The role of FHL2 in wound healing and inflammation.

The 4-and-a-half LIM domain protein 2 (FHL2) is a multifunctional adaptor protein that can interact with cell surface receptors, cytosolic adaptor and structural proteins, kinases, and nuclear transcription factors. It is involved in numerous functional activities, including the epithelial-mesenchymal transition, cell proliferation, apoptosis, adhesion, migration, structural stability, and gene expression. Despite this, FHL2-knockout (KO) mice are viable and fertile with no obvious abnormalities, rather suggesting a high capacity for fine-tuning adjustment and functional redundancy of FHL2. Indeed, challenging FHL2-KO cells or mice provided numerous evidences for the great functional significance of FHL2. In recent years, several reviews have been published describing the high capacity of FHL2 to bind diverse proteins as well as the versatile functions of FHL2, emphasizing in particular its role in cardiovascular diseases and carcinogenesis. Here, we view the function of FHL2 from a different perspective. We summarize the published data demonstrating the impact of FHL2 on wound healing and inflammation. FHL2 seems to be involved in numerous steps of these extremely complex and multidirectional but tightly regulated tissue remodeling processes, supporting tissue repair and coordinating inflammation. Deficiency of FHL2 not only slows down ongoing wound healing but also often turns it into a chronic condition.-Wixler, V. The role of FHL2 in wound healing and inflammation.

ISL1 predicts poor outcomes for patients with gastric cancer and drives tumor progression through binding to the ZEB1 promoter together with SETD7.

ISL1, a LIM-homeodomain transcription factor, serves as a biomarker of metastasis in multiple tumors. However, the function and underlying mechanisms of ISL1 in gastric cancer (GC) have not been fully elucidated. Here we found that ISL1 was frequently overexpressed in GC FFPE samples (104/196, 53.06%), and associated with worse clinical outcomes. Furthermore, the overexpression of ISL1 and loss-of-function of ISL1 influenced cell proliferation, invasion and migration in vitro and in vivo, including GC patient-derived xenograft models. We used ChIP-seq and RNA-seq to identify that ISL1 influenced the regulation of H3K4 methylation and bound to ZEB1, a key regulator of the epithelial-mesenchymal transition (EMT). Meanwhile, we validated ISL1 as activating ZEB1 promoter through influencing H3K4me3. We confirmed that a complex between ISL1 and SETD7 (a histone H3K4-specific methyltransferase) can directly bind to the ZEB1 promoter to activate its expression in GC cells by immunoprecipitation, mass spectrometry, and ChIP-re-ChIP. Moreover, ZEB1 expression was significantly positively correlated with ISL1 and was positively associated with a worse outcome in primary GC specimens. Our paper uncovers a molecular mechanism of ISL1 promoting metastasis of GC through binding to the ZEB1 promoter together with co-factor SETD7. ISL1 might be a potential prognostic biomarker of GC.

CTCF Governs the Identity and Migration of MGE-Derived Cortical Interneurons.

The CCCTC-binding factor (CTCF) is a central regulator of chromatin topology recently linked to neurodevelopmental disorders such as intellectual disability, autism, and schizophrenia. The aim of this study was to identify novel roles of CTCF in the developing mouse brain. We provide evidence that CTCF is required for the expression of the LIM homeodomain factor LHX6 involved in fate determination of cortical interneurons (CINs) that originate in the medial ganglionic eminence (MGE). Conditional Ctcf ablation in the MGE of mice of either sex leads to delayed tangential migration, abnormal distribution of CIN in the neocortex, a marked reduction of CINs expressing parvalbumin and somatostatin (Sst), and an increased number of MGE-derived cells expressing Lhx8 and other markers of basal forebrain projection neurons. Likewise, Ctcf-null MGE cells transplanted into the cortex of wild-type hosts generate fewer Sst-expressing CINs and exhibit lamination defects that are efficiently rescued upon reexpression of LHX6. Collectively, these data indicate that CTCF regulates the dichotomy between Lhx6 and Lhx8 to achieve correct specification and migration of MGE-derived CINs.SIGNIFICANCE STATEMENT This work provides evidence that CCCTC-binding factor (CTCF) controls an early fate decision point in the generation of cortical interneurons mediated at least in part by Lhx6. Importantly, the abnormalities described could reflect early molecular and cellular events that contribute to human neurological disorders previously linked to CTCF, including schizophrenia, autism, and intellectual disability.

The expression of TTF1, CDX2 and ISL1 in 74 poorly differentiated neuroendocrine carcinomas.

BACKGROUND: The expression profile of immunohistochemical markers of origin in poorly differentiated neuroendocrine carcinoma (PDNEC) is not well studied. MATERIALS AND METHODS: Seventy-four PDNECs from gastroenteropancreatic (GEP) organs and the lung, including 48 large cell NEC (LCNEC) and 26 small cell carcinomas (SmCC), were subject to immunohistochemical staining for CDX2, TTF1 and ISL1. The staining intensity (1 to 3) and percentage of positive tumor cells [0 (negative), 1 (<50%) and 2 (≥50%)] were assessed. The multiplicative index (maximum 6) was calculated and the average total score (aTS) was determined for each primary site and histologic subtype. RESULTS: In the 38 GEP and 36 lung PDNECs, CDX2, TTF1 and ISL1 staining was observed in 71% (aTS 2.8), 16% (aTS 0.4), 63% (aTS 1.9), and 22% (aTS 0.6), 72% (aTS 2.9) and 92% (aTS 3.8), respectively. GEP PDNECs showed a higher aTS for CDX2 and lower aTS for TTF1 and ISL1, compared to that of lung PDNECs (Student's t-test, p < 0.001). SmCC had a higher aTS for TTF1 and ISL1 (p < 0.001) and lower aTS for CDX2 (p < 0.002) than that of LCNEC. CONCLUSIONS: CDX2 and TTF1 demonstrate potential utility in suggesting the primary site of PDNEC. In addition, CDX2 may be useful in supporting the diagnosis of LCNEC in cases with overlapping or borderline morphology. Utility of ISL1 as an adjunctive diagnostic marker of SmCC remains to be studied.

Functional prediction of miR-3144-5p in human cardiac myocytes based on transcriptome sequencing and bioinformatics.

BACKGROUND: RAN guanine nucleotide release factor (RANGRF) encoding protein MOG1 plays an important role in cardiac arrhythmia, so we intended to investigate the regulatory miRNA of RANGRF and explore its potential regulatory mechanism in arrhythmogenesis. METHODS: Based on bioinformatic analysis, miR-3144-5p was predicted to be a regulatory miRNA of RANGRF, which were then validated through a dual-luciferase reporter plasmid assay. Subsequently, the expression level of miR-3144-5p in human cardiac myocytes (HCMs) was detected, followed by cell transfection with miR-3144-5p mimics. Transcriptome sequencing was then performed in HCMs with or without transfection. The sequencing results were subjected to bioinformatic analyses, including differentially expressed gene (DEG) analysis, functional enrichment analysis, protein-protein interaction (PPI) network analysis, miRNA-target gene analysis, and miRNA-transcription factor (TF)-target gene coregulatory network analysis. RESULTS: There really existed a regulatory relation between miR-3144-5p and RANGRF. The expression level of miR-3144-5p was low in HCMs. After cell transfection, miR-3144-5p expression level significantly increased in HCMs. Bioinformatic analyses of the transcriptome sequencing results identified 300 upregulated and 271 downregulated DEGs between miR-3144-5p mimic and control group. The upregulated genes ISL1 and neuregulin 1 (NRG1) were significantly enriched in cardiac muscle cell myoblast differentiation (GO:0060379). CCL21 was one of the hub genes in the PPI network and also a target gene of miR-3144-5p. Moreover, the TF of v-Myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN) was involved in the miR-3144-5p-TF-target gene coregulatory network and interacted with the target genes of miR-3144-5p. CONCLUSION: ISL1, NRG1, CCL21, and MYCN were differentially expressed in the miR-3144-5p mimic group, suggesting that miR-3144-5p overexpression plays a role in HCMs by regulating these genes and TF. This study may provide new insight into the mechanisms behind the progression of cardiac arrhythmia.

ADAM-17/FHL2 colocalisation suggests interaction and role of these proteins in colorectal cancer.

FHL2 is a multifunctional scaffolding protein; its expression is associated with poor prognosis in colorectal cancer. ADAM-17 is a metalloprotease implicated in ectodomain shedding. FHL2 regulates ADAM-17 plasma membrane localisation, and FHL2 deficiency leads to decreased activity of ADAM-17 in mouse macrophages. Presence and relationship of the ADAM-17/FHL2 complex with colorectal cancer progression is unknown. We studied FHL2 and ADAM-17 expression in several colon cancer cell lines by immunocytochemistry and western blot. To highlight the interaction between both molecules, we used the Duolink® kit for proximity ligation assay on SW480 cells. We also performed proximity ligation assay on biopsies and surgical specimens of colorectal adenocarcinoma and on matched normal mucosa. Furthermore, biopsies of colorectal adenoma with matched normal mucosa were selected. For quantification, pictures of the malignant, adenomatous and normal tissues were taken. Proximity ligation assay signals were quantified. Mean numbers of proximity ligation assay signals and of proximity ligation assay signals/nucleus were calculated. All cell lines showed FHL2 immunoreactivity; strongest positivity was observed in SW480 cells. ADAM-17 was expressed in all cell lines. Proximity ligation assay signals were present in SW480 cells. Quantitative analysis revealed that the interaction between FHL2 and ADAM-17 is more frequent in malignant than in normal tissue (p = 0.005). The mean number of ADAM-17/FHL2 proximity ligation assay signals was higher in colorectal adenocarcinoma than in adenoma with low-grade dysplasia (p = 0.0004). FHL2 interacts with ADAM-17 in normal, dysplastic and malignant colon epithelial cells. Colocalisation of these proteins is more frequent in malignant than in normal and dysplastic cells, suggesting a role for ADAM-17/FHL2 complex in the development of colorectal cancer.

Zearalenone exposure impairs ovarian primordial follicle formation via down-regulation of Lhx8 expression in vitro.

Zearalenone (ZEA) is an estrogenic mycotoxin mainly produced as a secondary metabolite by numerous species of Fusarium. Previous work showed that ZEA had a negative impact on domestic animals with regard to reproduction. The adverse effects and the mechanisms of ZEA on mammalian ovarian folliculogenesis remain largely unknown, particularly its effect on primordial follicle formation. Thus, we investigated the biological effects of ZEA exposure on murine ovarian germ cell cyst breakdown and primordial follicle assembly. Our results demonstrated that newborn mouse ovaries exposed to 10 or 30µM ZEA in vitro had significantly less germ cell numbers compared to the control group. Moreover, the presence of ZEA in vitro increased the numbers of TUNEL and γH2AX positive cells within mouse ovaries and the ratio of mRNA levels of the apoptotic genes Bax/Bcl-2. Furthermore, ZEA exposure reduced the mRNA of oocyte specific genes such as LIM homeobox 8 (Lhx8), newborn ovary homeobox (Nobox), spermatogenesis and oogenesis helix-loop-helix (Sohlh2), and factor in the germline alpha (Figlα) in a dose dependent manner. Exposure to ZEA led to remarkable changes in the Lhx8 3'-UTR DNA methylation dynamics in oocytes and severely impaired folliculogenesis in ovaries after transplantation under the kidney capsules of immunodeficient mice. In conclusion, ZEA exposure impairs mouse primordial follicle formation in vitro.

Transformation of jaw muscle satellite cells to cardiomyocytes.

In the embryo a population of progenitor cells known as the second heart field forms not just parts of the heart but also the jaw muscles of the head. Here we show that it is possible to take skeletal muscle satellite cells from jaw muscles of the adult mouse and to direct their differentiation to become heart muscle cells (cardiomyocytes). This is done by exposing the cells to extracellular factors similar to those which heart progenitors would experience during normal embryonic development. By contrast, cardiac differentiation does not occur at all from satellite cells isolated from trunk and limb muscles, which originate from the somites of the embryo. The cardiomyocytes arising from jaw muscle satellite cells express a range of specific marker proteins, beat spontaneously, display long action potentials with appropriate responses to nifedipine, norepinephrine and carbachol, and show synchronized calcium transients. Our results show the existence of a persistent cardiac developmental competence in satellite cells of the adult jaw muscles, associated with their origin from the second heart field of the embryo, and suggest a possible method of obtaining cardiomyocytes from individual patients without the need for a heart biopsy.

Expression of SOX11, PAX5, TTF-1 and ISL-1 in medulloblastoma.

The aim of our study was to evaluate the immunohistochemical expression of SOX11, PAX5, TTF-1 and ISL-1 in medulloblastoma (MB) to investigate their diagnostic usefulness. METHODS: Immunohistochemical expression of PAX5 (two antibodies: Dako, DAK-Pax5; and BD, clone 24), TTF-1 (Dako, 8G7G3/1), SOX11 (CL0142; Abcam) and ISL-1 (1 H9, Abcam) was analyzed using the h-score and Remmele score in 25 cases of MB. RESULTS: There were 18 MBs of classic and 7 of desmoplastic type. SOX11 was strongly expressed in all tumors. The expression of PAX5 was higher and more frequent in a case of DAK-Pax5 clone (25/25) than clone 24 (6/25). ISL-1 was positive in 11 (44%) and TTF-1 in 3 (12%) cases. ISL-1 expression correlated positively (p<0.001), while TTF-1 correlated negatively with the age of patients (p=0.039). PAX5 expression correlated with ISL-1 (p=0.039) and showed a trend toward higher expression in the desmoplastic subtype (p=0.069). CONCLUSIONS: SOX11 is strongly and robustly expressed in MBs. PAX5 expression pattern differs substantially among two antibody clones. TTF-1 and ISL-1 is associated with the age of patients.

Expression pattern of ISL-1, TTF-1 and PAX5 in olfactory neuroblastoma.

Olfactory neuroblastoma (ONB) is a rare neoplasm of the sinonasal area with neuroendocrine differentiation. ISL-1, TTF-1 and PAX5 are transcription factors that are frequently upregulated in tumors showing neuroendocrine differentiation. The aim of our study was to evaluate these markers in a group of ONBs. We included 11 ONBs from 4 large university hospitals. Immunohistochemical expression of TTF-1, PAX5 and ISL-1 was evaluated. TTF-1, ISL-1 and PAX5 were expressed in 3/11 cases (27.27%, h-score: 3-45), 7/11 cases (63.64%, h-score: 23-200), and in 3/11 cases (27.77%, h-score 3-85), respectively. The patient with the strongest PAX5 reactivity exhibited an aggressive clinical course with rapid dissemination to the spine and death shortly after the diagnosis. No significant correlation in the expression of PAX5 and TTF-1 ( = 0.43; p = 0.18) was observed. ISL-1 is widely expressed in tumors with neuroendocrine differentiation and therefore of limited value in their differential diagnosis. TTF-1 positivity does not exclude the diagnosis of primary ONB, although usually only a small percentage of cells are positive. PAX5 expression is infrequent (27.27%) in ONB; however, if present it can be associated with a very aggressive clinical course.

Expression patterns of homeobox genes in the mouse vomeronasal organ at postnatal stages.

Homeodomain proteins are encoded by homeobox genes and regulate development and differentiation in many neuronal systems. The mouse vomeronasal organ (VNO) generates in situ mature chemosensory neurons from stem cells. The roles of homeodomain proteins in neuronal differentiation in the VNO are poorly understood. Here we have characterized the expression patterns of 28 homeobox genes in the VNO of C57BL/6 mice at postnatal stages using multicolor fluorescent in situ hybridization. We identified 11 homeobox genes (Dlx3, Dlx4, Emx2, Lhx2, Meis1, Pbx3, Pknox2, Pou6f1, Tshz2, Zhx1, Zhx3) that were expressed exclusively in neurons; 4 homeobox genes (Pax6, Six1, Tgif1, Zfhx3) that were expressed in all non-neuronal cell populations, with Pax6, Six1 and Tgif1 also expressed in some neuronal progenitors and precursors; 12 homeobox genes (Adnp, Cux1, Dlx5, Dlx6, Meis2, Pbx2, Pknox1, Pou2f1, Satb1, Tshz1, Tshz3, Zhx2) with expression in both neuronal and non-neuronal cell populations; and one homeobox gene (Hopx) that was exclusively expressed in the non-sensory epithelium. We studied further in detail the expression of Emx2, Lhx2, Meis1, and Meis2. We found that expression of Emx2 and Lhx2 initiated between neuronal progenitor and neuronal precursor stages. As far as the sensory neurons of the VNO are concerned, Meis1 and Meis2 were only expressed in the apical layer, together with Gnai2, but not in the basal layer.

LMX1A inhibits metastasis of gastric cancer cells through negative regulation of ß-catenin.

Previously, we reported that the LIM homeobox transcription factor 1, alpha (LMX1A) presented tumor-suppressing roles in gastric AGS cells. Here, we showed that LMX1A also inhibits metastasis-related behaviors including migration and invasion of gastric cancer cells. Mechanistic study revealed that the role of LMX1A was mediated by ß-catenin, as knockdown of LMX1A upregulated the expression of ß-catenin and knockdown of ß-catenin reversed the effects of LMX1A silencing. ß-catenin is essential for the activation of WNT signaling pathway. Indeed, knockdown of LMX1A activated the expressions of WNT signaling target genes T cell-specific transcription factor 4 (TCF4) and matrix metalloproteinase-7 (MMP7). What is more, the expression of LMX1A was negatively correlated with WNT signaling target genes in two datasets of human gastric cancer tissues. Thus, our study revealed an anti-metastatic role of LMX1A in gastric cancer which is mediated by the negative regulation of ß-catenin signaling target genes.

Enhanced FHL2 and TGF-ß1 Expression Is Associated With Invasive Growth and Poor Survival in Malignant Melanomas.

OBJECTIVES: This study examines the expression and the role of four-and-a-half LIM domains protein 2 (FHL2) and transforming growth factor ß1 (TGF-ß1) in human malignant melanoma. It is determined whether both proteins influence melanoma survival time. METHODS: We analyzed the immunohistochemical staining intensities of FHL2 and TGF-ß1 in normal skin and in 50 malignant melanomas with different mutation status (BRAF-V600E, NRAS codon 61 mutation, and wild type). Survival data were available for 45 cases. RESULTS: In melanocytes of nonneoplastic human skin, FHL2 expression was absent. In contrast, 38 (76%) of 50 melanomas showed strong cytoplasmic and partly nuclear FHL2 expression. At the invasion front, cytoplasmic TGF-ß1 staining was observed in 32 (64%) of 50 melanomas, and a correlation of FHL2 and TGF-ß1 staining intensities was detectable. In follow-up analyses, enhanced FHL2 and TGF-ß1 staining intensities in the tumor invasion front were associated with poor survival. CONCLUSIONS: Enhanced FHL2 and TGF-ß1 expression is correlated with poor survival in human malignant melanoma. Protumorigenic effects of autocrine TGF-ß1 secretion might be exerted by induction of FHL2 expression in melanoma cells. Since melanomas treated with targeted therapies often do not show sufficient response rates, inhibition of FHL2 and/or TGF-ß1 might be a promising therapeutic approach.

'Neuroendocrine' middle ear adenomas: consistent expression of the transcription factor ISL1 further supports their neuroendocrine derivation.

AIMS: Neuroendocrine middle ear adenoma (MEA) is a rare epithelial neoplasm of uncertain histogenesis that frequently shows neuroendocrine features. To date, <120 cases have been reported. The aims of the current study were to describe our experience with neuroendocrine MEA, to assess the frequency of specific neuroendocrine differentiation, and to test these lesions for transcription factors known to be expressed in a variety of other neuroendocrine tumours. METHODS AND RESULTS: We investigated six cases of neuroendocrine MEA, and stained them, for the first time, for the transcription factors CDX2, TTF1, PAX8, and ISL1 (islet-1). The patients were four men and two women (mean age, 39 years; range, 27-53 years). Two of four patients with extended follow-up (4.5-22 years) experienced recurrence at 92 months, and at 9 and 22 years, respectively. One case extending into the external ear coexisted with cholesteatoma. Histological examination showed trabecular, solid, acinar, glandular, cribriform, organoid, nested, diffuse non-cohesive plasmacytoid and pseudoalveolar patterns in varying combinations. Immunohistochemistry showed consistent expression of vimentin (4/4), pancytokeratin (6/6), synaptophysin (6/6), CD56 (4/4), and ISL1 (6/6). A CK7 antibody stained scattered cells in two of five cases. The myoepithelial markers and transcription factors TTF1, CDX2 or PAX8 were not expressed in any of the cases. CONCLUSIONS: Middle ear adenoma is an indolent, locally recurring, but generally non-metastasizing neoplasm with uniform expression of synaptophysin and ISL1, indicating true neuroendocrine differentiation. Because of its highly varied cellular and architectural appearance, MEA should be distinguished from tympanic paraganglioma and a variety of rare benign and malignant lesions at this site.

FHL2 regulates the resolution of tissue damage in chronic inflammatory arthritis.

OBJECTIVE: We analysed the role of the adaptor molecule four-and-a-half Lin11, Isl-1 & Mec-3 (LIM) domain protein 2 (FHL2) in the activation of fibroblast-like synoviocytes in human rheumatoid arthritis (RA) and tumour necrosis factor α (TNFα)-dependent animal models of the disease. METHODS: Synovial tissues of patients with RA and osteoarthritis (OA) as well as hind paw sections from arthritic human TNFα transgenic (hTNFtg) mice and synovial fibroblasts from these were analysed. The effects of cytokines on the expression of FHL2 and disease-relevant matrixmetalloproteases (MMPs) were determined. Analyses of human tissue specimens from patients treated with anti-TNFα as well as anti-TNFα treatment of hTNFtg mice were performed to substantiate the TNFα effects on FHL2 levels. FHL2(-/-) mice and hTNFtg mice (with constitutive or inducible transgene expression) were crossbred to generate TNFα overexpressing FHL2-deficient animals. Signalling pathways were analysed in cells from these mice and in human cells after knock down of FHL2 by western blot. RESULTS: FHL2 levels were higher in RA than in OA and in hTNFtg than in wild-type mice. Surprisingly, while transforming growth factor (TGF)ß-induced FHL2 expression, TNFα suppressed FHL2. In vivo, anti-TNFα treatment led to higher FHL2 levels both in RA patients and hTNFtg mice. The loss of FHL2 increased joint destruction in hTNFtg mice, which was accompanied by elevated MMP-13. In vitro, TNFα-mediated MMP-13 was significantly higher in FHL2(-/-) cells and after knock down of FHL2, which was caused by prolonged p38 MAPK activation. CONCLUSIONS: These data suggest that FHL2 serves as a protective factor and that, rather than promoting the pathology, the upregulation of FHL2 in RA occurs in frame of a regenerative attempt.

Dopaminergic neuron-like cells derived from bone marrow mesenchymal stem cells by Lmx1α and neurturin overexpression for autologous cytotherapy in hemiparkinsonian rhesus monkeys.

Bone marrow-derived mesenchymal stem cells hold great potential for cytotherapeutics of neurodegenerative disorders, including Parkinson's disease. The neurotrophic factor neurturin can rescue dopaminergic neurons damaged during the disease process. Lmx1α can promote mesencephalic dopaminergic differentiation during embryogenesis. In this study, we tested a cytotherapeutic strategy combining NTN/Lmx1α gene therapy and cell transplantation to ameliorate disease progression in hemiparkinsonian rhesus. Rhesus BMSCs were prepared for autologous grafting by transfection with recombinant adenoviral vectors expressing secreted NTN and Lmx1α,and cultured in the presence of induce factors, particularly the Lmx1α regulatory factor sonic hedgehog, to guide dopaminergic differentiation. These induced rh-BMSCs exhibited gene/protein expression phenotypes resembling nigral dopaminergic neurons. They survived and retained dopaminergic function following stereotaxic injection into the MPTP-lesioned right-side substantia nigra as indicated by SPECT measurement of DAT activity. Injected cells preserved and supplemented the remaining endogenous population of dopamine neurons (TH-positive cell ipsilateral/contralateral ratio was 56.81% ± 7.28% vs. 3.86%±1.22% in vehicle-injected controls; p<0.05). Cell injection also partially restored motor function and reduce apomorphine-evoked rotation (p<0.05). Moreover, function recovery occurred earlier than in previous studies on injected BMSCs. Our findings demonstrate a promising strategy for restoration of PD-associated motor dysfunction by transplantation of autologous BMSCs overexpressing NTN/Lmx1α.

The terminal basal mitosis of chicken retinal Lim1 horizontal cells is not sensitive to cisplatin-induced cell cycle arrest.

For proper development, cells need to coordinate proliferation and cell cycle-exit. This is mediated by a cascade of proteins making sure that each phase of the cell cycle is controlled before the initiation of the next. Retinal progenitor cells divide during the process of interkinetic nuclear migration, where they undergo S-phase on the basal side, followed by mitoses on the apical side of the neuroepithelium. The final cell cycle of chicken retinal horizontal cells (HCs) is an exception to this general cell cycle behavior. Lim1 expressing (+) horizontal progenitor cells (HPCs) have a heterogenic final cell cycle, with some cells undergoing a terminal mitosis on the basal side of the retina. The results in this study show that this terminal basal mitosis of Lim1+ HPCs is not dependent on Chk1/2 for its regulation compared to retinal cells undergoing interkinetic nuclear migration. Neither activating nor blocking Chk1 had an effect on the basal mitosis of Lim1+ HPCs. Furthermore, the Lim1+ HPCs were not sensitive to cisplatin-induced DNA damage and were able to continue into mitosis in the presence of γ-H2AX without activation of caspase-3. However, Nutlin3a-induced expression of p21 did reduce the mitoses, suggesting the presence of a functional p53/p21 response in HPCs. In contrast, the apical mitoses were blocked upon activation of either Chk1/2 or p21, indicating the importance of these proteins during the process of interkinetic nuclear migration. Inhibiting Cdk1 blocked M-phase transition both for apical and basal mitoses. This confirmed that the cyclin B1-Cdk1 complex was active and functional during the basal mitosis of Lim1+ HPCs. The regulation of the final cell cycle of Lim1+ HPCs is of particular interest since it has been shown that the HCs are able to sustain persistent DNA damage, remain in the cell cycle for an extended period of time and, consequently, survive for months.

Lhx2 regulates bone remodeling in mice by modulating RANKL signaling in osteoclasts.

The LIM homeobox 2 (Lhx2) transcription factor Lhx2 has a variety of functions, including neural induction, morphogenesis, and hematopoiesis. Here we show the involvement of Lhx2 in osteoclast differentiation. Lhx2 was strongly expressed in osteoclast precursor cells but its expression was significantly reduced during receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclastogenesis. Overexpression of Lhx2 in bone marrow-derived monocyte/macrophage lineage cells (BMMs), which are osteoclast precursor cells, attenuated RANKL-induced osteoclast differentiation by inhibiting the induction of nuclear factor of activated T cells c1 (NFATc1). Interestingly, interaction of Lhx2 proteins with c-Fos attenuated the DNA-binding ability of c-Fos and thereby inhibited the transactivation of NFATc1. Furthermore, Lhx2 conditional knockout mice exhibited an osteoporotic bone phenotype, which was related with increased osteoclast formation in vivo. Taken together, our results suggest that Lhx2 acts as a negative regulator of osteoclast formation in vitro and in vivo. The anti-osteoclastogenic effect of Lhx2 may be useful for developing a therapeutic strategy for bone disease.

Mdm2 is required for maintenance of the nephrogenic niche.

The balance between nephron progenitor cell (NPC) renewal, survival and differentiation ultimately determines nephron endowment and thus susceptibile to chronic kidney disease and hypertension. Embryos lacking the p53-E3 ubiquitin ligase, Murine double minute 2 (Mdm2), die secondary to p53-mediated apoptosis and growth arrest, demonstrating the absolute requirement of Mdm2 in embryogenesis. Although Mdm2 is required in the maintenance of hematopoietic stem cells, its role in renewal and differentiation of stem/progenitor cells during kidney organogenesis is not well defined. Here we examine the role of the Mdm2-p53 pathway in NPC renewal and fate in mice. The Six2-GFP::Cre(tg/+) mediated inactivation of Mdm2 in the NPC (NPC(Mdm)2(-/-)) results in perinatal lethality. NPC(Mdm)2(-/-) neonates have hypo-dysplastic kidneys, patchy depletion of the nephrogenic zone and pockets of superficially placed, ectopic, well-differentiated proximal tubules. NPC(Mdm2-/-) metanephroi exhibit thinning of the progenitor GFP(+)/Six2(+) population and a marked reduction or loss of progenitor markers Amphiphysin, Cited1, Sall1 and Pax2. This is accompanied by aberrant accumulation of phospho-γH2AX and p53, and elevated apoptosis together with reduced cell proliferation. E13.5-E15.5 NPC(Mdm2-/-) kidneys show reduced expression of Eya1, Pax2 and Bmp7 while the few surviving nephron precursors maintain expression of Wnt4, Lhx1, Pax2, and Pax8. Lineage fate analysis and section immunofluorescence revealed that NPC(Mdm2-/-) kidneys have severely reduced renal parenchyma embedded in an expanded stroma. Six2-GFP::Cre(tg/+); Mdm2(f/f) mice bred into a p53 null background ensures survival of the GFP-positive, self-renewing progenitor mesenchyme and therefore restores normal renal development and postnatal survival of mice. In conclusion, the Mdm2-p53 pathway is essential to the maintenance of the nephron progenitor niche.

Upregulation of Lhx8 increase VAChT expression and ACh release in neuronal cell line SHSY5Y.

Lhx8 is a transcription factor for cholinergic differentiation. Our previous experiments found upregulation of Lhx8 promoted cholinergic neuronal differentiation of hippocampal neural stem/progenitor cells or hippocampal newborn neurons in vitro. However, the role of Lhx8 in VAChT expression and ACh release is still less understood. In this report, we transfected Lhx8 cDNA into neuronal cell line SHSY5Y by lentiviral vectors to acquire the cells which stably expressed high level of Lhx8. Using this cell model, we provided experimental evidence that increasing Lhx8 upregulated the expression of ChAT and VAChT, and also increased the ACh release in culture medium. We suggested that Lhx8 overexpression is a useful strategy to increase the release of ACh and maybe of therapeutic value to neurodegenerative diseases.