The genetics of early cerebellar development: networks not pathways.
Explore the source record for details and available documents.
Animals↗
SEARCH · PubMed Health
Results for “Engrailed 2 Protein”
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Ocular and cerebellar defects in zebrafish induced by overexpression of the LIM domains of the islet-3 LIM/homeodomain protein.
Islet-3 is an LIM/homeodomain protein that is expressed specifically in the eyes and the presumptive tectum in the central nervous system of zebrafish (Danio rerio) embryos. Overexpression of the protein (LIM(Isl-3)) consisting only of the Islet-3 LIM domains in embryos specifically prevented formation of the optic vesicles; caused abnormal termination of the expression of wnt1, engrailed2, and pax2 in the mesencephalic and metencephalic region between 14 hr and 20 hr postfertilization; and severely impaired morphogenetic movement in this region between 20 hr and 26 hr, which should normally lead to formation of the cerebellar primordium. Such defects were all rescued by simultaneous overexpression of Islet-3, suggesting that LIM(Isl-3) acted as a specific dominant-negative variant of Islet-3. These data, combined with the results of mosaic analyses, suggest that Islet-3 is activated by putative LIM-binding cofactors and functions to promote evagination of the optic vesicles and to maintain reciprocal interaction between the mesencephalon and the mesencephalic-metencephalic boundary essential for normal development of this region.
Animals↗
Direct regulation of the Xenopus engrailed-2 promoter by the Wnt signaling pathway, and a molecular screen for Wnt-responsive genes, confirm a role for Wnt signaling during neural patterning in Xenopus.
The co-activation of Wnt signaling and concomitant inhibition of BMP signaling has previously been implicated in vertebrate neural patterning, as evidenced by the combinatorial induction of engrailed-2 and krox-20 in Xenopus. However, screens have not previously been conducted to identify additional potential target genes. Using a PCR-based screening method we determined that XA-1, xCRISP, UVS.2, two UVS.2-related genes, and xONR1 are induced in response to Xwnt-3a and a BMP-antagonist, noggin. Two additional genes, connexin 30 and retinoic acid receptor gamma were induced by Xwnt-3a alone. To determine whether any of the induced genes are direct targets of Wnt signaling, we focussed on engrailed-2. In the present study we show that the Xenopus engrailed-2 promoter contains three consensus binding sites for LEF/TCF, which are HMG box transcription factors which bind to beta-catenin in response to activation of the Wnt- 1 signaling pathway. An engrailed-2 promoter luciferase reporter construct containing these LEF/TCF sites is induced in embryo explant assays by the combination of Xwnt-3a or beta-catenin and noggin. These LEF/TCF sites are required for expression of engrailed-2, as a dominant negative Xtcf-3 blocks expression of endogenous engrailed-2 as well as expression of the reporter construct. Moreover, mutation of these three LEF/TCF sites abrogates expression of the reporter construct in response to noggin and Xwnt-3a or beta-catenin. We conclude that the engrailed-2 gene is a direct target of the Wnt signaling pathway, and that Wnt signaling works with BMP antagonists to regulate gene expression during patterning of the developing nervous system of Xenopus.
Animals↗
Midbrain development induced by FGF8 in the chick embryo.
Vertebrate midbrain development depends on an organizing centre located at the isthmus, a constriction in the embryonic mid/hindbrain region. Isthmic tissue grafts transform chick caudal forebrain into an ectopic midbrain that is the mirror image of the normal midbrain. Here we report that FGF8 protein has the same midbrain-inducing and polarizing effect as isthmic tissue. Moreover, FGF8 induces ectopic expression in the forebrain of genes normally expressed in the isthmus, suggesting that the ectopic midbrain forms under the influence of signals from a new 'isthmus-like' organizing centre induced in the forebrain. Because Fgf8 itself is expressed in the isthmus, our results identify FGF8 as an important signalling molecule in normal midbrain development.
Animals↗
Photo-mediated gene activation using caged RNA/DNA in zebrafish embryos.
We report a new and simple technique for photo-mediated temporal and spatial control of gene activation in zebrafish embryos as an alternative to the gene 'knockdown' approach using antisense, morpholino-modified oligonucleotides (morpholinos). The synthetic compound 6-bromo-4-diazomethyl-7-hydroxycoumarin (Bhc-diazo) forms a covalent bond with the phosphate moiety of the sugar-phosphate backbone of RNA, a process known as caging. The 6-bromo-7-hydroxycoumarin-4-ylmethyl (Bhc) group binds to approximately 30 sites on the phosphate moieties per 1 kb of RNA sequence. Bhc-caged mRNA undergoes photolysis (uncaging) when exposed to long-wave ultraviolet light (350 to 365 nm). We show that Bhc-caged green fluorescent protein (Gfp) mRNA has severely reduced translational activity in vitro, whereas illumination of Bhc-caged mRNA with ultraviolet light leads to partial recovery of translational activity. Bhc-caged mRNA is highly stable in zebrafish embryos. In embryos injected with Bhc-caged Gfp mRNA at the one-cell stage, GFP protein expression and fluorescence is specifically induced by ultraviolet light. We also show that, consistent with results obtained using other methods, uncaging eng2a (which encodes the transcription factor Engrailed2a) in the head region during early development causes a severe reduction in the size of the eye and enhanced development of the midbrain and the midbrain-hindbrain boundary at the expense of the forebrain.
Animals↗
Crossregulation between En-2 and Wnt-1 in chick tectal development.
En-1, En-2 and Wnt-1 are proposed to be essential signals for the development of the optic tectum in chick embryos. Drosophila engrailed and wingless, homologs of En (En-1 and En-2) and Wnt-1, respectively, have been shown to crossregulate each other. In the present paper, it is reported that crossregulation between En-2 and Wnt-1 is preserved in the development of the chick optic tectum. When En-2 is overexpressed by the replication competent retroviral vector, Wnt-1 is expressed ectopically at the dorsal midline of the diencephalon. When Wnt-1 is introduced extrinsically either by ectopic transplantation of mesencephalon, or by implantation of Wnt-1 producing cells, En-2 is induced ectopically at the dorsal midline of the tel-diencephalic border. Thus, ectopic expression of En-2 and Wnt-1 leads to crossregulation of each other in the chick brain. As diencephalon transdifferentiates into the optic tectum by an appropriate signal, the crossregulation of En-2 and Wnt-1 in the diencephalon may mimic the relationship required for early development in the tectum.
Animals↗
Role of Pax-5 in the regulation of a mid-hindbrain organizer's activity.
The mes-metencephalic boundary (isthmus) has been suggested to act as an organizer in the development of the optic tectum. Pax-5 was cloned as a candidate for regulator of the organizing center. Isthmus-specific expression of Pax-5 and analogy with the genetic cascade in Drosophila suggest that Pax-5 may be at a higher hierarchical position in the gene regulation cascade of tectum development. To examine this possibility, a gain-of-function experiment on Pax-5 was carried out. In ovo electroporation on E2 chick brain with the eucaryotic expression vector that encodes chick Pax-5 cDNA was used. Not only was a considerable amount of Pax-5 expressed ectopically in the transfected brain, but irregular bulging of the neuroepithelium was induced in the diencephalon and mesencephalon. At Pax-5 misexpressing sites, uptake of BrdU was increased. Histological examination of E7 transfected brain revealed that Pax-5 caused transdifferentiation of diencephalon into the tectum-like structure. In the bulges of the E7 mesencephalon, differentiation of laminar structure was repressed when compared to the normal side. In transfected embryos, En-2, Wnt-1 and Fgf8 were up-regulated ectopically, and Otx2 was down-regulated in the diencephalon to mesencephalon. Moreover, Ephrin-A2, which is expressed specifically in the tectum with a gradient highest at the caudal end, is suggested to be involved in pathfinding of the retinal fibers, and was induced in the bulges. When the mouse Fgf8 expression vector was electroporated, Pax-5 and chick Fgf8 were also induced ectopically. These results suggest that Pax-5, together with Fgf8, hold a higher position in the genetic hierarchy of the isthmus organizing center and regulate its activity.
Amino Acid Sequence↗
Expression of the homeobox-containing genes EN1 and EN2 in human fetal midgestational medulla and cerebellum.
Homeobox-containing genes En-1 and En-2 have been implicated in the control of pattern formation during development of the central nervous system in experimental animals. In order to determine whether the expression of homologous human EN genes can be used as a developmental genetic marker of the arcuate nucleus of the medulla (a putative precerebellar nucleus that shows developmental deficiency in a subset of sudden infant death syndrome [SIDS]), we performed in situ hybridization with human EN1 and EN2 RNA probes in human fetal midgestational medulla and cerebellum (18-21 weeks gestational age, n=4). Expression of EN genes was demonstrated in all neuronal groups of the medulla and throughout the cerebellum. The RNA signal for both EN1 and EN2 was strongest in the cerebellar granule cell layers, white matter of the vermis and flocculus, inferior olive, arcuate nucleus, caudal raphe nuclei, corpus pontobulbare and nucleus ambiguus. Most of the structures that showed the strongest EN signal originate in the rhombic lip. Some of these structures are functionally interconnected, and show pathologic changes in the syndrome of infantile olivopontocerebellar hypoplasia/atrophy. Strong expression of EN signal in the arcuate nucleus could be used as a genetic marker of this nucleus in further developmental studies of the arcuate nucleus in SIDS. Although EN expression is not specific to the arcuate nucleus or to the rhombic lip derivatives, our results suggest that rhombic lip derivatives have the highest levels of EN RNA message among the medullary structures at midgestation.
Cerebellum↗
Knocking genes in instead of out.
Explore the source record for details and available documents.
Animals↗
Engrailed and Fgf8 act synergistically to maintain the boundary between diencephalon and mesencephalon.
Specification of the forebrain, midbrain and hindbrain primordia occurs during gastrulation in response to signals that pattern the gastrula embryo. Following establishment of the primordia, each brain part is thought to develop largely independently from the others under the influence of local organizing centers like the midbrain-hindbrain boundary (MHB, or isthmic) organizer. Mechanisms that maintain the integrity of brain subdivisions at later stages are not yet known. To examine such mechanisms in the anterior neural tube, we have studied the establishment and maintenance of the diencephalic-mesencephalic boundary (DMB). We show that maintenance of the DMB requires both the presence of a specified midbrain and a functional MHB organizer. Expression of pax6.1, a key regulator of forebrain development, is posteriorly suppressed by the Engrailed proteins, Eng2 and Eng3. Mis-expression of eng3 in the forebrain primordium causes downregulation of pax6.1, and forebrain cells correspondingly change their fate and acquire midbrain identity. Conversely, in embryos lacking both eng2 and eng3, the DMB shifts caudally into the midbrain territory. However, a patch of midbrain tissue remains between the forebrain and the hindbrain primordia in such embryos. This suggests that an additional factor maintains midbrain cell fate. We find that Fgf8 is a candidate for this signal, as it is both necessary and sufficient to repress pax6.1 and hence to shift the DMB anteriorly independently of the expression status of eng2/eng3. By examining small cell clones that are unable to receive an Fgf signal, we show that cells in the presumptive midbrain neural plate require an Fgf signal to keep them from following a forebrain fate. Combined loss of both Eng2/Eng3 and Fgf8 leads to complete loss of midbrain identity, resulting in fusion of the forebrain and the hindbrain primordia. Thus, Eng2/Eng3 and Fgf8 are necessary to maintain midbrain identity in the neural plate and thereby position the DMB. This provides an example of a mechanism needed to maintain the subdivision of the anterior neural plate into forebrain and midbrain.
Animals↗
A role for En-2 and other murine homologues of Drosophila segment polarity genes in regulating positional information in the developing cerebellum.
To gain insight into the molecular genetic basis of cerebellar patterning, the expression patterns of many vertebrate homologues of Drosophila segment polarity genes were examined during normal and abnormal cerebellar development, including members of the En, Wnt, Pax, Gli and Dvl gene families. Five of these genes were found to show transient, spatially restricted patterns of expression. Strikingly, expression of En-2, En-1, Wnt-7B and Pax-2 defined eleven similar sagittal domains at 17.5 dpc, reminiscent of the transient sagittal domains of expression of Purkinje cell markers which have been implicated in cerebellar afferent patterning. Postnatally, transient anterior/posterior differences in expression were observed for En-2, En-1, Gli and Wnt-7B dividing the cerebellum into anterior and posterior regions. The expression patterns of these genes were altered in cerebella of En-2 homozygous mutant mice, which show a cerebellar foliation patterning defect. Strikingly, four of the Wnt-7B expression domains that are adjacent to the En-2 domains are lost in En-2 mutant embryonic cerebella. These studies provide the first evidence of a potential network of regulatory genes that establish spatial cues in the developing cerebellum by dividing it into a grid of positional information required for patterning foliation and afferents. Taken together with previous gene expression studies, our data suggests that eleven sagittal domains and at least two anterior/posterior compartments are the basic elements of spatial information in the cerebellum.
Animals↗
Two Pax-binding sites are required for early embryonic brain expression of an Engrailed-2 transgene.
The temporally and spatially restricted expression of the mouse Engrailed (En) genes is essential for development of the midbrain and cerebellum. The regulation of En-2 expression was studied using in vitro protein-DNA binding assays and in vivo expression analysis in transgenic mice to gain insight into the genetic events that lead to regionalization of the developing brain. A minimum En-2 1.0 kb enhancer fragment was defined and found to contain multiple positive and negative regulatory elements that function in concert to establish the early embryonic mid-hindbrain expression. Furthermore, the mid-hindbrain regulatory sequences were shown to be structurally and functionally conserved in humans. The mouse paired-box-containing genes Pax-2, Pax-5 and Pax-8 show overlapping expression with the En genes in the developing brain. Significantly, two DNA-binding sites for Pax-2, Pax-5 and Pax-8 proteins were identified in the 1.0 kb En-2 regulatory sequences, and mutation of the binding sites disrupted initiation and maintenance of expression in transgenic mice. These results present strong molecular evidence that the Pax genes are direct upstream regulators of En-2 in the genetic cascade controlling mid-hindbrain development. These mouse studies, taken together with others in Drosophila and zebrafish on the role of Pax genes in controlling expression of En family members, indicate that a Pax-En genetic pathway has been conserved during evolution.
Animals↗
Evidence that FGF8 signalling from the midbrain-hindbrain junction regulates growth and polarity in the developing midbrain.
The developing vertebrate mesencephalon shows a rostrocaudal gradient in the expression of a number of molecular markers and in the cytoarchitectonic differentiation of the tectum, where cells cease proliferating and differentiate in a rostral to caudal progression. Tissue grafting experiments have implicated cell signalling by the mesencephalic-metencephalic (mid-hindbrain) junction (or isthmus) in orchestrating these events. We have explored the role of Wnt-1 and FGF8 signalling in the regulation of mesencephalic polarity. Wnt-1 is expressed in the caudal mesencephalon and Fgf8 in the most rostral metencephalon. Wnt-1 regulates Fgf8 expression in the adjacent metencephalon, most likely via a secondary mesencephalic signal. Ectopic expression of Fgf8 in the mesencephalon is sufficient to activate expression of Engrailed-2 (En-2) and ELF-1, two genes normally expressed in a decreasing caudal to rostral gradient in the posterior mesencephalon. Ectopic expression of Engrailed-1 (En-1), a functionally equivalent homologue of En-2 is sufficient to activate ELF-1 expression by itself. These results indicate the existence of a molecular hierarchy in which FGF8 signalling establishes the graded expression of En-2 within the tectum. This in turn may act to specify other aspects of A-P polarity such as graded ELF-1 expression. Our studies also reveal that FGF8 is a potent mitogen within the mesencephalon: when ectopically expressed, neural precursors continue to proliferate and neurogenesis is prevented. Taken together our results suggest that FGF8 signalling from the isthmus has a key role in coordinately regulating growth and polarity in the developing mesencephalon.
Animals↗
FGF8 can activate Gbx2 and transform regions of the rostral mouse brain into a hindbrain fate.
The mid/hindbrain junction region, which expresses Fgf8, can act as an organizer to transform caudal forebrain or hindbrain tissue into midbrain or cerebellar structures, respectively. FGF8-soaked beads placed in the chick forebrain can similarly induce ectopic expression of mid/hindbrain genes and development of midbrain structures (Crossley, P. H., Martinez, S. and Martin, G. R. (1996) Nature 380, 66-68). In contrast, ectopic expression of Fgf8a in the mouse midbrain and caudal forebrain using a Wnt1 regulatory element produced no apparent patterning defects in the embryos examined (Lee, S. M., Danielian, P. S., Fritzsch, B. and McMahon, A. P. (1997) Development 124, 959-969). We show here that FGF8b-soaked beads can not only induce expression of the mid/hindbrain genes En1, En2 and Pax5 in mouse embryonic day 9.5 (E9.5) caudal forebrain explants, but also can induce the hindbrain gene Gbx2 and alter the expression of Wnt1 in both midbrain and caudal forebrain explants. We also show that FGF8b-soaked beads can repress Otx2 in midbrain explants. Furthermore, Wnt1-Fgf8b transgenic embryos in which the same Wnt1 regulatory element is used to express Fgf8b, have ectopic expression of En1, En2, Pax5 and Gbx2 in the dorsal hindbrain and spinal cord at E10.5, as well as exencephaly and abnormal spinal cord morphology. More strikingly, Fgf8b expression in more rostral brain regions appears to transform the midbrain and caudal forebrain into an anterior hindbrain fate through expansion of the Gbx2 domain and repression of Otx2 as early as the 7-somite stage. These findings suggest that normal Fgf8 expression in the anterior hindbrain not only functions to maintain development of the entire mid/hindbrain by regulating genes like En1, En2 and Pax5, but also might function to maintain a metencephalic identity by regulating Gbx2 and Otx2 expression.
Animals↗
Selective disruption of "late onset" sagittal banding patterns by ectopic expression of engrailed-2 in cerebellar Purkinje cells.
To explore the role of Engrailed proteins in development of the cerebellum, Engrailed-2 (En-2) was ectopically expressed in cerebellar Purkinje cells from the late embryonic stage into adulthood. The fundamental organization of Purkinje cell sagittal zones as revealed by the "early onset" markers L7-beta-gal and cadherin-8 was found to be virtually identical to that in wild type. In contrast, "late onset" sagittal banding patterns revealed by Purkinje cell markers zebrin I, zebrin II, and 9-O-acetyl GD3 Ganglioside (P-Path), and the granule cell marker NADPH-diaphorase, were disrupted. In general, although some evidence of banding was still detectable, boundaries defined by the latter markers were poorly defined, and the patterns overall took on a diffuse appearance. In parallel with the changes in late onset markers, anterograde tracing of spinocerebellar axons revealed a general diffusion of the mossy fiber projection pattern in lobule VIII and the anterior lobe. These observations suggest that at least two separate mediolateral boundary systems exist in the cerebellum, and these are differentially affected by ectopic En-2 expression. Alternatively, one boundary system exists that remains primarily intact in the mutant, but recognition of this system by a set of late developmental events is perturbed.
Animals↗
Vertical regulation of En-2 expression and eye development by FGFs and BMPs.
The formation of the midbrain region depends mainly on the activity of a signalling center located in the isthmus region, on the border between the prospective mesencephalon and metencephalon. FGF-8 has been proposed as a signalling molecule responsible for this specification because of its expression pattern and its ability to elicit duplication of the midbrain region when expressed ectopically in the neuroepithelium. Here we present evidence that members of the FGF family of growth factors when released in the cephalic mesenchyme are able to extend the expression of the mesencephalic marker En-2 to both the anterior and the posterior regions of its original landmark. This alteration in the expression pattern of En-2 is not accompanied by a significant alteration in the later development of the midbrain-cerebellar anlage, although the eye development is severely altered. Members of the bone morphogenetic protein family ectopically released from the mesenchyme down-regulate the expression of En-2 and also have an effect on the development of the eye. These results demonstrate that growth factor molecules produced in the mesenchyme (vertical signalling) participate in the correct establishment of the antero-posterior patterning of the cephalic nervous system during development.
Animals↗
Midline structures and central nervous system coordinates in zebrafish.
The embryonic zebrafish provides a relatively simple and accessible experimental system for understanding the underlying Bauplan of a vertebrate central nervous system (CNS) and for uncovering interactions critical for patterning. We show that features of CNS organization in normal, mutant, and developmentally ventralized zebrafish embryos can be explained by a three-axis hypothesis: Specifications occur according to position relative to coordinates along the anterior-posterior, dorsal-ventral, and luminal-pial axes of the neural tube. Midline tissues may be responsible for directly establishing the dorsal-ventral coordinates, but may only be indirectly involved in patterning along the anterior-posterior axis.
Animals↗
Regional specification during cerebellar development.
At early phases of development (stage HH12 of chick and quail embryos) the rostral part of the cerebellar anlage extends into the caudal third of the mesencephalic vesicle. This region is characterized by its high level of expression of the Wnt-1 and engrailed genes, which encode, respectively, for a secreted molecule and for a homeodomain protein. In order to analyze the potentialities of various parts of the cerebellar anlage, pieces of quail or mouse embryonic neural tubes have been transplanted heterotopically in the prosencephalon of HH12 chick embryos. These experiments indicated that signals operating in the plane of the neural tube are still operating in the donor avian and murine embryos. The met- mes- and prosencephalic territories of HH12 chick embryos are still competent to change their fates under the influence of these signals. It was also found that the Wnt-1 expression domain, the caudal mesencephalic vesicle, has striking organizing properties until late stages of development in avian and rodent embryos. The exact role of Wnt-1 in these inductive interactions is still unknown. Wnt-1 influence on cell fate could be mediated by the engrailed genes. Observations suggesting a later role of en-2 in the regionalization of the cerebellar cortex are also reported.
Animals↗