The Engrailed homeobox genes determine the different foliation patterns in the vermis and hemispheres of the mammalian cerebellum.
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ABSTRACT:
SUBMITTER: Cheng Y
PROVIDER: S-EPMC2858911 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Underlying the seemingly uniform cellular composition of the adult mammalian cerebellum (Cb) are striking parasagittal stripes of gene expression along the medial-lateral (ML) axis that are organized with respect to the lobules that divide the Cb along the anterior-posterior (AP) axis. Although there is a clear correlation between the organization of gene expression stripes and Cb activity patterns, little is known about the genetic pathways that determine the intrinsic stripe molecular code. Here we establish that ML molecular code patterning is highly dependent on two homeobox transcription factors, Engrailed1 (En1) and En2, both of which are also required for patterning the lobules. Gene expression analysis of an allelic series of En1/2 mutant mice that have an intact Purkinje cell layer revealed severe patterning defects using three known components of the ML molecular code and a new marker of Hsp25 negative stripes (Neurofilament heavy chain, Nfh). Importantly, the complementary expression of ZebrinII/PhospholipaseC beta4 and Hsp25/Nfh changes in unison in each mutant. Furthermore, each En gene has unique as well as overlapping functions in patterning the ML molecular code and each En protein has dominant functions in different AP domains (subsets of lobules). Remarkably, in En1/2 mutants with almost normal foliation, ML molecular code patterning is severely disrupted. Thus, independent mechanisms that use En1/2 must pattern foliation and spatial gene expression separately. Our studies reveal that En1/2 are fundamental components of the genetic pathways that pattern the two intersecting coordinate systems of the Cb, morphological divisions and the molecular code.
| S-EPMC2864318 | biostudies-literature
Project description:The elucidation of epigenetic alterations in the autism brain has potential to provide new insights into the molecular mechanisms underlying abnormal gene expression in this disorder. Given strong evidence that engrailed-2 (EN-2) is a developmentally expressed gene relevant to cerebellar abnormalities and autism, the epigenetic evaluation of this candidate gene was undertaken in 26 case and control post-mortem cerebellar samples. Assessments included global DNA methylation, EN-2 promoter methylation, EN-2 gene expression and EN-2 protein levels. Chromatin immunoprecipitation was used to evaluate trimethylation status of histone H3 lysine 27 (H3K27) associated with gene downregulation and histone H3 lysine 4 (H3K4) associated with gene activation. The results revealed an unusual pattern of global and EN-2 promoter region DNA hypermethylation accompanied by significant increases in EN-2 gene expression and protein levels. Consistent with EN-2 overexpression, histone H3K27 trimethylation mark in the EN-2 promoter was significantly decreased in the autism samples relative to matched controls. Supporting a link between reduced histone H3K27 trimethylation and increased EN-2 gene expression, the mean level of histone H3K4 trimethylation was elevated in the autism cerebellar samples. Together, these results suggest that the normal EN-2 downregulation that signals Purkinje cell maturation during late prenatal and early-postnatal development may not have occurred in some individuals with autism and that the postnatal persistence of EN-2 overexpression may contribute to autism cerebellar abnormalities.
| S-EPMC3590998 | biostudies-other
Project description:BackgroundThe cerebellum has a striking morphology consisting of folia separated by fissures of different lengths. Since folia in mammals likely serve as a broad platform on which the anterior-posterior organization of the sensory-motor circuits of the cerebellum are built, it is important to understand how such complex morphology arises.ResultsUsing a combination of genetic inducible fate mapping, high-resolution cellular analysis and mutant studies in mouse, we demonstrate that a key event in initiation of foliation is the acquisition of a distinct cytoarchitecture in the regions that will become the base of each fissure. We term these regions 'anchoring centers'. We show that the first manifestation of anchoring centers when the cerebellar outer surface is smooth is an increase in proliferation and inward thickening of the granule cell precursors, which likely causes an associated slight invagination of the Purkinje cell layer. Thereafter, granule cell precursors within anchoring centers become distinctly elongated along the axis of the forming fissure. As the outer cerebellar surface begins to fold inwards, Bergmann glial fibers radiate in towards the base of the immature fissure in a fan shape. Once the anchoring center is formed, outgrowth of folia seems to proceed in a self-sustaining manner driven by granule cell migration along Bergmann glial fibers. Finally, by analyzing a cerebellum foliation mutant (Engrailed 2), we demonstrate that changing the timing of anchoring center formation leads to predictable changes in the shape and size of the surrounding folia.ConclusionWe present a new cellular model of the initial formation of cerebellar fissures with granule cells providing the driving physical force. Both the precise timing of the appearance of anchoring centers at the prospective base of each fissure and the subsequent coordinated action of granule cells and Bergmann glial fibers within the anchoring centers dictates the shape of the folia.
| S-EPMC2246128 | biostudies-literature
Project description:Adenosine kinase (ADK), the major adenosine-metabolizing enzyme, plays a key role in brain development and disease. In humans, mutations in the Adk gene have been linked to developmental delay, stunted growth, and intellectual disability. To better understand the role of ADK in brain development, it is important to dissect the specific roles of the two isoforms of the enzyme expressed in the cytoplasm (ADK-S) and cell nucleus (ADK-L). We, therefore, studied brain development in Adk-tg transgenic mice, which only express ADK-S in the absence of ADK-L throughout development. In the mutant animals, we found a reduction in the overall brain, body size, and weight during fetal and postnatal development. As a major developmental abnormality, we found a profound change in the foliation pattern of the cerebellum. Strikingly, our results indicated aberrant Purkinje cells arborization at P9 and accelerated cell death at P6 and P9. We found defects in cerebellar cell proliferation and migration using a bromodeoxyuridine (BrdU)-based cell proliferation assay at postnatal day 7. Our data demonstrate that dysregulation of ADK expression during brain development profoundly affects brain growth and differentiation.
| S-EPMC10645999 | biostudies-literature
Project description:The layered cortex of the cerebellum is folded along the anterior-posterior axis into lobules separated by fissures, allowing the large number of cells needed for advanced cerebellar functions to be packed into a small volume. During development, the cerebellum begins as a smooth ovoid structure with two progenitor zones, the ventricular zone and upper rhombic lip, which give rise to distinct cell types in the mature cerebellum. Initially, the cerebellar primordium is divided into five cardinal lobes, which are subsequently further subdivided by fissures. The cellular processes and genes that regulate the formation of a normal pattern of fissures are poorly understood. The engrailed genes (En1 and En2) are expressed in all cerebellar cell types and are critical for regulating formation of specific fissures. However, the cerebellar cell types that En1 and En2 act in to control growth and/or patterning of fissures has not been determined. We conditionally eliminated En2 or En1 and En2 either in both progenitor zones and their descendents or in the two complementary sets of cells derived from each progenitor zone. En2 was found to be required only transiently in the progenitor zones and their immediate descendents to regulate formation of three fissures and for general growth of the cerebellum. In contrast, En1 and En2 have overlapping functions in the cells derived from each progenitor zone in regulating formation of additional fissures and for extensive cerebellar growth. Furthermore, En1/2 function in ventricular zone-derived cells plays a more significant role in determining the timing of initiation and positioning of fissures, whereas in upper rhombic lip-derived cells the genes are more important in regulating cerebellar growth. Our studies reveal the complex manner in which the En genes control cerebellar growth and foliation in distinct cell types.
| S-EPMC4038292 | biostudies-other
Project description:The spatial organization of the cerebellar afferent map has remarkable correspondence to two aspects of intrinsic patterning within the cerebellum embodied by a series of lobules and Purkinje cell (PC)-striped gene expression. Using male and female mice, we tested whether the Engrailed (En) homeobox genes are a common genetic substrate regulating all three systems, since they are expressed in spatially restricted domains within the cerebellum and are critical for patterning PC gene expression and foliation. Indeed, we discovered that En1/2 are necessary for the precise targeting of mossy fibers to distinct lobules, as well as their subsequent resolution into discrete parasagittal bands. Moreover, each En gene coordinately regulates afferent targeting and the striped pattern of PC protein expression (e.g., ZebrinII/AldolaseC) independent of regulating foliation. We further found that En1/2, rather than the presence of a full complement of lobules, are critical for generating PC protein stripes and mossy fiber bands, and that PC striped gene expression is determined before afferent banding. Thus, the En transcription factors not only regulate cerebellum circuit topography, but they also link afferent and efferent neurons precisely enough that alterations in PC protein expression can be used as a read out for underlying defects in circuitry. In summary, our data suggest that En1/2 are master regulators of three-dimensional organization of the cerebellum and coordinately regulate morphology, patterned gene expression, and afferent topography.
| S-EPMC2921890 | biostudies-literature
Project description:Association analysis identified the homeobox transcription factor, ENGRAILED 2 (EN2), as a possible autism spectrum disorder (ASD) susceptibility gene (ASD [MIM 608636]; EN2 [MIM 131310]). The common alleles (underlined) of two intronic single nucleotide polymorphisms (SNPs), rs1861972 (A/G) and rs1861973 (C/T), are over-transmitted to affected individuals both singly and as a haplotype in three separate datasets (518 families total, haplotype p = .00000035).Further support that EN2 is a possible ASD susceptibility gene requires the identification of a risk allele, a DNA variant that is consistently associated with ASD but is also functional. To identify possible risk alleles, additional association analysis and linkage disequilibrium (LD) mapping were performed. Candidate polymorphisms were then tested for functional differences by luciferase (Luc) reporter transfections and electrophoretic mobility shift assays (EMSAs).Association analysis of additional EN2 polymorphisms and LD mapping with Hapmap SNPs identified the rs1861972-rs1861973 haplotype as the most appropriate candidate to test for functional differences. Luciferase reporters for the two common rs1861972-rs1861973 haplotypes (A-C and G-T) were then transfected into human and rat cell lines as well as primary mouse neuronal cultures. In all cases the A-C haplotype resulted in a significant increase in Luc levels (p < .005). The EMSAs were then performed, and nuclear factors were bound specifically to the A and C alleles of both SNPs.These data indicate that the A-C haplotype is functional and, together with the association and LD mapping results, supports EN2 as a likely ASD susceptibility gene and the A-C haplotype as a possible risk allele.
| S-EPMC2783416 | biostudies-literature
Project description:Engrailed variant-2 (EN2) has been suggested as a potential diagnostic biomarker; however, its presence and functional role in prostate cancer (PCa) cells is still controversial or unknown. Here, we analyzed 1) the expression/secretion profile of EN2 in five independent samples cohorts from PCa patients and controls (prostate tissues and/or urine) to determine its utility as a PCa biomarker; and 2) the functional role of EN2 in normal (RWPE1) and tumor (LNCaP/22Rv1/PC3) prostate cells to explore its potential value as therapeutic target. EN2 was overexpressed in our two cohorts of PCa tissues compared to control and in tumor cell lines compared with normal-like prostate cells. This profile was corroborated in silico in three independent data sets [The Cancer Genome Atlas(TCGA)/Memorial Sloan Kettering Cancer Center (MSKCC)/Grasso]. Consistently, urine EN2 levels were elevated and enabled discrimination between PCa and control patients. EN2 treatment increased cell proliferation in LNCaP/22Rv1/PC3 cells, migration in RWPE1/PC3 cells, and PSA secretion in LNCaP cells. These effects were associated, at least in the androgen-sensitive LNCaP cells, with increased AKT and androgen-receptor phosphorylation levels and with modulation of key cancer-associated genes. Consistently, EN2 treatment also regulated androgen-receptor activity (full-length and splicing variants) in androgen-sensitive 22Rv1 cells. Altogether, this study demonstrates the potential utility of EN2 as a non-invasive diagnostic biomarker for PCa and provides novel and valuable information to further investigate its putative utility to develop new therapeutic tools in PCa.
| S-EPMC6780828 | biostudies-other
Project description:Our previous research involving 167 nuclear families from the Autism Genetic Resource Exchange (AGRE) demonstrated that two intronic SNPs, rs1861972 and rs1861973, in the homeodomain transcription factor gene ENGRAILED 2 (EN2) are significantly associated with autism spectrum disorder (ASD). In this study, significant replication of association for rs1861972 and rs1861973 is reported for two additional data sets: an independent set of 222 AGRE families (rs1861972-rs1861973 haplotype, P=.0016) and a separate sample of 129 National Institutes of Mental Health families (rs1861972-rs1861973 haplotype, P=.0431). Association analysis of the haplotype in the combined sample of both AGRE data sets (389 families) produced a P value of .0000033, whereas combining all three data sets (518 families) produced a P value of .00000035. Population-attributable risk calculations for the associated haplotype, performed using the entire sample of 518 families, determined that the risk allele contributes to as many as 40% of ASD cases in the general population. Linkage disequilibrium (LD) mapping with the use of polymorphisms distributed throughout the gene has shown that only intronic SNPs are in strong LD with rs1861972 and rs1861973. Resequencing and association analysis of all intronic SNPs have identified alleles associated with ASD, which makes them candidates for future functional analysis. Finally, to begin defining the function of EN2 during development, mouse En2 was ectopically expressed in cortical precursors. Fewer En2-transfected cells than controls displayed a differentiated phenotype. Together, these data provide further genetic evidence that EN2 might act as an ASD susceptibility locus, and they suggest that a risk allele that perturbs the spatial/temporal expression of EN2 could significantly alter normal brain development.
| S-EPMC1271392 | biostudies-literature
Project description:During embryonic development, the telecephalon undergoes extensive growth and cleaves into right and left cerebral hemispheres. Although molecular signals have been implicated in this process and linked to congenital abnormalities, few studies have examined the role of mechanical forces. In this study, we quantified morphology, cell proliferation and tissue growth in the forebrain of chicken embryos during Hamburger-Hamilton stages 17-21. By altering embryonic cerebrospinal fluid pressure during development, we found that neuroepithelial growth depends on not only chemical morphogen gradients but also mechanical feedback. Using these data, as well as published information on morphogen activity, we developed a chemomechanical growth law to mathematically describe growth of the neuroepithelium. Finally, we constructed a three-dimensional computational model based on these laws, with all parameters based on experimental data. The resulting model predicts forebrain shapes consistent with observations in normal embryos, as well as observations under chemical or mechanical perturbation. These results suggest that molecular and mechanical signals play important roles in early forebrain morphogenesis and may contribute to the development of congenital malformations.
| S-EPMC6826035 | biostudies-literature