Expression Of Lmx1a Research Articles

Forced LMX1A expression induces dorsal neural fates and disrupts patterning of human embryonic stem cells into ventral midbrain dopaminergic neurons

The differentiation of human pluripotent stem cells into ventral mesencephalic dopaminergic (DA) fate is relevant for the treatment of Parkinson's disease. Shortcuts to obtaining DA cells through direct reprogramming often include forced expression of the transcription factor LMX1A. Although reprogramming with LMX1A can generate tyrosine hydroxylase (TH)-positive cells, their regional identity remains elusive. Using an invitro model of early human neural tube patterning, we report that forced LMX1A expression induced a ventral-to-dorsal fate shift along the entire neuroaxis with the emergence of roof plate fates despite the presence of ventralizing molecules. The LMX1A-expressing progenitors gave rise to grafts containing roof plate-derived choroid plexus cysts as well as ectopically induced TH-positive neurons of a forebrain identity. Early activation of LMX1A prior to floor plate specification was necessary for the dorsalizing effect. Our work suggests using caution in employing LMX1A for the induction of DA fate, as this factor may generate roof plate rather than midbrain fates.

Recombinant Human Interferon α-2b Nanoparticles in the Treatment of Human Papillomavirus Infection Model and Detection of LMX 1A Expression in Cervical Exhumation Cells

Interferon (INF) is a kind of multifunctional and highly active protein-like cytokines produced by animal cells after stimulation, which exerts the broad-spectrum antiviral, anti-tumor, and immunomodulatory effects. Preparing the nanoparticles (NPs) loaded drug can improve the stability of protein polypeptides in vivo, prolong the biological half-life period, and improve the bioavailability. In this research, recombinant human interferon (RHUINF) α-2b NPs were prepared to explore their therapeutic effect on human papillomavirus (HPV) infection model. Firstly, the RHUINF α-2b poly (lactic acid-glycolic acid) copolymer (PLGA) of composite bioactive glass was prepared by electrostatic spray technology. The in vitro quality, stability, and pharmacokinetic characteristics in vivo of the NPs were measured. Then, a mouse model infected with HPV16 pseudovirus infection (NC group) was prepared. Next, they were treated by painting imiquimod (IMQ) (IMQ group), intramuscularly injecting the RHUINF α-2b (Intron A), intramuscularly injecting the RHUINF α-2b-Plga NP suspension (Intra/PLGA), and intramuscularly injecting the RHUINF α-2B-bioactive glass-PLGA NP suspension (Intron A/BAG/PLGA), respectively. In addition, the differences in serum inflammatory cytokines, vaginal biofluorescence activity (VBA), HPV load, and LMX 1A expressions in cervical exhumation cells (CECs) were analyzed. The average particle sizes (PSs) of Intron A/PLGA and Intron A/BAG/PLGA NPs were 352.3 nm and 385.1 nm, respectively, and no great differences were observed in encapsulation rate (ER), drug loading (DL), and in vivo release (IVR). The maximum peak times (MPTs) of Intron A, Intron A/PLGA, and Intron A/BAG/PLGA were 1.3 h, 6.2 h, and 6.5 h, respectively, and their maintenance durations were 5.5 h, 120.7 h, and 245.6 h, respectively. Based on the NC group, IFN-γ, IL-2, and TNF-α in the IMQ group, Intron A group, Intron A/PLGA group, Intron A/PLGA group, and Intron A/BAG/PLGA group were decreased, VBA and HPV load were decreased, and mRNA of LMX 1A in ECEs was increased (p < 0.05). Based on the Intron A group, the Intron A/PLGA and Intron A/BAG/PLGA groups exhibited decreased serum inflammatory factors (IFFs), VBA, HPV load, and LMX 1A mRNA in the CECs were increased (p < 0.05). By taking the Intron A/PLGA group as reference, the Intron A/BAG/PLGA group showed greatly reduced serum IFFs, VBA, HPV load, and LMX 1A mRNA expression in CECs (p<0.05). Preparing RHUINF α-2b NPs could prolong the half-life period of RHUINF α-2b in vivo, reduce the inflammatory response and load of HPV-DNA in HPV infection models, and upregulate LMX 1A in CECs. In addition, RHUINF α-2b NPs could improve the therapeutic effect of RHUINF α-2b.

NOTCH1 signaling regulates the latent neurogenic program in adult reactive astrocytes after spinal cord injury.

Background: Direct reprogramming of astrocytes into neurons opens up a new avenue for neuroregenerative medicine. However, the poor understanding of the molecular mechanisms underpinning the latent neurogenic program in astrocytes has largely restricted this strategy towards safe and effective clinical therapies.Methods: Immunocytochemistry, immunohistochemistry, western blotting, qRT-PCR, gene knockdown and fate-mapping are performed to analyze the role of NOTCH1 signaling in regulation of the latent neurogenic program in reactive astrocytes after spinal cord injury.Results: Western blotting analysis highlights that NOTCH1 is a key signaling mediating Ascl1- and Neurog2-driven astrocyte-to-neuron conversion. Inhibition of NOTCH1 signaling in cultured astrocytes by shRNA or DAPT (a NOTCH1 inhibitor) is sufficient to reprogram them into neurons by upregulating the expression of pro-neural transcription factors, including NeuroD1, NeuroD2, Pax6, Lmx1a and Lhx6. In the spinal cord of adult mouse, the expression of Notch1 is detected in resident astrocytes, which was significantly increased after spinal cord injury (SCI). Genetical knockdown of NOTCH1 signaling alone successfully triggers endogenous reactive astrocytes reprogramming into neurons in the injured adult spinal cord. Importantly, pharmacologically blocking NOTCH1 signaling with small molecule DAPT alone can also induce in situ astrocyte-to-neuron conversion after SCI.Conclusions: We identify NOTCH1 as a key common signaling pathway in reactive astrocyte that provides a barrier for cell fate conversion. This proof-of-principle study will significantly expand our molecular understanding of astroglial-lineage reprogramming and overcoming the NOTCH1 gatekeeper with small molecules may provide a transgene-free approach for in vivo chemical neuronal reprogramming with potential clinical application in neuroregeneration.

Caveolin-1 downregulation promotes the dopaminergic neuron-like differentiation of human adipose-derived mesenchymal stem cells.

Previous studies have shown that caveolin-1 is involved in regulating the differentiation of mesenchymal stem cells. However, its role in the differentiation of human adipose mesenchymal stem cells into dopaminergic neurons remains unclear. The aim of this study was to investigate whether caveolin-1 regulates the differentiation of human adipose mesenchymal stem cells into dopaminergic-like neurons. We also examined whether the expression of caveolin-1 could be modulated by RNA interference technology to promote the differentiation of human adipose mesenchymal stem cells into dopaminergic-like neurons. The differentiation of human adipose mesenchymal stem cells into dopaminergic neurons was evaluated morphologically and by examining expression of the markers tyrosine hydroxylase, Lmx1a and Nurr1. The analyses revealed that during the differentiation of human adipose mesenchymal stem cells into dopaminergic neurons, the expression of caveolin-1 is decreased. Notably, the downregulation of caveolin-1 promoted the differentiation of human adipose mesenchymal stem cells into dopaminergic-like neurons, and it increased the expression of tyrosine hydroxylase, Lmx1a and Nurr1. Together, our findings suggest that caveolin-1 plays a negative regulatory role in the differentiation of dopaminergic-like neurons from stem cells, and it may therefore be a potential molecular target for strategies for regulating the differentiation of these cells. This study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Dalian Medical University of China (approval No. PJ-KS-KY-2020-54) on March 7, 2017.

Epigenetic Silencing of LMX1A Contributes to Cancer Progression in Lung Cancer Cells.

Epigenetic modification is considered a major mechanism of the inactivation of tumor suppressor genes that finally contributes to carcinogenesis. LIM homeobox transcription factor 1α (LMX1A) is one of the LIM-homeobox-containing genes that is a critical regulator of growth and differentiation. Recently, LMX1A was shown to be hypermethylated and functioned as a tumor suppressor in cervical cancer, ovarian cancer, and gastric cancer. However, its role in lung cancer has not yet been clarified. In this study, we used public databases, methylation-specific PCR (MSP), reverse transcription PCR (RT-PCR), and bisulfite genomic sequencing to show that LMX1A was downregulated or silenced due to promoter hypermethylation in lung cancers. Treatment of lung cancer cells with the demethylating agent 5-aza-2’-deoxycytidine restored LMX1A expression. In the lung cancer cell lines H23 and H1299, overexpression of LMX1A did not affect cell proliferation but suppressed colony formation and invasion. These suppressive effects were reversed after inhibition of LMX1A expression in an inducible expression system in H23 cells. The quantitative RT-PCR (qRT-PCR) data showed that LMX1A could modulate epithelial mesenchymal transition (EMT) through E-cadherin (CDH1) and fibronectin (FN1). NanoString gene expression analysis revealed that all aberrantly expressed genes were associated with processes related to cancer progression, including angiogenesis, extracellular matrix (ECM) remodeling, EMT, cancer metastasis, and hypoxia-related gene expression. Taken together, these data demonstrated that LMX1A is inactivated through promoter hypermethylation and functions as a tumor suppressor. Furthermore, LMX1A inhibits non-small cell lung cancer (NSCLC) cell invasion partly through modulation of EMT, angiogenesis, and ECM remodeling.

Single-Cell RNA Analysis of Type I Spiral Ganglion Neurons Reveals a Lmx1a Population in the Cochlea.

In the mature cochlea, each inner hair cell (IHC) is innervated by multiple spiral ganglion neurons of type I (SGNI). SGNIs are morphologically and electro-physiologically diverse. Also, they differ in their susceptibility to noise insult. However, the molecular underpinnings of their identity and physiological differences remain poorly understood. In this study, we developed a novel triple transgenic mouse, which enabled the isolation of pure populations of SGNIs and the analysis of a 96-gene panel via single-cell qPCR. We found three distinct populations of Type I SGNs, which were marked by their exclusive expression of Lmx1a, Slc4a4, or Mfap4/Fzd2, respectively, at postnatal days P3, P8, and P12. Our data suggest that afferent SGN subtypes are established genetically before the onset of hearing and that the expression of key physiological markers, such as ion channels, is heterogeneous and may be underlying the heterogeneous firing proprieties of SGNIs.

Interaction with ectopic cochlear crista sensory epithelium disrupts basal cochlear sensory epithelium development in Lmx1a mutant mice.

The LIM homeodomain transcription factor Lmx1a shows a dynamic expression in the developing mouse ear that stabilizes in the non-sensory epithelium. Previous work showed that Lmx1a functional null mutants have an additional sensory hair cell patch in the posterior wall of a cochlear duct and have a mix of vestibular and cochlear hair cells in the basal cochlear sensory epithelium. In E13.5 mutants, Sox2-expressing posterior canal crista is continuous with an ectopic "crista sensory epithelium" located in the outer spiral sulcus of the basal cochlear duct. The medial margin of cochlear crista is in contact with the adjacent Sox2-expressing basal cochlear sensory epithelium. By E17.5, this contact has been interrupted by the formation of an intervening non-sensory epithelium, and Atoh1 is expressed in the hair cells of both the cochlear crista and the basal cochlear sensory epithelium. Where cochlear crista was formerly associated with the basal cochlear sensory epithelium, the basal cochlear sensory epithelium lacks an outer hair cell band, and gaps are present in its associated Bmp4 expression. Further apically, where cochlear crista was never present, the cochlear sensory epithelium forms a poorly ordered but complete organ of Corti. We propose that the core prosensory posterior crista is enlarged in the mutant when the absence of Lmx1a expression allows JAG1-NOTCH signaling to propagate into the adjacent epithelium and down the posterior wall of the cochlear duct. We suggest that the cochlear crista propagates in the mutant outer spiral sulcus because it expresses Lmo4 in the absence of Lmx1a.

LncRNA KCNQ1OT1 regulates microRNA-9-LMX1A expression and inhibits gastric cancer cell progression.

LMX1A (LIM homeobox transcription factor 1α) is a tumor suppressor protein. Our previous study has shown that microRNA-9 (“miR-9”), being upregulated in human gastric cancer (GC), targets LMX1A to promote GC cell progression. Through searching long non-coding RNA (LncRNA) database, we identified that LncRNA KCNQ1OT1 is the competing endogenous RNA (ceRNA) of miR-9. KCNQ1OT1 putatively targets miR-9. Its level is downregulated in human GC tissues. In AGS cells and primary human GC cells, forced overexpression of KCNQ1OT1, by a lentiviral construct, induced miR-9 downregulation and LMX1A upregulation. Furthermore, KCNQ1OT1 overexpression inhibited GC cell survival, proliferation, migration and invasion, but inducing apoptosis activation. Contrarily, KCNQ1OT1 silencing, by targeted siRNAs, induced miR-9 accumulation and LMX1A downregulation. Consequently, GC cell proliferation, migration and invasion were enhanced. Importantly, KCNQ1OT1 overexpression or silencing was ineffective in LMX1A knockout AGC cells. Taken together, KCNQ1OT1 inhibits GC cell progression via regulating miR-9 and LMX1A expression.

Circular RNA circMTO1 Suppresses RCC Cancer Cell Progression via miR9/LMX1A Axis.

Renal cell carcinoma is one of the most common kidney cancer, which accounts almost 90% of the adult renal malignancies worldwide. In recent years, a new class of endogenous noncoding RNAs, circular RNAs, exert important roles in cell function and certain types of pathological responses, especially in cancers, generally by acting as a microRNA sponge. Circular RNAs could act as sponge to regulate the microRNA and the target genes. However, the knowledge about circular RNAs in renal cell carcinoma remains unclear so far. In the research, we selected a highly expressed novel circular RNAs named circMTO1 in renal cell carcinomas. We investigated the roles of circMTO1 and found that circMTO1 overexpression could suppress cell proliferation and metastases in both A497 and 786-O renal cancer cells, while silencing of circMTO1 could promote the progression in SN12C and OS-RC-2 renal cancer cells. The study showed that circMTO1 acted as miR9 and miR223 sponge and inhibited their levels. Furthermore, silencing of circMTO1 in renal cell carcinoma could downregulate LMX1A, the target of miR-9, resulting in the promotion of renal cell carcinoma cell proliferation and invasion. In addition, LMX1A expression suppression induced by transfection of miR9 mimics confirmed that miR9 exerted its function in renal cell carcinoma by regulating LMX1A expression. What’s more, miR9 inhibitor and LMX1A overexpression could block the tumor-promoting effect of circMTO1 silencing. In conclusion, circMTO1 suppresses renal cell carcinoma progression by circMTO1/miR9/ LMX1A, indicating that circMTO1 may be a potential target in renal cell carcinoma therapy.

Pathogenic Pathways in Early-Onset Autosomal Recessive Parkinson's Disease Discovered Using Isogenic Human Dopaminergic Neurons.

SummaryParkinson's disease (PD) is a complex and highly variable neurodegenerative disease. Familial PD is caused by mutations in several genes with diverse and mostly unknown functions. It is unclear how dysregulation of these genes results in the relatively selective death of nigral dopaminergic neurons (DNs). To address this question, we modeled PD by knocking out the PD genes PARKIN (PRKN), DJ-1 (PARK7), and ATP13A2 (PARK9) in independent isogenic human pluripotent stem cell (hPSC) lines. We found increased levels of oxidative stress in all PD lines. Increased death of DNs upon differentiation was found only in the PARKIN knockout line. Using quantitative proteomics, we observed dysregulation of mitochondrial and lysosomal function in all of the lines, as well as common and distinct molecular defects caused by the different PD genes. Our results suggest that precise delineation of PD subtypes will require evaluation of molecular and clinical data.

The Substantia Nigra Is Permissive and Gains Inductive Signals When Lesioned for Dopaminergic Differentiation of Embryonic Stem Cells

Transplantation of dopaminergic (DA) cells into the striatum can rescue from dopamine deficiency in a Parkinson's disease condition, but this is not a suitable procedure for regaining the full control of motor activity. The minimal condition toward recovering the nigrostriatal pathway is the proper innervation of transplanted DA neurons or their precursors from the substancia nigra pars compacta (SNpc) to their target areas. However, functional integration of transplanted cells would require first that the host SNpc is suitable for their survival and/or differentiation. We recently reported that the intact adult SNpc holds a strong neurogenic environment, but primed embryonic stem cells (ie, embryoid body cells, EBCs) could not derive into DA neurons. In this study, we transplanted into the intact or lesioned SNpc, EBCs derived from embryonic stem cells that were prompt to differentiate into DA neurons by the forced expression of Lmx1a in neural precursor cells (R1B5/NesE-Lmx1a). We observed that, 6 days posttransplantation (dpt), R1B5 or R1B5/NesE-Lmx1a EBCs gave rise to Nes+ and Dcx+ cells within the host SNpc, but a large number of Th+ cells derived only from EBCs exogenously expressing Lmx1a. In contrast, when transplantation was carried out into the 6-hydroxidopamine-lesioned SNpc, the emergence of Th+ cells from EBCs was independent of exogenous Lmx1a expression, although these cells were not found by 15 dpt. These results suggest that the adult SNpc is not only a permissive niche for initiation of DA differentiation of non-neuralized cells but also releases factors upon damage that promote the acquisition of DA characteristics by transplanted EBCs.

Maternal Vitamin D Prevents Abnormal Dopaminergic Development and Function in a Mouse Model of Prenatal Immune Activation

Dysfunction in dopamine (DA) systems is a prominent feature in schizophrenia patients and may result from the abnormal development of mesencephalic (mes)DA systems. Maternal immune activation (MIA) and developmental vitamin D (DVD)-deficiency both induce schizophrenia-relevant dopaminergic abnormalities in adult offspring. In this study, we investigated whether maternal administration of the vitamin D hormone (1,25OHD, VITD) could prevent MIA-induced abnormalities in DA-related behaviors and mesDA development. We administrated the viral mimetic polyriboinosinic-polyribocytidylic (poly (I:C)) simultaneously with 1,25OHD and/or their vehicles, to pregnant mouse dams at gestational day 9. Maternal treatment with VITD prevented MIA-induced hypersensitivity to acute DA stimulation induced by amphetamine, whereas it failed to block prepulse inhibition deficiency in MIA-exposed offspring. MIA and VITD both reduced fetal mesDA progenitor (Lmx1a + Sox2+) cells, while VITD treatment increased the number of mature (Nurr1 + TH+) mesDA neurons. Single-cell quantification of protein expression showed that VITD treatment increased the expression of Lmx1a, Nurr1 and TH in individual mesDA cells and restored normal mesDA positioning. Our data demonstrate that VITD prevents abnormal dopaminergic phenotypes in MIA offspring possibly via its early neuroprotective actions on fetal mesDA neurons. Maternal supplementation with the dietary form of vitamin D, cholecalciferol may become a valuable strategy for the prevention of MIA-induced neurodevelopmental abnormalities.

Tcf12 Is Involved in Early Cell-Fate Determination and Subset Specification of Midbrain Dopamine Neurons.

The basic helix-loop-helix (bHLH) protein family has previously been shown to be involved in the development of mesodiencephalic dopaminergic (mdDA) neurons in the murine midbrain. Specifically, Ngn2 and Mash1 are known to have a role in the specification of neural progenitors in the ventricular zone (VZ) of the midbrain towards an mdDA neuronal cell-fate. Furthermore, other members of the bHLH protein family, the E-box factors, are expressed in the developing midbrain and are thought to have a role in neuronal differentiation. Here we show that the E-box factor Tcf12 is implicated in early and late development of mdDA neurons. Tcf12 is expressed in the midbrain and in young TH-expressing mdDA neurons throughout development. Tcf12lox/lox;En1cre/+ embryos, that lose Tcf12 at ~embryonic day (E)9 throughout the En1 expression domain, have a changed spatial expression of Lmx1a and Nurr1 and a consistent loss of rostral TH expression. Expression of the subset marker Ahd2 is initially delayed, but recovers during development, eventually showing an ~10% increase in AHD2-expressing cells at postnatal day (P)30. Tcf12lox/lox;Pitx3cre/+ embryos, that lose Tcf12 at ~E12 in post-mitotic mdDA neurons, show no effect on the amount of TH-expressing neurons in the developing midbrain. However, similar as to Tcf12lox/lox;En1cre/+ embryos, subset specification is delayed during development. Taken together, we have identified Tcf12 as a novel factor in mdDA neuronal development. It serves a dual function; one in early cell-fate commitment of neural progenitors and one late in subset specification.

Lmx1a and Lmx1b regulate mitochondrial functions and survival of adult midbrain dopaminergic neurons

The LIM-homeodomain transcription factors Lmx1a and Lmx1b play critical roles during the development of midbrain dopaminergic progenitors, but their functions in the adult brain remain poorly understood. We show here that sustained expression of Lmx1a and Lmx1b is required for the survival of adult midbrain dopaminergic neurons. Strikingly, inactivation of Lmx1a and Lmx1b recreates cellular features observed in Parkinson's disease. We found that Lmx1a/b control the expression of key genes involved in mitochondrial functions, and their ablation results in impaired respiratory chain activity, increased oxidative stress, and mitochondrial DNA damage. Lmx1a/b deficiency caused axonal pathology characterized by α-synuclein(+) inclusions, followed by a progressive loss of dopaminergic neurons. These results reveal the key role of these transcription factors beyond the early developmental stages and provide mechanistic links between mitochondrial dysfunctions, α-synuclein aggregation, and the survival of dopaminergic neurons.

Dickkopf 3 Promotes the Differentiation of a Rostrolateral Midbrain Dopaminergic Neuronal Subset In Vivo and from Pluripotent Stem Cells In Vitro in the Mouse.

Wingless-related MMTV integration site 1 (WNT1)/β-catenin signaling plays a crucial role in the generation of mesodiencephalic dopaminergic (mdDA) neurons, including the substantia nigra pars compacta (SNc) subpopulation that preferentially degenerates in Parkinson's disease (PD). However, the precise functions of WNT1/β-catenin signaling in this context remain unknown. Stem cell-based regenerative (transplantation) therapies for PD have not been implemented widely in the clinical context, among other reasons because of the heterogeneity and incomplete differentiation of the transplanted cells. This might result in tumor formation and poor integration of the transplanted cells into the dopaminergic circuitry of the brain. Dickkopf 3 (DKK3) is a secreted glycoprotein implicated in the modulation of WNT/β-catenin signaling. Using mutant mice, primary ventral midbrain cells, and pluripotent stem cells, we show that DKK3 is necessary and sufficient for the correct differentiation of a rostrolateral mdDA neuron subset. Dkk3 transcription in the murine ventral midbrain coincides with the onset of mdDA neurogenesis and is required for the activation and/or maintenance of LMX1A (LIM homeobox transcription factor 1α) and PITX3 (paired-like homeodomain transcription factor 3) expression in the corresponding mdDA precursor subset, without affecting the proliferation or specification of their progenitors. Notably, the treatment of differentiating pluripotent stem cells with recombinant DKK3 and WNT1 proteins also increases the proportion of mdDA neurons with molecular SNc DA cell characteristics in these cultures. The specific effects of DKK3 on the differentiation of rostrolateral mdDA neurons in the murine ventral midbrain, together with its known prosurvival and anti-tumorigenic properties, make it a good candidate for the improvement of regenerative and neuroprotective strategies in the treatment of PD. Significance statement: We show here that Dickkopf 3 (DKK3), a secreted modulator of WNT (Wingless-related MMTV integration site)/β-catenin signaling, is both necessary and sufficient for the proper differentiation and survival of a rostrolateral (parabrachial pigmented nucleus and dorsomedial substantia nigra pars compacta) mesodiencephalic dopaminergic neuron subset, using Dkk3 mutant mice and murine primary ventral midbrain and pluripotent stem cells. The progressive loss of these dopamine-producing mesodiencephalic neurons is a hallmark of human Parkinson's disease, which can up to now not be halted by clinical treatments of this disease. Thus, the soluble DKK3 protein might be a promising new agent for the improvement of current protocols for the directed differentiation of pluripotent and multipotent stem cells into mesodiencephalic dopaminergic neurons and for the promotion of their survival in situ.

Islet1 and its co-factor Ldb1 are expressed in quiescent cells of mouse intestinal epithelium.

Islet1 belongs to Lim homeobox (Lhx) gene family which encodes transcription factors that have been conserved in evolution. They form complexes with other transcriptional regulators, among them obligatory co-factors encoded by Ldb genes. Isl1 (Islet1), Lhx and Ldb1 genes play a crucial role in organ patterning, cell fate determination and cell differentiation in both embryonic and adult tissues. In this study we analyzed expression pattern of Isl1 and its co-factor Ldb1 in small intestine. We also studied the biological role of Ldb1 in gut endoderm. Quantitative PCR analysis revealed a relatively high level of expression of Lhx1, Isl1, Isl2, Lmx1a, Ldb1 and Ldb2 mRNAs in the gut tissue as compared to the level of less abundant detectable Lmx1b mRNA. Immunohistochemical studies demonstrated a unique pattern of Ldb1 and Islet1 proteins in the crypt compartment. Ldb1 is produced at a low level in majority of crypt cells; but, its abundant expression was demonstrated for some single cells. Islet1 is also expressed in single cells of the crypt. Double staining experiments with Ldb1 and Isl1 antibodies showed that both genes are co-expressed in certain cells of the crypt. Further analysis revealed the Ldb1-expressing cells in the gut are both of endodermal and mesodermal origin. Proliferation studies using antibodies to phospho-histone H3 and Ki-67 antigens, as well as long-term BrdU labeling, showed that cells prominently expressing Ldb1/Islet1 are quiescent but do not belong to any known terminally differentiated cell lineages. They may represent a group of stem-like cells in the crypt. Further experiments by cell lineage tracing should be performed to better characterize this cell population. Functional studies of mice with Ldb1 gene ablated in gut endoderm revealed no specific role of Ldb1 in that tissue.

Engineering of Midbrain Organoids Containing Long-Lived Dopaminergic Neurons

The possibility to generate dopaminergic (DA) neurons from pluripotent stem cells represents an unlimited source of material for tissue engineering and cell therapy for neurodegenerative disease. We set up a protocol based on the generation of size-calibrated neurospheres for a rapid production (3 weeks) of a high amount of DA neurons (>60%) oriented toward a midbrain-like phenotype, characterized by the expression of FOXA2, LMX1A, tyrosine hydroxylase (TH), NURR1, and EN1. By using γ-secretase inhibitors and varying culture time of neurospheres, we controlled maturation and cellular composition of a three-dimensional (3D) engineered nervous tissue (ENT). ENT contained neurons and glial cells expressing various markers of maturity, such as synaptophysin, neuronal nuclei-specific protein (NeuN), and glial fibrillary acidic protein (GFAP), and were electrophysiologically active. We found that 3-week-old neurospheres were optimal to generate 3D tissue containing DA neurons with typical A9 morphology. ENT generated from 4-week-old neurospheres launched glial cell type since astrocytes and myelin could be detected massively at the expense of TH-immunoreactive neurons. All γ-secretase inhibitors were not equivalent; compound E was more efficient than DAPT in generating DA neurons. This DA tissue provides a tool for drug screening, and toxicology. It should also become a useful biomaterial for studies on Parkinson's disease.

DNA-Dependent Protein Kinase Is a Context Dependent Regulator of Lmx1a and Midbrain Specification

The identification of small molecules capable of directing pluripotent cell differentiation towards specific lineages is highly desirable to both reduce cost, and increase efficiency. Within neural progenitors, LIM homeobox transcription factor 1 alpha (Lmx1a) is required for proper development of roof plate and cortical hem structures of the forebrain, as well as the development of floor plate and midbrain dopaminergic neurons. In this study we generated homologous recombinant cell lines expressing either luciferase or β-lactamase under the control of the Lmx1a promoter, and used these cell lines to investigate kinase-mediated regulation of Lmx1a activity during neuronal differentiation. A screen of 143 small molecule tyrosine kinase inhibitors yielded 16 compounds that positively or negatively modulated Lmx1a activity. Inhibition of EGF, VEGF and DNA-dependent protein kinase (DNA-PK) signaling significantly upregulated Lmx1a activity whereas MEK inhibition strongly downregulated its activity. Quantitative FACS analysis revealed that the DNA-PK inhibitor significantly increased the number of Lmx1a+ progenitors while subsequent qPCR showed an upregulation of Notch effectors, the basic helix-loop-helix genes, Hes5 and Hey1. FACS further revealed that DNA-PK-mediated regulation of Lmx1a+ cells is dependent on the rapamycin-sensitive complex, mTORC1. Interestingly, this DNA-PK inhibitor effect was preserved in a co-culture differentiation protocol. Terminal differentiation assays showed that DNA-PK inhibition shifted development of neurons from forebrain toward midbrain character as assessed by Pitx3/TH immunolabeling and corresponding upregulation of midbrain (En1), but not forebrain (FoxG1) transcripts. These studies show that Lmx1a signaling in mouse embryonic stem cells contributes to a molecular cascade establishing neuronal specification. The data presented here identifies a novel regulatory pathway where signaling from DNA-PK appears to suppress midbrain-specific Lmx1a expression.

The Correlations of LMX1A and Osteopontin Expression to the Clinicopathologic Stages in Pancreatic Adenocarcinoma

To test the association of LMX1A and osteopontin (OPN) expression with histologic differentiation or pathologic stage in pancreatic ductal adenocarcinoma. Immunohistochemical analysis was performed to determine LMX1A and OPN expression in 100 surgical specimens obtained from Chinese patients with well-differentiated (n=15), moderately differentiated (n=65), and poorly differentiated (n=20) pancreatic ductal adenocarcinomas. LMX1A and OPN immunoreactivities were undetectable in normal pancreatic glandular epithelia. Stronger immunostaining for LMX1A and OPN was associated with advanced nuclear grades of pancreatic ductal adenocarcinomas (70.7 and 87.1 for grade I, 109.8 and 118.3 for grade II, and 171.3 and 183.8 for grade III), and advanced TNM and American Joint Committee on Cancer stages of pancreatic ductal adenocarcinomas. A higher expression of LMX1A and OPN is well correlated with histologic grade and pathologic stage of pancreatic ductal adenocarcinomas.

The role of Sp1 and EZH2 in the regulation of LMX1A in cervical cancer cells

We have reported previously that LIM homeobox transcription factor 1α (LMX1A) is hypermethylated and functions as a metastasis suppressor in cervical cancer cells. However, the regulation of LMX1A in carcinogenesis has not been reported. We aim to clarify whether specificity protein 1 (Sp1) and enhancer of zeste homolog 2 (EZH2) are involved in the regulation of LMX1A in cervical cancer. First we characterized the LMX1A promoter and used overexpression, knockdown, and reporter assays to show that Sp1 increased LMX1A promoter activity. Next, we used site-directed mutagenesis and electrophoresis mobility shift assays (EMSAs) to demonstrate that Sp1-binding sites were important for Sp1-mediated activation of the LMX1A promoter. Chromatin immunoprecipitation data demonstrated that Sp1 could bind directly to the LMX1A promoter and activate endogenous LMX1A expression in cells pretreated with 5-aza-2′-deoxycytidine (5-aza-dC). Knockdown of EZH2 decreased H3K27me3 histone modification but was insufficient to restore LMX1A expression. To explore the effect of EZH2 on the endogenous LMX1A promoter, we treated EZH2-knockdown cells with 5-aza-dC and trichostatin A (TSA) and then depleted the cells of drugs for 3days. H3K14ac was enriched at the LMX1A promoter in EZH2-knockdown cells and LMX1A mRNA was still expressed. Taken together, these data imply that Sp1 may activate LMX1A expression upon oncogenic stress during cervical cancer development. Moreover, suppression of EZH2 may delay resilencing of LMX1A after the removal of 5-aza-dC and TSA.