut 1 (OC-1) factor that permits Isl1 expression in LMCm neurons. Interestingly, the core region of E1 has been conserved in evolution, even in the lamprey, a jawless vertebrate with primitive motor neurons. All E1 sequences from lamprey to mouse responded equally well to Phox2a and the Isl1-Lhx3 complex. Conversely, E2, the enhancer for limb-innervating motor neurons, was only found in tetrapod animals. This suggests that evolutionarily-conserved enhancers permit the diversification of motor neurons.

MicroRNA library screening identifies growth-suppressive microRNAs that regulate genes involved in cell cycle progression and apoptosis.

Micro(mi)RNAs play important and varied roles in tumorigenesis; however, the full repertoire of miRNAs that affect cancer cell growth is not known. In this study, an miRNA library was screened to identify those that affect the growth of A549 tumor cells. Among 300 miRNAs, miR-28-5p, -323-5p, -510-5p, -552-3p, and -608 were the most effective in inhibiting cell growth. More specifically, overexpressing miR-28-5p, -323-5p, and -510-5p induced G1 arrest, as determined by flow cytometry, whereas that of miR-608 induced cell death in a caspase-dependent manner. Moreover, several genes involved in apoptosis and cell cycle progression were downregulated upon overexpression of each of the five miRNAs, with the functional targets of miR-552-3p and miR-608 confirmed by microarray, quantitative real-time PCR, and luciferase reporter assay. In miR-608-transfected cells, B cell lymphoma 2-like 1 (BCL2L1), D-type cyclin 1 (CCND1), CCND3, cytochrome b5 reductase 3 (CYB5R3), phosphoinositide 3-kinase regulatory subunit 2 (PIK3R2), specificity protein 1 (SP1), and phosphorylated Akt were all downregulated, while Bcl-2-interacting killer (BIK) was upregulated. Moreover, miR-608 was determined to have a suppressive function on tumor growth in an NCI-H460 xenograft model. These findings provide insights into the roles of five miRNAs in growth inhibition and their potential function as cancer therapeutics.