Table of contents

Mouse Engrailed: Function in neural development

During mouse development, the homeobox gene En-1 is specifically expressed across the mid-hindbrain junction, the ventral ectoderm of the limb buds, and in regions of the hindbrain, spinal cord, somites and somite-derived tissues. Knock-out mice homozygous for a targeted deletion of En-1 are missing large parts of the brain including most of the colliculi and cerebellum, and the third and fourth cranial nerves. Patterning of the forelimb paws and sternum is disrupted, and the 13th ribs are truncated. Thus En-1 has roles in generation of mid-hindbrain precursor cells and in signaling normal development of the limbs and sternum (Wurst, 1994).

The temporally and spatially restricted expression of the mouse Engrailed (En) genes is essential for development of the midbrain and cerebellum. A minimum En-2 1.0 kb enhancer fragment contains multiple positive and negative regulatory elements that function in concert to establish the early embryonic mid-hindbrain expression. These mid-hindbrain regulatory sequences are 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 have been identified in the 1.0 kb En-2 regulatory sequences: mutation of the binding sites disrupts 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 (Song, 1996).

Axons from the vertebrate eye project to the tectum, a structure in the midbrain that processess visual information. A rostralcaudal gradient exists for both Engrailed homologs correlated with the polarity of the retinotectal projection map in the vertebrate tectum. Scattered engrailed expression, caused by retroviral gene transfer, perturbs this retinotectal order. This suggests that the Engrailed gradient defines positional identity in the tectum (Itasaki, 1996). The Engrailed homolog drives the expression of genes responsible for intrinsic tectal axonal patterning, but not for tectal recognition by retinal axons (Retaux, 1996).

In the mouse, Engrailed-1 is a target of Wnt-1 (See Drosophila Wingless) signaling in the midbrain. In Wnt-1 knockout mice, En1 and En2 are first expressed normally, but subsequently domains of En expression are lost, concomitant with a failure of midbrain and anterior hindbrain (cerebellum) development. Although neither single En mutant has a severe a phenotype, compound mutants have a similar midbrain and anterior hindbrain phenotype to that of Wnt-1 knockouts. An Engrailed-1 transgene can completely or partially rescue Wnt-1 mutants morphologically, and the expression domains of Pax-5, Fgf-8, En, and in a few cases, Wnt-1 are only slightly reduced relative to wild-type littermates. Nevertheless, two cranial motor nerves, III (oculomotor) and IV (trochlear), which normally develop adjacent to Wnt-1-expressing cells, are not present in Wnt-1 knockouts rescued with Engrailed-1. Thus there may be additional functions of Wnt-1 signaling that cannot be replaced by En-1 (Danielian, 1996).

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 signaling by the mesencephalic-metencephalic (mid-hindbrain) junction (or isthmus) in orchestrating these events. The role of Wnt-1 and FGF8 (Drosophila homolog: Branchless) signaling has been explored in the regulation of mesencephalic polarity. FGF8 is expressed in cardiac mesoderm underlying the presumptive mesencephalic/metencephalic region and may play a role in mesencephalic induction. Fgf8 is also expressed in the neural plate itself, in the most rostral metencephalon. Wnt-1 is expressed in the caudal mesencephalon. 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 and ELF-1, two genes normally expressed in a decreasing caudal to rostral gradient in the posterior mesencephalon. ELF-1 is a ligand for a EPH-like receptor tyrosine kinase expressed in rostrocaudally increasing gradients across the caudal tectum and may function to inhibit temporal axon ingrowth and/or to attract nasal axons. Ectopic expression of Engrailed-1, a functionally equivalent homolog of En-2 is sufficient to activate ELF-1 expression by itself. These results indicate the existence of a molecular hierarchy in which FGF8 signaling establishes the graded expression of En-2 within the tectum (Lee, 1997).

Null alleles of the mouse Engrailed-2 gene, a molecular homolog of the fly gene engrailed, have demonstrable effects on the anteroposterior (A/P) patterning of cerebellum as reflected in the disruption of the normal process of foliation of the cerebellar cortex and the alteration of transgene expression boundaries in the adult. Engrailed-2 also affects the transient mediolateral (M/L) pattern of En-1 and Wnt-7b expression seen in late embryogenesis. Three markers of cerebellar compartmentation were examined in En-2 mutant mice: the Zebrin II and Ppath monoclonal antibodies and the transgene L7lacZ. In En-2 mutants, the normal temporal pattern of expression is preserved for all three markers, although the size and spatial location of various bands differ from those of the wild type. Unlike the foliation abnormalities, the M/L pattern disturbances that are found occur in nearly all cerebellar regions. Cell counts reveal that all major cell types of the olivocerebellar circuit are reduced by 30-40%. It is proposed that these results are best explained by a model in which the Engrailed-2 gene is involved in the early specification of the cerebellar field, including the number of progenitors. Because each of these progenitors gives rise to a clone of defined size, Engrailed-2 helps specify adult cell number. It is further postulated that the configuration of the seven Zebrin bands as well as the shapes and locations of the cerebellar lobules are set up by a second patterning event that occurs after neurogenesis is complete (Kuemerle, 1997).

Members of the En and Wnt gene families seem to play a key role in the early specification of the midhindbrain junction, the brain territory that gives rise to the cerebellum. To analyze the possible continuous role of the En and Wnt signaling pathway in later cerebellar patterning and function, En-2 was expressed ectopically in Purkinje cells during late embryonic and postnatal cerebellar development. As a result of this expression, the cerebellum is greatly reduced in size, and Purkinje cell numbers throughout the cerebellum are reduced by more than one-third relative to normal animals. Detailed analysis of both adult and developing cerebella reveals a pattern of selectivity for the loss of Purkinje cells and other cerebellar neurons. This is observed as a general loss of prominence of cerebellar fissures, highlighted by a total loss of sublobular fissures. In contrast, mediolateral patterning is generally only subtly affected. That En-2 overexpression selectively affects Purkinje cells in the transition zone between lobules is evidenced by direct observation of selective Purkinje cell loss in certain fissures and by the observation that growth and migration of the external germinal layer (EGL) is selectively retarded in the deep fissures during early postnatal development. Thus, in addition to demonstrating the critical role of Purkinje cells in the generation and migration of granule cells, the heterogeneous distribution of cellular effects induced by ectopic En expression suggests a relatively late morphogenetic role for this and other segment polarity proteins, mainly oriented at lobule junctions (Baader, 1998).

Genes encoding fibroblast growth factors (FGFs) are expressed in early Xenopus neurulae in the prospective midbrain--hindbrain boundary (MHB) region of the neural plate. These expression domains overlap those of XWnt-1 and XEn-2, raising the question of the role of FGF signaling in the regulation of these genes, and more generally about the function of FGF during Xenopus midbrain development. Explants from the prospective MHB grafted into the anterior neural plate in midneurula stage embryos induce XWnt-1 expression and, at a lower frequency, XEn-2 expression in the vicinity of the graft. Such a process is likely to involve FGF signaling. Implantation of FGF4- or FGF8-soaked beads in the prospective forebrain at neurula and tailbud stages causes the up-regulation of XWnt-1 and XEn-2 in the dorsal and lateral region of the anterior midbrain. This effect is not relayed by endogenous FGF genes since exogenous FGFs inhibit the expression of endogenous XFGF3 or XFGF8. However, consequences of grafting MHB or implanting FGF4 or FGF8 beads on tadpole brain development are different. MHB grafts induce ectopic mesencephalic structures, strongly suggesting that a region homologous to the isthmic organizer of amniotes is specified as early as the midneurula stage. In contrast, exogenous FGFs do not cause the formation of ectopic mesencephalic structures but an overgrowth of mesencephalon and diencephalon. It is proposed that FGF signals from the prospective MHB play a crucial role in the spatial regulation of XWnt-1 and XEn-2 expression in the posterior midbrain, but that the full organizing activity of the MHB involves other factors in combination with FGF (Riou, 1998).

In vertebrates, the engrailed genes are expressed at early neurula stage in a narrow stripe encompassing the midbrain-hindbrain boundary (MHB), a region from which a peculiar structure, the isthmus, is formed. Knock-out experiments in mice have demonstrated that these genes are essential for the development of this structure and of its derivatives. In contrast, little is known about the effect of an overexpression of engrailed genes in vertebrate development. The isolation of Ol-eng2, a medaka fish (Oryzias latipes) engrailed gene is reported here. The effects of its widespread expression following mRNA injections in 1- and 2-cell medaka and Xenopus embryos is reported. The ectopic expression of Ol-eng2 predominantly results in an altered development of the anterior brain, including an inhibition of optic vesicle formation. No change in the patterns of mesencephalic and telencephalic markers was observed. In contrast, expressions of markers of the diencephalon are strongly repressed in injected embryos. Furthermore, the endogenous Ol-eng2, Pax2, Wnt1 and Fgf8, which are essential components of the MHB genetic cascade, are ectopically expressed in this region. Therefore, it is proposed that Ol-eng2 induces de novo formation of an isthmus-like structure, which correlates with the development of ectopic midbrain structures, including optic tectum. A competence of the diencephalon to change to a midbrain fate has been demonstrated in isthmic graft experiments. These data demonstrate that this change can be mimicked by ectopic engrailed expression alone (Ristoratore, 1999).

Thus, Ol-eng2 overexpression leads to the ectopic activation of MHB marker genes. In order to better understand the temporal sequence that leads to this transformation, the expression patterns of several genes expressed sequentially in this region were studied. Otx 1/2 and Gbx2 start to be expressed during gastrulation and have been shown by genetic analysis to be required for the normal onset of the MHB region. However, at the stages examined, the patterns of expression of these two genes appear unchanged in injected medaka embryos. This indicates that Ol-eng2 overexpression does not interfere with the development of the anterior region at this stage. Similarly, the MHB Pax2 expression domain is not modified in late gastrula-stage embryos. Accordingly, it has been proposed that, in zebrafish, an initial phase in MHB establishment is the activation of several independent pathways, involving Pax2 and Wnt1, which act in parallel to activate engrailed genes and set up midbrain and hindbrain primordia. The lack of ectopic activation of MHB genes at such stages shows that molecular pathways that act in the diencephalon are not influenced by ectopic Ol-eng2 and that a time-dependent competence exists in this region. In contrast, when the expression of MHB genes was monitored later (early somitogenesis), an ectopic domain of Pax2 and Wnt1 expression is observed in embryos overexpressing Ol-eng2. Therefore Ol-eng2, when ectopically produced in the diencephalon, is sufficient to activate the expression of other genes in the MHB genetic cascade, but only during a late phase of MHB establishment. In this context, it has been proposed that during a late maintenance phase, expressions of Wnt1, Pax2 and eng2/3 become mutually dependent. This result is also reminiscent of those obtained in Drosophila embryos at postblastoderm stage, where engrailed and wingless gene expression becomes dependent on the expression of one another in adjacent cells. These reciprocal interactions normally observed in the MHB would therefore be forced by the ectopic presence of the Ol-eng2 protein in the diencephalon at early stages (Ristoratore, 1999).

Transcriptional factors and signaling molecules are responsible for regionalization of the central nervous system. In the early stage of neural development, Pax6 is expressed in the prosencephalon, while En1 and Pax2 are expressed in the mesencephalon. Pax6 was misexpressed in the mesencephalon to elucidate the mechanism of the di-mesencephalic boundary formation. Histological analysis, expression patterns of diencephalic marker genes, and fiber trajectory of the posterior commissure indicate that Pax6 misexpression causes a caudal shift of the di-mesencephalic boundary. Pax6 represses En1, Pax2 and other tectum (mesencephalon)-related genes such as En2, Pax5, Pax7, but induces Tcf4, a diencephalon marker gene. To know how Pax6 represses En1 and Pax2, a dominant-active or negative form of Pax6 was ectopically expressed. The dominant-active form of Pax6 shows a similar but more severe phenotype than Pax6, while the dominant-negative form shows an opposite phenotype, suggesting that Pax6 acts as a transcriptional activator. Thus Pax6 may repress tectum-related genes by activating an intervening repressor. The results of misexpression experiments, together with normal expression patterns of Pax6, En1 and Pax2, suggest that repressive interaction between Pax6 and En1/Pax2 defines the di-mesencephalic boundary (Matsunaga, 2000).

Mutant cells result in a reduction or loss of expression of Rpx/Hesx1, Wnt1, R-cadherin and ephrin-A2, while expression of En2 and Six3 is rescued by surrounding wild-type cells. Forebrain Otx2 mutant cells subsequently undergo apoptosis. In the forebrain, Otx2 is required to activate the expression of the homeobox gene Rpx and maintain the expression of another homeobox gene, Six3. To determine if Otx2 is required cell autonomously or non-cell autonomously to regulate expression of these genes, the forebrain of moderate chimeric embryos was analyzed in double-labelling experiments, using histochemical staining for beta-galactosidase activity to distinguish WT from Otx2 mutant cells, and whole-mount RNA in situ hybridization to characterize Rpx or Six3 expression. Rpx is expressed in the forebrain of control embryos at E8.5. In moderate chimeras, Rpx expression is absent from the patches of Otx2 mutant cells, but is present in the surrounding WT forebrain cells. At the border of the mutant cell patches, Otx2 mutant cells fail to express Rpx while neighboring WT forebrain cells maintain expression of the gene. The strict correlation at the cellular level between lack of Otx2 activity and loss of Rpx expression demonstrates that Otx2 is required cell autonomously for expression of this gene in the forebrain. In contrast, Six3, another homeobox gene expressed in the forebrain, is expressed in groups of Otx2 mutant cells as in surrounding WT cells in moderate chimeras at E8.5, indicating that Otx2 is required non-cell autonomously for maintenance of Six3 expression. Thus, Otx2 regulates expression of different regulatory genes in the forebrain through distinct pathways. Similar results were obtained for the regulation of gene expression in the mid-hindbrain region. Otx2 is required for the activation of expression of the signaling molecule Wnt1 and for the maintenance of expression of the homeobox gene En2. Wnt1 expression is observed in WT midbrain cells in control embryos and moderate chimeras but is not detected in any Otx2 mutant cells in the midbrain of moderate chimeras, including those in contact with WT cells. This result demonstrates that Otx2 is required cell autonomously in midbrain cells to activate Wnt1 expression. In contrast, En2 expression in Otx2 mutant cells in the mid-hindbrain of moderate chimeras is rescued by the presence of surrounding WT cells, demonstrating a non-cell autonomous function for Otx2 in regulating En2 expression. Therefore, Otx2 also regulates the expression of mid-hindbrain genes through different mechanisms. Altogether, this study demonstrates that Otx2 is an important regulator of brain patterning and morphogenesis, through its regulation of candidate target genes such as Rpx/Hesx1, Wnt1, R-cadherin and ephrin-A2 (Rhinn, 1999).

Fgf8, which is expressed at the embryonic mid/hindbrain junction, is required for and sufficient to induce the formation of midbrain and cerebellar structures. To address the genetic pathways through which FGF8 acts, the epistatic relationships of mid/hindbrain genes that respond to FGF8 were examined, using a novel mouse brain explant culture system. En2 and Gbx2 are the first genes to be induced by FGF8 in wild-type E9.5 diencephalic and midbrain explants treated with FGF8-soaked beads. By examining gene expression in En1/2 double mutant mouse embryos, it was found that Fgf8, Wnt1 and Pax5 do not require the En genes for initiation of expression, but do for their maintenance, and Pax6 expression is expanded caudally into the midbrain in the absence of EN function. Since E9.5 En1/2 double mutants lack the mid/hindbrain region, forebrain mutant explants were treated with FGF8 and, significantly, the EN transcription factors were found to be required for induction of Pax5. Thus, FGF8-regulated expression of Pax5 is dependent on EN proteins, and a factor other than FGF8 could be involved in initiating normal Pax5 expression in the mesencephalon/metencephalon. The En genes also play an important, but not absolute, role in repression of Pax6 in forebrain explants by FGF8. Gbx2 gain-of-function studies have shown that misexpression of Gbx2 in the midbrain can lead to repression of Otx2. However, in the absence of Gbx2, FGF8 can nevertheless repress Otx2 expression in midbrain explants. In contrast, Wnt1 is initially broadly induced in Gbx2 mutant explants, as in wild-type explants, but not subsequently repressed in cells near FGF8 that normally express Gbx2. Thus GBX2 acts upstream of, or parallel to, FGF8 in repressing Otx2, and acts downstream of FGF8 in repression of Wnt1. This is the first such epistatic study performed in mouse that combines gain-of-function and loss-of-function approaches to reveal aspects of mouse gene regulation in the mesencephalon/metencephalon that have been difficult to address using either approach alone (Liu, 2001).

Deficiencies in neurotransmitter-specific cell groups in the midbrain result in prominent neural disorders, including Parkinson's disease, which is caused by the loss of dopaminergic neurons of the substantia nigra. The role of the Engrailed homeodomain transcription factors, En-1 and En-2, in controlling the developmental fate of murine midbrain dopaminergic neurons has been investigated. En-1 is highly expressed by essentially all dopaminergic neurons in the substantia nigra and ventral tegmentum, whereas En-2 is highly expressed by a subset of them. These neurons are generated and differentiate their dopaminergic phenotype in En-1/En-2 double null mutants, but disappear soon thereafter. Use of an En-1/tau-LacZ knock-in mouse as an autonomous marker for these neurons indicates that they are lost, rather than that they change their neurotransmitter phenotype. A single allele of En-1 on an En-2 null background is sufficient to produce a wild type-like substantia nigra and ventral tegmentum, whereas in contrast a single allele of En-2 on an En-1 null background results in the survival of only a small proportion of these dopaminergic neurons, a finding that relates to the differential expression of En-1 and En-2. Additional findings indicate that En-1 and En-2 regulate expression of alpha-synuclein, a gene that is genetically linked to Parkinson's disease. These findings show that the engrailed genes are expressed by midbrain dopaminergic neurons from their generation to adulthood but are not required for their specification. However, the engrailed genes control the survival of midbrain dopaminergic neurons in a gene dose-dependent manner. These findings also suggest a link between engrailed and Parkinson's disease (Simon, 2001).

Interneurons in the ventral spinal cord are essential for coordinated locomotion in vertebrates. During embryogenesis, the V0 and V1 classes of ventral interneurons are defined by expression of the homeodomain transcription factors Evx1/2 and En1, respectively. Evx1 V0 interneurons are locally projecting intersegmental commissural neurons. In Evx1 mutant embryos, the majority of V0 interneurons fail to extend commissural axons. Instead, they adopt an En1-like ipsilateral axonal projection and ectopically express En1, indicating that V0 interneurons are transfated to a V1 identity. Conversely, misexpression of Evx1 represses En1, suggesting that Evx1 may suppress the V1 interneuron differentiation program. These findings demonstrate that Evx1 is a postmitotic determinant of V0 interneuron identity and reveal a critical postmitotic phase for neuronal determination in the developing spinal cord (Moran-Rivard, 2001).

The pax2.1 gene encodes a paired-box transcription factor that is one of the earliest genes to be specifically activated in development of the midbrain and midbrain-hindbrain boundary (MHB), and is required for the development and organizer activity of this territory. To understand how this spatially restricted transcriptional activity of pax2.1 is achieved, the pax2.1-promoter has been isolated and characterized using a lacZ and a GFP reporter gene in transient injection assays and transgenic lines. Stable transgenic expression of this reporter gene shows that a 5.3-kb fragment of the 5' region contains most, but not all, elements required for driving pax2.1 expression. The expressing tissues include the MHB, hindbrain, spinal cord, ear and pronephros. Transgene activation in the pronephros and developing ear suggests that these pax2.1-expressing tissues are composed of independently regulated subdomains. In addition, ectopic but spatially restricted activation of the reporter genes in rhombomeres 3 and 5 and in the forebrain, none of which normally express endogenous pax2.1, demonstrates the importance of negative regulation of pax2.1. Comparison of transgene expression in wild-type and homozygous pax2.1 mutant no isthmus (noi) embryos reveals that the transgene contains control element(s) for a novel, positive transcriptional feedback loop in MHB development. Transcription of endogenous pax2.1 at the MHB is known to be initially Pax2.1 independent, during activation in late gastrulation. In contrast, transgene expression requires the endogenous Pax2.1 function. Transplantations, mRNA injections and morpholino knock-down experiments show that this feedback regulation of pax2.1 transcription occurs cell-autonomously, and that it requires the engrailed-type genes eng2 and eng3 as known targets for Pax2.1 regulation. eng2 and eng3 are necessary to maintain, but not initiate, expression of pax2.1 at the MHB. It is suggested that this novel feedback loop may allow continuation of pax2.1 expression, and hence development of the MHB organizer, to become independent of the patterning machinery of the gastrula embryo (Picke, 2002).

The ectopic activation of placZ5.3 in the fore- and hind-brain, in addition to the pax2.1-like domain at the MHB, is especially evident at the 20-somite stage where the overall expression pattern has a 'multiple-stripe' appearance, akin to a segmental pattern. The Drosophila Pax-2 ortholog shaven (DPax2), is at embryonic stages expressed in a segmental pattern in the developing external sensory organs of the CNS and PNS. There is as yet no direct evidence for a similar metameric expression pattern of Pax2 orthologs in other organisms, although it has been previously noted that pax2.1-expressing interneurons in the hindbrain and spinal cord form repetitive clusters along the AP axis of the embryo. However, the Drosophila engrailed ortholog AmphiEn of the basal chordate Amphioxus is expressed in 'metameric' stripes along the AP axis of the segmentally organized mesoderm, suggesting a relationship between segmentation in protostomes and deuterostomes. Although it is unclear whether metameric subdivisions exist in the midbrain and isthmic regions, metamerism is a well established concept for the vertebrate hindbrain. Therefore, an interesting possibility is presented: that the partially metameric pattern produced by the pax2.1 promoter/enhancer fragment reflects an ancestral state of pax2 gene regulation in a 'metameric' pattern, which in modern vertebrates, possibly through evolution of additional silencer elements, became restricted to one stripe at the MHB (Picke, 2002).

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, the establishment and maintenance of the diencephalic-mesencephalic boundary (DMB) was studied. 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. Fgf8 is a candidate for this signal, because it is both necessary and sufficient to repress pax6.1 and hence to shift the DMB to the anterior, independent of the expression status of eng2/eng3. By examining small cell clones that are unable to receive an Fgf signal, it has been shown 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 (Scholpp, 2003).

The neuropathological hallmark of Parkinson’s disease is the loss of dopaminergic neurons in the substantia nigra pars compacta, presumably mediated by apoptosis. The homeobox transcription factors engrailed 1 and engrailed 2 are expressed by this neuronal population from early in development to adulthood. Despite a large mid-hindbrain deletion in double mutants null for both genes, mesencephalic dopaminergic (mDA) neurons are induced, become postmitotic and acquire their neurotransmitter phenotype. However, at birth, no mDA neurons are left. The entire population of these neurons is lost by E14 in the mutant animals, earlier than in any other described genetic model system for Parkinson’s disease. This disappearance is caused by apoptosis revealed by the presence of activated caspase 3 in the dying tyrosine hydroxylase-positive mutant cells. Furthermore, using in vitro cell mixing experiments and RNA interference on primary cell culture of ventral midbrain, the demise of mDA neurons in the mutant mice was shown to be due to a cell-autonomously requirement of the engrailed genes and not a result of the missing mid-hindbrain tissue. Gene silencing in the postmitotic neurons by RNA interference activates caspase 3 and induces apoptosis in less than 24 hours. This rapid induction of cell death in mDA neurons suggests that the engrailed genes participate directly in the regulation of apoptosis, a proposed mechanism for Parkinson’s disease (Alberi, 2004).

Pbx proteins are a family of TALE-class transcription factors that are well characterized as Hox co-factors acting to impart segmental identity to the hindbrain rhombomeres. However, no role for Pbx in establishing more anterior neural compartments has been demonstrated. Studies done in Drosophila show that Engrailed requires Exd (Pbx orthologue) for its biological activity. Evidence is presented that zebrafish Pbx proteins cooperate with Engrailed to compartmentalize the midbrain by regulating the maintenance of the midbrain–hindbrain boundary (MHB) and the diencephalic–mesencephalic boundary (DMB). Embryos lacking Pbx function correctly initiate midbrain patterning, but fail to maintain eng2a, pax2a, fgf8, gbx2, and wnt1 expression at the MHB. Formation of the DMB is also defective as shown by a caudal expansion of diencephalic epha4a and pax6a expression into midbrain territory. These phenotypes are similar to the phenotype of an Engrailed loss-of-function embryo, supporting the hypothesis that Pbx and Engrailed act together on a common genetic pathway. Consistent with this model, it has been demonstrated that zebrafish Engrailed and Pbx interact in vitro and that this interaction is required for both the eng2a overexpression phenotype and Engrailed's role in patterning the MHB. The data support a novel model of midbrain development in which Pbx and Engrailed proteins cooperatively pattern the mesencephalic region of the neural tube (Erickson, 2007).

Engrailed and the genetic subdivision of the tectum and cerebellum

The genetic pathways that partition the developing nervous system into functional systems are largely unknown. The engrailed (En) homeobox transcription factors are candidate regulators of this process in the dorsal midbrain (tectum) and anterior hindbrain (cerebellum). En1 mutants lack most of the tectum and cerebellum and die at birth, whereas En2 mutants are viable with a smaller cerebellum and foliation defects. The difference in phenotypes is due to the earlier expression of En1 as compared with En2, rather than differences in protein function, since knock-in mice expressing En2 in place of En1 have a normal brain. This study uncovered a wider spectrum of functions for the En genes by generating a series of En mutant mice. First, using a conditional allele it was demonstrated that En1 is required for cerebellum development only before embryonic day 9, but plays a sustained role in forming the tectum. Second, by removing the endogenous En2 gene in the background of En1 knock-in alleles, it was shown that Drosophila en (En1Denki that express Drosophila en in place of En1) is not sufficient to sustain midbrain and cerebellum development in the absence of En2, whereas En2 is more potent than En1 in cerebellum development. Third, based on a differential sensitivity to the dose of En1/2, these studies reveal a genetic subdivision of the tectum into its two functional systems and the medial cerebellum into four regions that have distinct circuitry and molecular coding. This study suggests that an 'engrailed code' is integral to partitioning the tectum and cerebellum into functional domains (Sgaier, 2007).

This study analyzed a series of mouse En conditional knock-in and null mutants to decipher the overlapping and individual functions of the two highly conserved En genes in mes/r1 development. Overall, it was found that the inferior colliculus of the tectum and three regions of the cerebellum (Cb) are particularly sensitive to the level of En genes. Interestingly, the anterior vermis and tectum defects were observed in En1flox/cre, En1Denki/Denki;En2-/+ and En1-/+;En2-/+ mutant mice have similarities to Fgf8-/+;Fgf17-/- mutants, raising the possibility that a key role of En1/2 is to maintain Fgf8 expression. Fgf8 expression is maintained as long as one allele of En1 is present, although there are subtle decreases in Fgf8/17 expression. Since En1/2 expression persists after E12.5, when Fgf8 expression is terminated, and En1-/+;En2-/- mutants have a much more severe loss of the tectum and Cb folia than Fgf8-/+;Fgf17-/- mutants, it is possible that En1/2 do not control mes/r1 development solely through regulating Fgf8/17 expression (Sgaier, 2007).

By determining the fate of the En1-expressing cells at ~E11, which normally give rise to the vermis and inferior colliculus, in En1-/+;En2-/- mutants using genetic inducible fate mapping (GIFM), an unexpected differential role was uncovered for En1/2 in regulating growth and survival of cells in the tectum versus the Cb. In En1/2 mutants, the posterior mes cells marked at E12.5 do not expand normally and this results in a smaller inferior colliculus in the adult. By contrast, the anterior r1 cells marked at E12.5 are not only retained but contribute to more lateral regions of the vermis than normal. If the lineage restriction at the mes/r1 border that restricts mes and r1 cells from mixing is disrupted in En1/2 mutants, then it is possible that the population of marked mes cells in En1CreERT1/+;En2-/-;R26R/+ embryos move into r1 and expand the marked population in the Cb. Another possibility is that the precursors of the lateral Cb are selectively lost in the mutants. However, this is not in accordance with the observation that the hemispheres of En1-/+;En2-/- adults are less compromised than the vermis. Although the ultimate overall loss of cells in the mes and r1 of En1-/+;En2-/- mutants could be accounted for by cell death, similar to the situation in En1 mutants, fate mapping study shows that it is not as simple as the cells being lost equally on either side of the isthmus (Sgaier, 2007).

Consistent with the En1 expression domain being encompassed by the En2 domain after E9, it was found that En1CreERT1/flox conditional mutants are viable and have a normal Cb and superior colliculus. However, despite strong expression of En2 in the posterior mes after E9 when En1 is deleted in these mutants, the inferior colliculus does not develop normally in En1flox/Cre mutants. These results indicate that the En1 protein has a different function from En2, or that En1 and En2 are expressed differently in the tectum after E9. The latter is the case, because the tectum develops normally when En2 is produced from the En1 locus in the absence of endogenous En2 (En1En2ki/En2ki;En2-/- mice). Based on expression analysis, the crucial difference must be that En1 is transiently expressed around E9 in a broader domain than En2, or that En1 is later produced at a higher level than En2. Regardless, these studies have uncovered a differential requirement for the two En genes in the superior and inferior colliculi (Sgaier, 2007).

Analysis of En1/2 double-mutant combinations (null, knock-in and conditional) uncovered additional differential requirements for En1 and En2 in specific regions of the Cb. En1/2 functions are normally uncoupled in the hemispheres as only En2 is required to divide the posterior region into two folds (crusII and paramedian). However, the partial rescue of the hemisphere phenotype in rare En1-/+;En2-/- and En1Denki/+;En2-/- mutants indicates that En1 can support hemisphere development when expressed more laterally than normal. A comparison of the phenotypes of these mice with En1flox/Cre;En2+/+ mutants (which have normal posterior foliation) indicated that En2 plays a greater role than En1 in formation of folium VIII. This difference is not owing to a difference in gene expression, but instead to a difference in protein activity because the vermis foliation defect seen in En1+/+;En2-/- mutants is rescued in En1En2ki/En2ki;En2-/- mice. Furthermore, En1En2ki/-;En2-/- mice have a milder phenotype than En1-/+;En2-/- mutants. Thus, En2 appears to be more effective in promoting development of the vermis (folia I-V and VIII) than En1. It was further discovered that the two En genes act concomitantly to divide the anterior Cb into five folia. En1-/+;En2-/+ double heterozygotes and the majority of En1flox/Cre;En2+/+ mutants have a fusion of the anterior three folia (I-III) and the anterior defect is greatly exaggerated in En1-/+;En2-/- mutants (fusion of folia I-V), despite En2-/- mutants having normal anterior foliation. Since some En1flox/Cre mice have normal anterior folia, this indicates a crucial requirement for expression of En1 only at ~E9, when En1 expression is fading out in the mutants and En2 is initiating. This is the first evidence that the pattern of Cb folia can be influenced by genetic events that occur at such an early embryonic stage (Sgaier, 2007).

It is revealing to compare the early En1/2 expression patterns and the broad regions of the mes/r1 that differentially require the two genes. Based fate map of the mes/r1 using GIFM, the anterior and posterior mes give rise to the superior and inferior colliculi, respectively. Consistent with strong and sustained expression of the En genes in the primordium of the inferior colliculus, this region is most sensitive to the dose of En genes, and in particular to that of En1. However, by using the sensitive assay of En12ki knock-in alleles combined with removal of the endogenous En2 gene, it was found that En2 is at least as potent as En1 at promoting inferior colliculus development. Given the transient expression of the En genes in the superior colliculus, it is perhaps surprising that this region is dependent at all on the combination of the two genes. This indicates a requirement for a short burst of En function (En1 or En2) before E9.5. The remaining inferior colliculus tissue in En mutants is likely to correlate with tectum cells that are normally in the low end of the En gradient, suggesting that they are least sensitive to loss of En alleles. There also is a general correlation between the domains of En gene expression and the requirement for each gene in the Cb (vermis versus hemispheres). After E9.5, En1 is maintained in only anterior r1 and the medial Cb primordium (the anlage of the vermis), consistent with no function in the hemispheres. The limit of the En2 expression domain extends more posterior early in r1 and laterally later in the CbP, correlating with a role in the hemispheres. It is not clear, however, why the En genes do not play a major role in the anterior hemispheres or in folia VI/VII and IX/X in the vermis (Sgaier, 2007).

Taken together, this analysis of a series of En mutants provides evidence that functional domains of the Cb are genetically encoded by the engrailed genes; specific regions of the tectum and Cb have differential sensitivities to reducing En gene dosage. The phenotypes of multiple mutants point to a genetic division of the tectum into two regions and of the Cb into six. It is proposed that this represents an 'En code' that is used to partition the mes/r1 region into domains that in the adult regulate related neural functions. The two functional divisions of the tectum, the inferior and superior colliculi, are delineated based on a temporal requirement for En1 and sensitivity to the overall dose of En protein. Within the vermis of the Cb, the anterior five folia (I-V) and folium VIII are particularly sensitive to a reduction of En genes, and preferentially to En2, thus dividing the vermis into four broad regions (folia I-V, VI/VII, VIII, IX/X). Strikingly, this division of the Cb is very similar to the transverse zones recently proposed based on four different domains of parasagittal gene expression. The fact that two independent genetic measures of regionalization of the vermis (mutant phenotypes and gene expression) point to the same subdivisions of the vermis strongly argues that patterning of the folia is fundamental to organization of Cb function. Consistent with this, each transverse zone receives afferent inputs from distinct regions of the spinal cord and/or particular hindbrain nuclei. It is predicted that, likewise, the division of the hemispheres into regions based on a need for En2 only in two folia (crusII and paramedian) represents genetic partitioning into related functional systems (Sgaier, 2007).

Little is known about the genetic pathways and cellular processes responsible for regional differences in cerebellum foliation, which interestingly are accompanied by regionally distinct afferent circuitry. The Engrailed (En) homeobox genes have been identified as being crucial to producing the distinct medial vermis and lateral hemisphere foliation patterns in mammalian cerebella. By producing a series of temporal conditional mutants in En1 and/or En2, it was demonstrated that both En genes are required to ensure that folia exclusive to the vermis or hemispheres form in the appropriate mediolateral position. Furthermore, En1/En2 continue to regulate foliation after embryonic day 14, at which time Fgf8 isthmic organizer activity is complete and the major output cells of the cerebellar cortex have been specified. Changes in spatially restricted gene expression occur prior to foliation in mutants, and foliation is altered from the onset and is accompanied by changes in the thickness of the layer of proliferating granule cell precursors. In addition, the positioning and timing of fissure formation are altered. Thus, the En genes represent a new class of genes that are fundamental to patterning cerebellum foliation throughout the mediolateral axis and that act late in development (Cheng, 2010).

A key question in developmental biology is how tissues acquire their overall shape, as proper tissue morphology underlies normal organ function. The anatomy of the cerebellum exemplifies the relationship between differences in morphology and neural circuitry. In most vertebrates, the outer surface of the cerebellum folds during development creating mediolateral (ML) fissures surrounding folia (called lobules). The enlarged surface area created by the lobules allows for an increase in the number of neurons organized into a layered surface cytoarchitecture (called the cortex), and thus in the complexity of neural circuits and the range of behaviors controlled by the cerebellum. Whereas the cerebellum of most vertebrates has one foliation pattern along the ML axis, mammals have a central core, called the vermis, which has a foliation pattern that is distinct from the two surrounding hemispheres and the most lateral floculi-paraflocculi. Furthermore, each region of the cerebellum receives distinct neural inputs. Most of the proprioceptive and sensory inputs project to the vermis, whereas the hemispheres are enriched with circuits arising from the cortex. Moreover, in humans, compared with in mouse, the hemispheres are much larger than the vermis, possibly reflecting a greater cerebellum involvement in cortex-associated functions. It is crucial to determine how the distinct morphology of each region is regulated during development (Cheng, 2010).

Most mammals have a basic pattern of ten major lobules in the vermis (I-X anterior to posterior) and four in the hemispheres (Simplex, CrusI, CrusII and Paramedian). The foliation pattern rapidly transitions from the vermis to the hemispheres in the paravermis. The extent to which the major lobules are further subdivided by fissures varies between species and to a small degree between mouse strains. There is a degree of continuity between the hemispheres and the vermis, as two of the vermis lobules (VI and VII) are continuous with the four lobules of the hemispheres. However, the morphology of the lobules is distinct in each ML region, and so is the degree of subdivision of the lobules. The genetic pathways that regulate the formation of distinct lobules in the vermis and the hemispheres are not known (Cheng, 2010).

The cerebellum cortex consists of a dense layer of granule cells, an overlying monolayer of Purkinje cells and Bergmann glia, and an outer cell-sparse molecular layer. Although the Purkinje cells are derived from the ventricular zone of dorsal rhombomere 1 by embryonic day (E) 13.5 in mouse, the granule cells are generated from E18.5 to postnatal day (P) 16.5 by a progenitor layer covering the surface of the cerebellum (the external granule layer, EGL). Foliation occurs simultaneously with, and is dependent on, granule cell production. At the base of each fissure, Purkinje cells and Bergmann glial fibers have a distinct cellular arrangement that could allow them to act as 'anchoring centers', so that proliferation of granule cells between anchoring centers would result in outward growth of lobules. An important question is what genes regulate the formation of anchoring centers in the appropriate spatial and temporal manner to produce a normal foliation pattern (Cheng, 2010).

Of the factors known to be required for cerebellum development, fibroblast growth factor 8 (Fgf8) is expressed from E8.5 to E12.5 by the isthmus organizer, and is required to specify the cerebellum primordium. Sonic hedgehog (Shh) secreted by the Purkinje cells after E16.5 is required to maintain granule cell precursor proliferation. By contrast, there is evidence that the two engrailed (En) homeobox transcription factors regulate the pattern of at least some lobules. In En2 mutants, vermis lobule VIII is abnormally shifted posterior and only three lobules form in the hemispheres. By contrast, En1 null mutants on most genetic backgrounds are perinatal lethal and lack the cerebellum. However, when En1 is conditionally ablated at ~E9, the cerebellum forms and some mutants (En1lox/Cre) have normal foliation. These results raised the question of whether En1 is required for the initial specification of the cerebellum primordium and En2 is required subsequently for foliation. An alternative is that in En1lox/Cre mutants En2 compensates for the lack of En1 in foliation, as En2 can replace En1 in specification of the cerebellum. Furthermore, lobules I-V and VIII are greatly reduced in size in En1+/-; En2-/- mutants. The extent to which the two En genes regulate foliation throughout the ML axis of the cerebellum is not clear because of the early requirement for En1 in specification of the cerebellum (Cheng, 2010).

A temporal series of En2 and En1/En2 conditional mutant mice were produced in this study, and the two genes were found to act together to preferentially promote formation of lobules that are specific to the hemispheres and the vermis. Furthermore, En1/En2 are required even after Fgf8 expression has ended for cerebellum foliation to be patterned normally. En1/En2 regulate both the position and the timing of formation of fissures in the vermis. Also, the normal regional differences in the thickness of the EGL along the anteroposteior (AP) axis are altered in unison with changes in AP spatially restricted gene expression patterns. Thus, En1/En2 are high in the genetic hierarchy that regulates the morphology of the entire cerebellum (Cheng, 2010).

Mouse Engrailed and axon guidance

During early development, multiple classes of interneurons are generated in the spinal cord including association interneurons that synapse with motor neurons and regulate their activity. Very little is known about the molecular mechanisms that generate these interneuron cell types, nor is it known how axons from association interneurons are guided toward somatic motor neurons.

EN1 is a prototypic cell-type-specific transcription factor that is expressed in a restricted population of early postmitotic ventral neurons that are located in two bilateral columns, dorsal to the motor neurons. The expression of EN1 in these ventral neurons is controlled by inductive signals that pattern the ventral neural tube. EN1 expression in the ventral spinal cord is dependent on the activity of the PAX6 transcription factor, which is expressed in ventral progenitors that give rise to EN1 interneurons, and EN1 is no longer expressed in the ventral spinal cord of Small eye (Pax6-) mutant embryos. The restricted expression of EN1 in early postmitotic neurons, together with the specific loss of these cells in Pax6 mutant mice has led to the hypothesis that EN1 marks a subclass of ventral interneurons and that this interneuron subclass may be specified in part by EN1. However, the interneurons in the embryonic spinal cord that express EN1 have not been characterized in detail, nor has the function of En1 in these neurons been determined. By targeting the axonal reporter gene tau-lacZ to the En1 locus, it has been shown that the cell-type-specific transcription factor Engrailed-1 (EN1) defines a population of association neurons that project locally to somatic motor neurons. These EN1 interneurons are born early and their axons pioneer an ipsilateral longitudinal projection in the ventral spinal cord. The EN1 interneurons extend axons in a stereotypic manner, first ventrally, then rostrally for one to two segments where their axons terminate close to motor neurons. The growth of EN1 axons along a ventrolateral pathway toward motor neurons is dependent on netrin-1 signaling. In addition, this study demonstrates that En1 regulates pathfinding and fasciculation during the second phase of EN1 axon growth in the ventrolateral funiculus (VLF); however, En1 is not required for the early specification of ventral interneuron cell types in the embryonic spinal cord (Saueressig, 1999).

A series of gain- or loss-of-function experiments performed in different vertebrate species have demonstrated that the Engrailed genes play multiple roles during brain development. In particular, they have been implicated in the determination of the mid/hindbrain domain, in cell proliferation and survival, in neurite formation, tissue polarization and axonal pathfinding. The consequences of a local gain of En function within or adjacent to the endogenous expression domain has been examined in mouse and chick embryos. In WEXPZ.En1 transgenic mice several genes are induced as a consequence of ectopic expression of En1 in the diencephalic roof (but in a pattern inconsistent with a local di- to mes-encephalon fate change). The development of several structures with secretory function, generated from the dorsal neuroepithelium, is severely compromised. The choroid plexus, subcommissural organ and pineal gland either fail to form or are atrophic. These defects are preceded by an increase in cell death at the dorsal midline. Comparison with the phenotype of Wnt1sw/sw (swaying) mutants suggests that subcommissural organ failure is the main cause of prenatal hydrocephalus observed in both strains. The formation of the posterior commissure is also delayed, and errors in axonal pathfinding are frequent. In chick, ectopic expression of En by in ovo electroporation, affects growth and differentiation of the choroid plexus (Louvi, 2000).

Secretion and extracellular function of Engrailed

Chicken Engrailed 2 homeoprotein is transported between cells in culture. This intercellular transfer is based on unconventional secretion and internalization mechanisms: Engrailed 2 has access to vesicles but lacks a signal sequence for secretion and is internalised by a non-endocytic process. Phosphorylation of a serine-rich domain within Engrailed 2 by the protein kinase CK2 specifically inhibits Engrailed 2 secretion. The availability of the serine-rich domain to CK2 is highly increased when it is displaced from its normal position to the C terminus of Engrailed 2, leading to a constitutive blockage of Engrailed 2 intercellular transfer. This indicates that intercellular transfer of Engrailed 2 is a highly regulated process (Maize, 2002).

Engrailed transcription factors regulate the expression of guidance cues that pattern retinal axon terminals in the dorsal midbrain. They also act directly to guide axon growth in vitro. This study shows that an extracellular En gradient exists in the tectum along the anterior-posterior axis. Neutralizing extracellular Engrailed in vivo, with genetically encoded secreted single-chain antihomeoprotein antibodies expressed in the tectum, causes temporal axons to map aberrantly to the posterior tectum in chick and Xenopus. Furthermore, posterior membranes from wild-type tecta incubated with anti-Engrailed antibodies or posterior membranes from Engrailed-1 knockout mice exhibit diminished repulsive activity for temporal axons. Since EphrinAs play a major role in anterior-posterior mapping, tests were performed to see whether Engrailed cooperates with EphrinA5 in vitro. It was found that Engrailed restores full repulsion to axons given subthreshold doses of EphrinA5. Collectively, these results indicate that extracellular Engrailed contributes to retinotectal mapping in vivo by modulating the sensitivity of growth cones to EphrinA (Wizenmann, 2009).

Table of contents

engrailed: Biological Overview | Transcriptional regulation | Targets of activity | Protein Interactions | Developmental Biology | Effects of mutation | References

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