Engrailed 2 triggers the activation of multiple phosphorylation-induced signaling pathways in both transcription-dependent and -independent manners.

Homeodomain (HD)-containing proteins are typically recognized as transcription factors. Engrailed 2 (EN2) is an HD-containing protein that is highly expressed in various types of cancers, however, the mechanism underlying the biological function of EN2 is not fully understood. Here, we report a transcription-independent function of EN2 in addition to its role as a transcription factor. EN2 expression leads to the activation of multiple signaling pathways mediated by phosphorylation cascades. A phosphoproteomic analysis revealed that the phosphorylation status of numerous protein sites was altered after EN2 is expressed. Notably, EN2 was shown to interact with a myriad of proteins implicated in phosphorylation signaling cascades, as determined by immunoprecipitation-mass spectrometry (IP-MS). We validated the interaction between EN2 and B55α, the regulatory subunit of the PP2A-B55α complex, and confirmed that the phosphatase activity of the complex was suppressed by EN2 binding. To target EN2-induced malignancy, two kinds of small molecules were utilized to inhibit the EN2-activated NF-κB and AKT signaling pathways. A clear synergistic effect was observed when the activation of the two pathways was simultaneously blocked. Collectively, the data show that EN2 functions in a transcription-independent manner in addition to its role as a transcription factor. This finding may have therapeutic implications in treating esophageal squamous cell carcinoma (ESCC).

Novel sensitive immunosensor for the selective detection of Engrailed 2 urinary prostate cancer biomarker.

Engrailed 2 (EN2) is a homeodomain-containing transcription factor expressed in prostate cancer (PCa) cell lines and is secreted into the urines. It is nowadays considered as a promising non-invasive biomarker for PCa early diagnosis. Herein, we report the design of an electrochemical immunosensor for EN2 detection. The biosensor fabrication involved a covalent immobilization of anti-EN2 antibodies onto a poly para amino benzoic acid (PABA) film electropolymerized on a gold electrode. Square wave voltammetry was investigated for EN2 detection in a phosphate buffer solution in a concentration range of 10-5 ng/mL to 1 µg/mL. The limit of detection of the designed sensor was equal to 10-5 ng/mL and the sensitivity was of order of (29 ± 2) µL/ng. The dissociation constant Kd of the "complex" EN2/anti-EN2, estimated from a Hill model, was of order of (0.9 ± 0.2) fM. Experimental results revealed that the immunosensor enabled selective detection of EN2 in a mixture of three proteins which can be found in men' urine: human serum albumin (HSA), prostate-specific antigen (PSA) and immunoglobulin G (IgG). Tests in artificial urine, with an ionic strength of 0.18 M, have been done and results were found comparable to those obtained in PBS (0.16 M). These encouraging results show a potentially promising future for the development of an electrochemical biosensor for robust and accurate urinary biomarkers detection.

Engrailed 2 deficiency and chronic stress alter avoidance and motivation behaviors.

Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder characterized by impairments in social interaction, cognition, and communication, as well as the presence of repetitive or stereotyped behaviors and interests. ASD is most often studied as a neurodevelopmental disease, but it is a lifelong disorder. Adults with ASD experience more stressful life events and greater perceived stress, and frequently have comorbid mood disorders such as anxiety and depression. It remains unclear whether adult exposure to chronic stress can exacerbate the behavioral and neurodevelopmental phenotypes associated with ASD. To address this issue, we first investigated whether adult male and female Engrailed-2 deficient (En2-KO, En2-/-) mice, which display behavioral disturbances in avoidance tasks and dysregulated monoaminergic neurotransmitter levels, also display impairments in instrumental behaviors associated with motivation, such as the progressive ratio task. We then exposed adult En2-KO mice to chronic environmental stress (CSDS, chronic social defeat stress), to determine if stress exacerbated the behavioral and neuroanatomical effects of En2 deletion. En2-/- mice showed impaired instrumental acquisition and significantly lower breakpoints in a progressive ratio test, demonstrating En2 deficiency decreases motivation to exert effort for reward. Furthermore, adult CSDS exposure increased avoidance behaviors in En2-KO mice. Interestingly, adult CSDS exposure also exacerbated the deleterious effects of En2 deficiency on forebrain-projecting monoaminergic fibers. Our findings thus suggest that adult exposure to stress may exacerbate behavioral and neuroanatomical phenotypes associated with developmental effects of genetic En2 deficiency.

Engrailed-2 is a cell autonomous regulator of neurogenesis in cultured hippocampal neural stem cells.

Abnormalities in genes that regulate early brain development are known risk factors for neurodevelopmental disorders. Engrailed-2 (En2) is a homeodomain transcription factor with established roles in cerebellar patterning. En2 is highly expressed in the developing mid-hindbrain region, and En2 knockout (KO) mice exhibit major deficits in mid-hindbrain structures. However, En2 is also expressed in forebrain regions including the hippocampus, but its function is unknown. Previous studies have shown that the hippocampus of En2-KO mice exhibits reductions in its volume and cell numbers due to aberrant neurogenesis. Aberrant neurogenesis is due, in part, to noncell autonomous effects, specifically, reductions of innervating norepinephrine fibers from the locus coeruleus. In this study, we investigate possible cell autonomous roles of En2 in hippocampal neurogenesis. We examine proliferation, survival, and differentiation using cultures of hippocampal neurospheres of P7 wild-type (WT) and En2-KO hippocampal neural progenitor cells (NPCs). At 7 days, En2-KO neurospheres were larger on average than WT spheres and exhibited 2.5-fold greater proliferation and 2-fold increase in apoptotic cells, similar to in vivo KO phenotype. Further, En2-KO cultures exhibited 40% less cells with neurite projections, suggesting decreased differentiation. Lastly, reestablishing En2 expression in En2-KO NPCs rescued excess proliferation. These results indicate that En2 functions in hippocampal NPCs by inhibiting proliferation and promoting survival and differentiation in a cell autonomous manner. More broadly, this study suggests that En2 impacts brain structure and function in diverse regions outside of the mid-hindbrain.

Altered staining patterns and expression level of Engrailed-2 in benign prostatic hyperplasia and prostate Cancer predict prostatic disease progression.

BACKGROUND: Prostate cancer (PC), a common malignant tumor, is the second-leading cause of cancer death among American men. Its successful treatment greatly relies on the early diagnose. Engrailed-2 (EN2) has been confirmed being existed with a high level in the urine of PC patients. In this study, to explore the application of EN2 in PC, we detected the immunohistochemical staining difference and EN2 expression level between benign prostatic hyperplasia (BPH) and PC. METHODS: We developed a monoclonal antibody against the helix 3 in EN2 and confirmed its specificity with Western blotting (WB) and immunofluorescence detecting the subcellular localization of endogenous and exogenous EN2 in three PC cell lines (LNCap, PC3, and DU145). We conducted immunohistochemical staining using this homemade antibody, and RT-PCR to detect the expression of EN2 in 25 PC and 25 BPH cases, and analyzed the correlation of EN2 expression and PC clinical staging. RESULTS: The results of WB and immunofluorescence showed our homemade EN2 monoclonal antibody could specifically bind endogenous and exogenous EN2 protein in three different PC cell lines. Endogenous EN2 was generally expressed in the cytoplasm and exogenous EN2 mostly existed in the nucleus of these cell lines. Immunohistochemical staining in PC had extremely stronger signals than that in BPH, suggesting a higher EN2 expression level in PC, which was confirmed by RT-PCR. Interestingly, the stained areas in BPH tissues were mainly in nucleus and cytoplasm, while in PC tissues were mainly on cytomembrane. Moreover, the expression level of EN2 was positively correlated with the PC clinical staging. CONCLUSION: Using our homemade EN2 antibody, we have found different staining patterns and expression level of EN2 in BPH and PC,which may be helpful to predict prostatic disease progression.

Bidirectional transfer of homeoprotein EN2 across the plasma membrane requires PIP2.

Homeoproteins are a class of transcription factors sharing the unexpected property of intercellular trafficking that confers to homeoproteins a paracrine mode of action. Homeoprotein paracrine action participates in the control of patterning processes, including axonal guidance, brain plasticity and boundary formation. Internalization and secretion, the two steps of intercellular transfer, rely on unconventional mechanisms, but the cellular mechanisms at stake still need to be fully characterized. Thanks to the design of new quantitative and sensitive assays dedicated to the study of homeoprotein transfer within HeLa cells in culture, we demonstrate a core role of phosphatidylinositol (4,5)-bisphosphate (PIP2) together with cholesterol in the translocation of the homeobox protein engrailed-2 (EN2) across the plasma membrane. By using drug and enzyme treatments, we show that both secretion and internalization are regulated according to PIP2 levels. The requirement for PIP2 and cholesterol in EN2 trafficking correlates with their selective affinity for this protein in artificial bilayers, which is drastically decreased in a paracrine-deficient mutant of EN2. We propose that the bidirectional plasma membrane translocation events that occur during homeoprotein secretion and internalization are parts of a common process.

Engrailed-2 promotes a malignant phenotype of esophageal squamous cell carcinoma through upregulating the expression of pro-oncogenic genes.

BACKGROUND: A number of homeobox genes have been implicated in the development of various cancers. However, the role of engrailed 2 (EN2), a member of the homeobox gene superfamily, in esophageal squamous cell carcinoma (ESCC) remains unknown. METHODS: The expression of EN2 was examined using quantitative real-time PCR and immunohistochemistry. A stable cell line was established to express exogenous EN2 using a lentivirus system. The malignant phenotype was analyzed with proliferation, clonogenicity, wound-healing and invasion assays. The CRISPR/Cas9 system was adopted to deplete endogenous EN2. RNA profiling was performed using gene expression microarray. The ShRNA-mediated method was used to knock down the expression of SPARC. The structure-function relationship was determined using site-directed mutagenesis. RESULTS: EN2 is highly expressed in ESCC. The malignant phenotype of the ESCC cell line was amplified by an overexpression of EN2 but was attenuated by a disruption of EN2. RNA profiling analysis revealed that distinct sets of genes were modulated by the expression of EN2 in various ESCC cell lines and oncogenes were among these. EN2 greatly increased the expression of SPARC in Eca109. Site-directed mutagenesis revealed that the induction of SPARC was closely correlated with the protumor function of EN2. ShRNA-mediated knockdown of SPARC attenuated the malignant phenotype of EN2-infected cells. These data suggest that SPARC is crucial for mediating the protumor function of EN2. DISCUSSION: EN2 has an oncogenic function in ESCC that is mediated by upregulating the expression of pro-oncogenic genes downstream. EN2 may potentially act as a diagnostic marker or therapeutic target for ESCC treatment in the future.

Is Engrailed-2 (EN2) a truly promising biomarker in prostate cancer detection?

Purpose: Prostate-specific antigen (PSA) is a sensitive but unspecific marker for prostate cancer (PC) detection, which may result in harms including overdiagnosis and overtreatment. Therefore, the development of new markers is of absolute value. The urinary level of engrailed-2 (EN2) protein has been recently suggested as a promising PC biomarker, correlating with tumour volume and stage. This study evaluated EN2 and its potential use in clinical practice.Materials and methods: Urinary EN2 was assessed by different commercially available enzyme-linked immunosorbent assay kits. The study sample included 90 patients with clinically localized PC compared to 30 healthy controls, and a group of 40 patients indicated for prostate biopsy due to an elevated PSA level where both pre- and post-digital rectal examination urine samples were collected.Results: No statistical difference between the patient group and the control group was obtained in all measured variables. There was no significant correlation between urinary EN2 and serum PSA, tumour staging and grading. Attentive DRE did not lead to significant changes of urinary EN2 or impact on its predictive power.Conclusions: Our results show that EN2 as a PC biomarker brings no additional value to the current use of PSA in clinical practice.

Engrailed-2 (EN2) - a novel biomarker in epithelial ovarian cancer.

BACKGROUND: Epithelial ovarian cancer is a common malignancy, with no clinically approved diagnostic biomarker. Engrailed-2 (EN2) is a homeodomain-containing transcription factor, essential during embryological neural development, which is dysregulated in several cancer types. We evaluated the expression of EN2 in Epithelial ovarian cancer, and reviewed its role as a biomarker. METHODS: We evaluated 8 Epithelial ovarian cancer cell lines, along with > 100 surgical specimens from the Royal Surrey County Hospital (2009-2014). In total, 108 tumours and 5 normal tissue specimens were collected. En2 mRNA was evaluated by semi-quantitative RT-PCR. Histological sub-type, and platinum-sensitive/-resistant status were compared. Protein expression was assessed in cell lines (immunofluorescence), and in > 150 tumours (immunohistochemistry). RESULTS: En2 mRNA expression was elevated in serous ovarian tumours compared with normal ovary (p < 0.001), particularly in high-grade serous ovarian cancer (p < 0.0001) and in platinum-resistant tumours (p = 0.0232). Median Overall Survival and Progression-free Survival were reduced with high En2 expression (OS = 28 vs 42 months, p = 0.0329; PFS = 8 vs 27 months; p = 0.0004). Positive cytoplasmic EN2 staining was demonstrated in 78% of Epithelial ovarian cancers, with absence in normal ovary. EN2 positive high-grade serous ovarian cancer patients had a shorter PFS (10 vs 17.5 months; p = 0.0103). CONCLUSION: The EN2 transcription factor is a novel ovarian cancer biomarker. It demonstrates prognostic value, correlating with worse Overall Survival and Progression-free Survival. It is hoped that further work will validate its use as a biomarker, and provide insight into the role of EN2 in the development, progression and spread of ovarian cancer.

Clinical Phenotypes Associated to Engrailed 2 Gene Alterations in a Series of Neuropediatric Patients.

The engrailed homeobox protein (EN) plays an important role in the regionalization of the neural tube. EN distribution regulates the cerebellum and midbrain morphogenesis, as well as retinotectal synaptogenesis. In humans, the EN1 and EN2 genes code for the EN family of transcription factors. Genetic alterations in the expression of EN2 have been related to different neurologic conditions and more particularly to autism spectrum disorders (ASD). We aimed to study and compare the phenotypes of three series of patients: (1) patients with encephalic structural anomalies (ESA) and abnormalities in the genomic (DNA) and/or transcriptomic (RNAm) of EN2 (EN2-g), (2) ESA patients having other gene mutations (OG-g), and (3) ESA patients free of these mutations (NM-g). Subjects and Methods: We have performed a descriptive study on 109 patients who suffer from mental retardation (MR), cerebral palsy (CP), epilepsy (EP), and behavioral disorders (BD), showing also ESA in their encephalic MRI. We studied genomic DNA and transcriptional analysis (cDNA) on EN2 gene (EN2), and in other genes (OG): LIS1, PTAFR, PAFAH1B2, PAFAH1B3, FGF8, PAX2, D17S379, D17S1866, and SMG6 (D17S5), as a routine genetic diagnosis in ESA patients. Results: From 109 patients, fifteen meet the exclusion criteria. From the remaining 94 patients, 12 (12.8%) showed mutations in EN2 (EN2-g), 20 showed mutations in other studied genes (OG-g), and 62 did not showed any mutation (NM-g). All EN2-g patients, suffered from MR, nine EP, seven BD and four CP. The proportions of these phenotypes in EN2-g did not differ from those in the OG-g, but it was significantly higher when comparing EN2-g with NM-g (MR: p = 0.013; EP: p = 0.001; BD: p = 0.0001; CP: p = 0.07, ns). Groups EN2-g and OG-g showed a 100 and a 70% of comorbidity, respectively, being significantly (p = 0.04) greater than NM-group (62.9%). Conclusion: Our series reflects a significant effect of EN2 gene alterations in neurodevelopmental abnormalities associated to ESA. Conversely, although these EN2 related anomalies might represent a predisposition to develop brain diseases, our results did not support direct relationship between EN2 mutations and specific clinical phenotypes.

Engrailed-2 promoter hyper-methylation is associated with its downregulation in clear cell renal cell carcinoma.

In a previous study by the present authors, it was identified that the expression of engrailed-2 (EN2) gene was downregulated in clear cell renal cell carcinoma (cc-RCC). The aim of the present study was to determine whether aberrant methylation was the mechanism underlying the silencing of EN2 gene in cc-RCC. A total of forty paired cc-RCC tissues, four cc-RCC cell lines and one normal human proximal tubule epithelial cell line were evaluated for EN2 gene methylation status using methylation-specific polymerase chain reaction (PCR). Following treatment with 5-Aza-dc, reverse transcription-quantitative PCR and western blot analysis were performed to examine the expression of EN2. Furthermore, cell proliferation, apoptosis and invasion assays were conducted to analyze the inhibitory effects of EN2 re-expression in 786-O cells. The results of the present study demonstrated that hyper-methylation of EN2 was identified in 12/40 cc-RCC tissues and all cc-RCC cell lines. The methylation status of the EN2 gene was revealed to be associated with histological grade and tumor size in cc-RCC. Following 5-Aza-dc treatment, demethylation of the EN2 gene was identified in 786-O cells, in conjunction with EN2 re-expression. Furthermore, re-activation of the EN2 gene markedly inhibited the proliferative and invasive capacities of cc-RCC. The results of the present study demonstrated that the EN2 gene promoter was hyper-methylated in cc-RCC, which may underlie the silencing of the EN2 gene in cc-RCC.

Hsa-miR-27b is up-regulated in cytomegalovirus-infected human glioma cells, targets engrailed-2 and inhibits its expression.

Human cytomegalovirus (HCMV) dormant infection can alter the expression of the hosts' microRNAs (miRNAs) and impact on the regulation of target genes. To investigate the differentially expressed miRNAs induced by HCMV in human glioma U251 cells, a comprehensive miRNA screen was performed. As a result, 19 up-regulated and 14 down-regulated miRNAs were determined. Of these, hsa-miR-27b (miR-27b) attracted our attention. MiR-27b levels in U251 cells increased 7.70-fold, 8.64-fold, and 4.78-fold, respectively, post 24 h, 48 h, and 72 h HCMV infection, compared to those in the mimic-infected cells, and this up-regulation was further confirmed by quantitative RT-PCR. The bioinformatic analyses show that miR-27b targets engrailed-2 (EN2) gene; however, the effect of miR-27b on EN2 is rarely encountered. In this study, we initially conducted dual luciferase assay to validate the target function of miR-27b on EN2. The results manifested that EN2 is a novel target of miR-27b, which could directly target the 3' untranslated region (3'-UTR) of the gene. We further found that the miR-27b transfected glioma U251 cells exhibited longer cell bodies with more synapses and multiple-angle shapes; moreover, Western blot detection revealed that the EN2 protein levels in these cells were significantly low. In conclusion, our study originally reports the up-regulation of miR-27b in HCMV-infected glioma cells. Our study also provides the first experimental evidence that miR-27b could affect glioma cells' growth, target EN2 and inhibit its expression in glioma cells. Our data indicate that miR-27b may be related to the development of neurological disorders with HCMV infection. The newly identified miR-27b/EN2 signal pathway may provide new insights into the glioma pathogenesis and a novel target for glioma therapy. Impact statement Our study is the first to demonstrate that the HCMV infection could alter the expression of cellular microRNAs of the host glioma cells, which may develop an understanding of the pathogenesis of the HCMV infection in the microRNA level. Recently, HCMV infection and engrailed-2 have been reported to be related to the autism spectrum disorder (ASD). In this study, we confirmed that engrailed-2 is the target of hsa-miR-27b. As far as we know, our findings of the hsa-miR-27b up-regulation in the HCMV-infected glioma cells, targeting engrailed-2 and inhibiting its expression have never been reported or documented. Our data indicate that miR-27b may be related to the development of neurological disorders with the HCMV infection. The newly identified miR-27b/EN2 signal pathway may provide new insights into the glioma pathogenesis and a novel target for glioma therapy.

Biological function and mechanism of miR-33a in prostate cancer survival and metastasis: via downregulating Engrailed-2.

OBJECTIVE: Recent studies have identified Engrailed-2 (EN-2), a homeobox-containing transcription factor, as a candidate oncogene in prostate cancer (PC). Therapeutic targeting on EN-2, however, is limited because the mechanism underlying EN-2 overexpression in prostatic cancer cells is unknown. This study was to investigate the potential regulatory role of miR-33a on EN-2 expression and explore this signaling axis in ability of prostate cancer survival and metastasis. METHODS: The relative expression of miR-33a and EN-2 in paired prostate cancer tissue and adjacent normal tissue as well as in prostate cancer cell lines, PC3 and DU145, was determined using quantitative real-time PCR or western blot, respectively. Cells survival, migration and invasion were evaluated by assays of MTT, TUNEL and Boyden chamber assays, respectively. Direct regulation of EN-2 by miR-33a was examined by luciferase reporter assay. RESULTS: The data showed that miR-33a was upregulated and EN-2 was downregulated in both prostate cancer tissue and prostate cancer cells. miR-33a overexpression suppresses prostate cancer cell survival and metastasis. miR-33a can directly act on EN-2 expression by binding to 3'UTR of its mRNA. Also, miR-33a negatively regulated EN-2 mRNA and protein expression. In pcDNA-EN-2 and miR-33a mimic co-transfected PC3 and DU145 cells, EN-2 overexpression reverses the anti-cell survival and metastasis actions of miR-33a overexpression. The pivotal role of miR-33a in inhibiting prostate tumor growth was confirmed in xenograft models of prostate cancer. CONCLUSION: Our data suggest that the functional interaction of miR-33a and EN-2 is involved in tumorigenesis of prostate cancer. Also in this process EN-2 serves as a negative responder for miR-33a.

Expression of Engrailed-2 in hepatocellular carcinoma and effect of silencing Engrailed-2 gene on the proliferation and invasion of hepatocellular carcinoma cells / 实用医学杂志

Objective To investigate the role and implication of Engrailed-2 (EN2) in human hepatocellular carcinoma (HCC) and the effect of silencing EN2 genes on the proliferation and invasion of HepG2 cells by RNA interference.Methods Immunohistochemistry was used to detect the expression of EN2 in the HCC tissues and corresponding non-cancerous adjacent liver tissues in 126 patients with HCC.The expressions of EN2 and the relationship between EN2 expression and clinicopathological features and prognosis were analyzed using the Chi-square test.The survival curve was drawn using the Kaplan-Meier method and the survival was analyzed using the log-rank test.EN2-small interfering RNA (siRNA) was transfected into HepG2 cell lines mediated by LipofectamineTM 2000,and the expression of EN2 were detected by Western blotting assay.The cell proliferation and invasion were measured by methyl thiazol tetrazolium (MTT) and Transwell assays.Results EN2 positive expression rate was high (62.7％) in HCC tissue but low in the corresponding peritumoral tissue (23.5％,P ＜ 0.01).The high expression of EN2 was strongly correlated with tumor size,metastasis and AJCC TNM stage.The cumulative 5-year survival rate was 33.9％ in the low EN2 expression group,whereas it was 5.1％ in the high EN2 expression group (P ＜ 0.01).Expression of EN2 in EN2 siRNA group was significantly lower than that in control siRNA group and control group (P ＜ 0.05),respectively.The cell proliferation and invasion ability were significantly reduced in the EN2 siRNA group.Conclusion The expression of EN2 is highly up-regulated in HCC tissues and down-regulation of EN2 could inhibit proliferation and invasion of HepG2 cells,which indicating that EN2 is involved in the process of HCC carcinogenesis and progression and may serve as a biomarker for predicting prognosis of HCC patients.

Engrailed-2 might play an anti-oncogenic role in clear-cell renal cell carcinoma.

Our preliminary study indicated that Engrailed-2 (EN2) is downregulated but also ectopically expressed in clear-cell renal cell carcinoma (CCRCC), and the absence of EN2 expression was associated with poor histological grade. However, the specific roles of EN2 in CCRCC have yet to be elucidated. In the present study, we examined the effects of inhibiting EN2 expression by human renal tubular epithelial cells (HK-2) and overexpressing EN2 by human clear-cell renal cells (786-O). Results showed that EN2 inhibition accelerated HK-2 cell proliferation, shortened the cell cycle, reduced apoptosis, and acted more invasively. By contrast, EN2 overexpression in 786-O cells decelerated the proliferative ability of 786-O, increased the percentage of cell apoptosis, and weakened the invasive ability. Overall, the results demonstrated that EN2 might play an anti-oncogenic role in oncogenesis and development of CCRCC, thereby maintaining the normal growth of human renal tubular epithelial cells.

Morphogenesis of the cerebellum and cerebellum-related structures in the shark Scyliorhinus canicula: insights on the ground pattern of the cerebellar ontogeny.

Because the cerebellum emerged at the agnathan-gnathostome transition and cartilaginous fishes are at the base of the gnathostome lineage, this group is crucial to determine the basic developmental pattern of the cerebellum and to gain insights into its origin. We have systematically analyzed key events in the development of cerebellum and cerebellum-related structures of the shark Scyliorhinus canicula. Three developmental periods are distinguished based on anatomical observations combined with molecular analysis. We present neurochemical and genoarchitectonic evidence on the onset of cerebellar development, the rostral and caudal cerebellar boundaries, the compartmentalization of the cerebellum, and correspondence of cerebellar domains to rhombomeric segmentation of the rostral hindbrain. Our observations, mainly based on the expression pattern of ScHoxA2, support the origin of both the upper and lower auricular leaves from r1 and exclude any cerebellar origin from r2. Correlation between subrhombomeres r1a/r1b and cerebellar domains is proposed based on the ScEn2 expression. The ScEn2 and ScOtx2 expression patterns revealed an antero-posterior cerebellar compartmentalization similar to that of mammals, and supported certain fissures (commonly used to define cerebellar domains) as reliable anatomical landmarks. At difference from mammals, the expression of ScEn2 along the cerebellar median-lateral axis does not reveal a multiple-banded pattern. The present study provides an atlas of cerebellar development in one of the most basal extant gnathostome lineages and emphasizes the importance of combining classic descriptive with modern molecular studies to gain knowledge on the ancestral condition of cerebellar developmental processes and the origins and evolution of the cerebellum.

EN2 in Prostate Cancer.

Despite extensive efforts to identify a clinically useful diagnostic biomarker in prostate cancer, no new test has been approved by regulatory authorities. As a result, this unmet need has shifted to biomarkers that additionally indicate presence or absence of "significant" disease. EN2 is a homeodomain-containing transcription factor secreted by prostate cancer into the urine and can be detected by enzyme-linked immunoassay. EN2 may be an ideal biomarker because normal prostate tissue and benign prostatic hypertrophic cells do not secrete EN2. This review discusses the enormous potential of EN2 to address this unmet need and provide the urologist with a simple, inexpensive, and reliable prostate cancer biomarker.

Serotonin abnormalities in Engrailed-2 knockout mice: New insight relevant for a model of Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a congenital neurodevelopmental behavioral disorder that appears in early childhood. Recent human genetic studies identified the homeobox transcription factor, Engrailed 2 (EN2), as a possible ASD susceptibility gene. En2 knockout mice (En2-/-) display subtle cerebellar neuropathological changes and reduced levels of tyrosine hydroxylase, noradrenaline and serotonin in the hippocampus and cerebral cortex similar to those ones which have been observed in the ASD brain. Furthermore other similarities link En2 knockout mice to ASD patients. Several lines of evidence suggest that serotonin may play an important role in the pathophysiology of the disease. In the present study we measured, by using an HPLC, the 5-HT levels in different brain areas and at different ages in En2-/- mice. In the frontal and occipital cortex, the content of 5HT was reduced in En2-/- 1 and 3 months old mice; in 6 month old mice, the difference was still present, but it was not statistically significant. The 5-HT content of cerebellar cortex was significantly reduced at 1 month old but significantly high when the KO mice reached 3 months of age. The increase was present even at 6 months of age. A similar trend was highlighted by SERT immunolabeling in En2-/- mice compared to control in the same areas and age analyzed. Our findings, in agreement with the current knowledge on the 5-HT system alterations in ASD, confirm the early neurotransmitter deficit with a late compensatory recovery in En2 KO-mice further suggesting that this experimental animal may be considered a good predictive model for the human disease.

Control of brain patterning by Engrailed paracrine transfer: a new function of the Pbx interaction domain.

Homeoproteins of the Engrailed family are involved in the patterning of mesencephalic boundaries through a mechanism classically ascribed to their transcriptional functions. In light of recent reports on the paracrine activity of homeoproteins, including Engrailed, we asked whether Engrailed intercellular transfer was also involved in brain patterning and boundary formation. Using time-controlled activation of Engrailed combined with tools that block its transfer, we show that the positioning of the diencephalic-mesencephalic boundary (DMB) requires Engrailed paracrine activity. Both zebrafish Eng2a and Eng2b are competent for intercellular transfer in vivo, but only extracellular endogenous Eng2b, and not Eng2a, participates in DMB positioning. In addition, disruption of the Pbx-interacting motif in Engrailed, known to strongly reduce the gain-of-function phenotype, also downregulates Engrailed transfer, thus revealing an unsuspected participation of the Pbx interaction domain in this pathway.

Ultrasensitive electrochemical detection of engrailed-2 based on homeodomain-specific DNA probe recognition for the diagnosis of prostate cancer.

It is well known that the engrailed-2 (EN2) protein, a biomarker for prostate cancer, strongly binds to a specific DNA sequence (5'-TAATTA-3') to regulate transcription. Based on this intrinsic property, DNA probes with additional flanked sequences were designed and optimized. Various measurements, such as electrophoresis mobility shift assay, surface plasmon resonance, and quantitative fluorescence assay were performed to investigate the feasibility of the DNA probes. Then, the affinities of the DNA probes to the target protein were quantitatively determined using FAM-modified DNA probes and magnetic beads, resulting in dissociation constants ranging from 61.03 to 98.84nM. To develop an early diagnosis platform for prostate cancer, an ultrasensitive electrochemical biosensor based on the electrodeposition of gold nanoparticles was designed. The EN2 protein was quantitatively detected using the electrochemical biosensor, and the calculated detection limit was found to be 5.62fM. Finally, the specificity and applicability of the biosensor were verified using several proteins and an artificial urine medium. The impedance signals increased in the cases of EN2, suggesting that the system exhibited high selectivity to only EN2.