ventral nervous system defective: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - ventral nervous system defective

Synonyms - NK2

Cytological map position - 1C1-5

Function - transcription factor

Keywords - CNS

Symbol - vnd

FlyBase ID:FBgn0261930

Genetic map position - 1-[0.0]

Classification - homeodomain - NK-2 class

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

vnd/NK2 is the first neural gene expressed. It functions early in neural system development forming a prepattern, even before the proneural genes begin to function. It is required for the formation of a subset of segmental neuroblasts, and possibly as a neuroectodermal committment gene. vnd transcription is upstream of proneural achaete-scute complex. It is responsible for the induction of achaete and scute in a subset of neuroblasts. It also regulates genes of the Enhancer of split complex (Kramatschek, 1994).

It is clear that E(spl)-C gene expression is dependent on lateral inhibition and the Notch pathway acting through Suppressor of Hairless. The role of VND in the transcriptional activation of E(spl)-C genes is currently unclear. Perhaps VND activates proneural genes which in turn activate E(spl)-C genes. Physical interaction of VND with the E(spl)-C has not yet been verified.

The specificity of neuron function in regulating different segmentally repeated muscle groups has its origin in the establishment of a pattern of neuron differentiation in the neuroectoderm. This pattern is determined by a combination of pair rule and segment polarity genes, the former responsible for segmentation and the latter for subdivision of cell fates within segments. The appropriately named dorsoventral polarity genes establish gene expression along the DV axis. By stage eight of embryonic development, each ventral hemisegment (the left or right half of a segment) is subdivided into three longitudinal columns from the ventral midline: medial, intermediate and lateral. They are also divided in the anterior to posterior axis into rows A through D.

Expression of achaete-scute is dependent on VND/NK2 in two proneural clusters of the medial column, rows B and D, while VND/NK2 shows little or no effect on gene expression in rows A and C, and no effect on gene expression in the intermediate and lateral columns. Thus VND/NK2 is required along precise domains of both the anterior-posterior axis and the dorsal-ventral axis. Using Snail or Hunchback proteins as markers, it has been shown that medial neuroblasts from rows B and D are not formed in VND/NK1 mutants. Do other genes specify the remaining neuroblasts? To date these regulators have not been identified (Skeath, 1994).

It is suggested that muscle segment homeobox functions in a similar fashion to vnd/NK2 in neural patterning, and that these roles are conserved in insects and vertebrates. Interaction between DPP and Short gastrulation (vertebrate homologs BMPs and chordin) restrict msh (vertebrate homologs the Msx genes) expression to the lateral most column of proneural clusters (in vertebrates the lateral-most portions of the neural plate). In a similar fashion vnd is restricted to the medial column of proneural clusters. In vertebrates the vnd homolog Nkx-2 is restricted to the medial region of the neural plate. Both msh and vnd serve a similar conserved function, regulation of expression of the achaete-scute complex to particular neuroblasts. Likewise Msx and Nkx-2 regulate the expression of vertebrate achaete-scute homolog (ash) in the developing neural column (D'Alessio, 1996).

To assay CNS cell fates in embryos lacking vnd function (vnd embryos), markers were used that specifically label ventral, intermediate, or dorsal column neuroectoderm and neuroblasts. Three ventral column markers were used: Achaete, Odd-skipped (Odd), and Prospero. Achaete labels ventral and dorsal column neuroectoderm and neuroblasts of rows 3 and 7; Odd labels the ventral and dorsal column neuroblasts of row 1, whereas Prospero labels the ventral column neuroblast in row 3 (MP2). Two intermediate column markers were used: Ind and Huckebein. Intermediate neuroblast defective labels all intermediate column neuroectoderm and neuroblasts, whereas Huckebein labels the intermediate neuroectoderm and neuroblasts of row 3 (e.g., NB 4-2) and the ventral and intermediate neuroectoderm of rows 1 and 5. The dorsal marker Msh labels all dorsal column neuroectoderm and neuroblasts. Using these markers, an investigation was carried out of to determine whether vnd is necessary or sufficient to specify ventral column identity within the developing CNS. In vnd mutant embryos, Achaete, Odd, and Prospero are not detected in the ventral column neuroectoderm or neuroblasts. This phenotype is the result of a lack of gene expression in the neuroectoderm and in some neuroblasts, as well as a failure in neuroblast formation. For example, the ventral column NB 1-1 forms >80% of the time, yet it is never Odd+. However, absence of a Prospero+ MP2 is the result of a failure in MP2 formation. It is concluded that vnd is necessary to specify ventral column neuroectoderm and neuroblast identity, and to form specific ventral column neuroblasts (McDonald, 1998).

The ventral column in vnd embryos could be fully or partially transformed to a different columnar identity (intermediate or dorsal) or could assume a novel identity. To distinguish between these two possibilities, intermediate and dorsal column markers were examined in vnd embryos. In vnd embryos, intermediate column markers are ectopically expressed in the ventral column. Ind is detected in both ventral and intermediate column neuroectoderm and neuroblasts, although because some ventral neuroblasts do not form in vnd embryos, the intermediate column neuroblasts often shift to a more ventral position. The intermediate column marker Huckebein is also detected in both the ventral and intermediate columns of row 3 neuroectoderm and neuroblasts (13% in the ventral column). Expression of Msh in dorsal column neuroectoderm and neuroblasts is normal in vnd embryos. In addition, dorsal column expression of Achaete and Odd are unchanged. Taken together, these data show that vnd is necessary for the specification of ventral column identity and the repression of intermediate column identity within the CNS (McDonald, 1998).

During the course of this gene expression analysis, it was noticed that the ventral column neuroectoderm has a distinctive cellular morphology. The Vnd+ ventral column cells are frequently elongated along the DV axis to give them an asymmetry ratio of equal or >1.5 (long axis divided by short axis), whereas the Ind+ intermediate column cells are more often round, with an asymmetry ratio closer to 1.0. In vnd embryos most ventral column cells fail to assume an elongated morphology, instead showing a round morphology characteristic of intermediate column cells. Taken together, this molecular marker and cell morphology analyses show that vnd is required to establish ventral column gene expression profiles (perhaps by direct transcriptional activation and/or repression) as well as to induce a cell shape change characteristic of the ventral column neuroectoderm (McDonald, 1998).

To determine whether vnd is sufficient to specify ventral column fate, an hsp70-vnd transgene was used to ectopically express vnd in the intermediate and dorsal columns of neuroectoderm. In embryos carrying the hsp70-vnd transgene that are heat shocked to induce ubiquitous vnd expression (hs-vnd embryos), ventral markers are ectopically expressed in the intermediate and dorsal columns of the neuroectoderm and neuroblasts, and intermediate and dorsal markers are lost. However, the transformation of intermediate to ventral cell fate is more complete than that of dorsal to ventral cell fate transition. In hs-vnd embryos, the ventral column marker Achaete expands into the intermediate column, leading to Achaete expression extending continuously across ventral, intermediate, and dorsal columns of neuroectoderm and neuroblasts. Similarly, the ventral neuroblast markers Prospero and Odd also show ectopic expression in the intermediate column in hs-vnd embryos. Conversely, hs-vnd embryos show a loss of intermediate column marker expression. The intermediate column marker Ind is strongly repressed or completely abolished in hs-vnd embryos. The row 3 intermediate column marker Huckebein is also repressed in hs-vnd embryos; Huckebein row 5 expression appears unaffected, which is not surprising because both the ventral and intermediate columns express Huckebein in wild-type row 5 neuroectoderm. These results show that ectopic vnd results in a transformation of intermediate column to ventral column identity within both neuroectoderm and neuroblast cell types (McDonald, 1998).

The dorsal column is mis-specified in hs-vnd embryos, but is not fully transformed into ventral column identity. In hs-vnd embryos, the row 1 ventral column marker Huckebein is ectopically detected in the dorsal neuroectoderm, and the dorsal column marker Msh is partially repressed. However, the ventral column marker Prospero is ectopically expressed in the intermediate column but not in the dorsal column. Thus, ectopic vnd is sufficient to partially transform dorsal column to ventral column identity (McDonald, 1998).

To determine the extent to which Vnd controls ventral column neuroblast identity, a neuroblast cell lineage marker, Even-skipped (Eve), was used to assay the development of specific ventral and intermediate column neuroblasts. Eve labels the progeny of two ventral column neuroblasts (aCC/pCC neurons from NB 1-1; U/CQ neurons from NB 7-1) and the progeny of one intermediate column neuroblast (RP2/RP2sib neurons from NB 4-2). The pattern of Eve is a sensitive indicator for normal cell fates within these neuroblast cell lineages. In vnd embryos, the Eve+ aCC/pCC and U/CQ neurons, derived from ventral column neuroblasts, are never detected. NB 1-1 forms and produces Prospero+, Eve GMCs; thus loss of Eve from the aCC/pCC neurons is caused by an alteration in NB 1-1 identity or cell lineage. In contrast, the absence of the Eve+ U/CQ neurons is the result of failure of their parental NB 7-1 to form. In addition, vnd embryos show a partially penetrant duplication of the Eve+ RP2/RP2sib neurons derived from the intermediate column NB 4-2. This phenotype is the result of a transformation of a Huckebein ventral column neuroblast into a duplicate Huckebein+ intermediate column NB 4-2 (McDonald, 1998).

Is vnd sufficient to induce ventral neuroblast cell lineages in the intermediate or dorsal column neuroblasts? In hs-vnd embryos, an excess number of the ventral column Eve+ aCC/pCC and U/CQ neurons develop. Because hs-vnd produces intermediate to ventral transformations of neuroblast identity, it is likely that the excess aCC/pCC and U/CQ neurons develop from duplicated ventral column neuroblasts (NBs 1-1 and 7-1). However, the possibility cannot be ruled out that ectopic vnd triggers duplication of GMC identities or excess rounds of cell division. It is concluded that loss of vnd results in a transformation of ventral to intermediate column neuroblast identity that is maintained in the cell lineage of at least three neuroblasts (NBs 1-1, 7-1, and 4-2), and ectopic vnd results in the converse transformation of intermediate to ventral column neuroblast identity that is maintained in the cell lineage of at least two neuroblasts (NBs 1-1 and 7-1) (McDonald, 1998).

Defects in the formation of ventral and intermediate column neuroblasts occur in vnd embryos. There is a loss of ventral column neuroblasts, particularly MP2 and NB 7-1. In addition, in hs-vnd embryos there is precocious formation of neuroblasts in the intermediate column. In wild-type embryos, intermediate column neuroblasts form at mid-stage 9, but in hs-vnd embryos these neuroblasts form earlier, at early stage 9, about the time the adjacent ventral column neuroblasts are forming. These data provide additional evidence for a transformation of intermediate to ventral column identity (McDonald, 1998).

vnd also regulates dorsoventral patterning of the procephalic neuroectoderm. vnd, msh, and ind are each expressed in the procephalic ectoderm: Vnd in a ventral domain, Ind in three small clusters of cells at intermediate positions, and Msh in a dorsal domain. There are two differences in gene expression and regulation in the procephalic region compared with the thoracic and abdominal neuroectoderm: (1) Vnd and Msh share an extensive border, only interrupted by two small islands of Ind+ cells. In vnd embryos, Msh expands into the ventral domain of the procephalic neuroectoderm, showing that Vnd is required to repress msh expression in the head. Consistent with this result, misexpression of vnd leads to repression of msh. (2) The Ind+ anterior cell cluster 1 appears to coexpress Vnd; coexpression of Vnd and Ind is never observed in the thoracic and abdominal neuroectoderm. Surprisingly, vnd embryos show a loss of the Ind+ cluster 1, and misexpression of vnd does not affect Ind expression in cluster 1; thus, in this domain of the embryo, Vnd is required for the development of the Ind+ cluster 1. Because the Ind+ cells of cluster 1 are primarily restricted to neuroblasts, one possibility is that loss of vnd in the neuroectoderm leads to a failure of neuroblast formation and thus to a loss of Ind+ cells, rather than that Vnd directly activates ind transcription in this domain. The remaining two Ind+ cell clusters (2 and 3) are expressed and regulated in a manner consistent with the thoracic and abdominal neuroectoderm. Both Ind+ cell clusters 2 and 3 directly abut Vnd+ cells but do not express Vnd. In vnd embryos, the Ind+ cluster 3 expands ventrally into the domain normally expressing vnd, whereas Ind+ cluster 2 appears unaffected. Misexpression of vnd represses ind expression in clusters 2 and 3. Thus, vnd can both activate ind (cluster 1) or repress ind (clusters 2 and 3) depending on the position within the procephalic neuroectoderm (McDonald, 1998).

Role of en and novel interactions between msh, ind, and vnd in dorsoventral patterning of the Drosophila brain and ventral nerve cord

Subdivision of the neuroectoderm into discrete gene expression domains is essential for the correct specification of neural stem cells (neuroblasts) during central nervous system development. This study extends knowledge on dorsoventral (DV) patterning of the Drosophila embryonic brain and uncovers novel genetic interactions that control expression of the evolutionary conserved homeobox genes ventral nervous system defective (vnd), intermediate neuroblasts defective (ind), and muscle segment homeobox (msh). Cross-repression between Ind and Msh was shown to stabilize the border between intermediate and dorsal tritocerebrum and deutocerebrum, and both transcription factors are competent to inhibit vnd expression. Conversely, Vnd segment-specifically affects ind expression; it represses ind in the tritocerebrum but positively regulates ind in the deutocerebrum by suppressing Msh. These data provide further evidence that in the brain, in contrast to the trunk, the precise boundaries between DV gene expression domains are largely established through mutual inhibition. Moreover, it was found that the segment-polarity gene engrailed (en) regulates the expression of vnd, ind, and msh in a segment-specific manner. En represses msh and ind but maintains vnd expression in the deutocerebrum, is required for down-regulation of Msh in the tritocerebrum to allow activation of ind, and is necessary for maintenance of Ind in truncal segments. These results indicate that input from the anteroposterior patterning system is needed for the spatially restricted expression of DV genes in the brain and ventral nerve cord (Seibert, 2010)

The spatial and temporal order in which the DV genes (vnd, ind, and msh) are activated in neuromeres of the brain differs from their appearance in the trunk neuroectoderm, and those differences seem to be basic for the segment-specific regulation of vnd, ind, and msh expression. In the early trito- and deutocerebrum, Vnd is expressed not only in the ventral but also in the intermediate neuroectoderm, where cross-repression between Vnd and dorsally expressed Msh establishes the border between intermediate and dorsal neuroectoderm. Since Msh was found to be an ind repressor, the repression of msh via Vnd is a prerequisite for ind to become activated in the intermediate tritocerebrum (anterior) and deutocerebrum. In the trunk, ind expression in the intermediate neuroectoderm starts before that of msh in the dorsal neuroectoderm, and msh and vnd domains do not abut; accordingly, repressive interaction between Msh and Vnd is not required (Seibert, 2010)

In the tritocerebrum, Vnd not only acts as repressor of msh but also of ind, in contrast to the deutocerebrum. When the level of Vnd protein in the intermediate tritocerebrum declines with time (down-regulated through the activity of Ems), ind becomes subsequently activated. In the trito- and deutocerebrum, instead of Vnd, increasing levels of Ind, together with the recently uncovered msh-repressor Nkx6, still keep msh expression limited to the dorsal neuroectoderm. Since it was found that Nkx6 expression starts earlier and persists longer than that of ind in both brain neuromeres, and additionally, that msh is expanded into the intermediate neuroectoderm in Nkx6 but not in ind mutants, it is proposes that Nkx6 represses msh more efficiently (Seibert, 2010)

The most striking difference in DV gene regulation leads to the question how vnd and ind can be co-expressed in the anterior deutocerebrum (during stages 6–9), if Vnd is a repressor of ind and, vice versa, Ind is also capable of preventing vnd expression in the neuroectoderm. It has been reported recently that the repressor activity of Ind on vnd seems to be stage-specific, not taking place before stage 9. By contrast, Vnd repression of ind seems independent of the developmental period. In this context, it is interesting that activity of Vnd can be modified by EGFR signalling, which is supposed to affect the selective interaction of Vnd with co-factors necessary to mediate repression or activation of target genes. Availability of co-factors might also account for the specific situation of vnd and ind co-expression in the anterior deutocerebrum that was observed specifically during early stages of development (Seibert, 2010)

Involvement of en, which can act as transcriptional repressor as well as activator, has been implicated in diverse developmental processes in Drosophila such as compartmentalization in the early embryo, modulation of Hox gene expression, or regulation of molecules that directly govern axon growth (e.g. frazzled). This study demonstratea a novel function for En in the early embryo, that is to control the spatially restricted expression of the DV genes in the neuromeres of the posterior brain (trito- and deutocerebrum) and ventral nerve cord. In the posterior compartment of the deutocerebrum, En represses expression of msh and ind, but maintains expression of vnd. Since it was found that Ind (later) becomes a vnd repressor, this indicates that En maintains expression of vnd by repressing ind. In the posterior compartment of the tritocerebrum, En is also required for down-regulation of Msh, but opposite to the deutocerebrum, En is necessary for activation of ind. This study shows that Msh is an ind repressor, its repression by En seems to allow for activation of ind; yet, it cannot be excluded that En in addition directly activates ind expression. Similar to the situation in the tritocerebrum, En seems to negatively regulate expression of msh and to positively regulate expression of ind (as a maintenance factor) in the neuroectoderm of the ventral nerve cord. Together, these data suggest that the AP patterning gene engrailed is crucially involved in fine-tuning the regionalized expression of distinct DV genes in the posterior compartment of neuromeres in the brain and ventral nerve cord. En may act as a positive or negative transcriptional regulator depending on the gene that is regulated and on the segmental context. For DV genes it is known that they control formation and specification of brain neuroblasts. Since all the genetic interactions between En and DV genes take place during the period when neuroblasts develop, it is likely that En, via regulation of DV genes, controls formation and fate specification of neuroblasts in the brain (Seibert, 2010)

It was observed that cross-repressive interaction between pairs of DV gene factors in the brain (i.e. in trito- and deutocerebrum) is essential for the establishment and maintenance of discrete DV gene expression domains. Early, cross-repression between Ems/Vnd pre-patterns the ventral and intermediate neuroectoderm in both neuromeres. Mutual repression between Msh/Nkx6 and Msh/Ind maintains the dorsal/intermediate neuroectodermal border in trito- and deutocerebrum, and between Ind/Vnd the intermediate/ventral border in the tritocerebrum. All these genetic interactions, and the observation that Msh and Vnd act as mutual repressors, are not in compliance with the concept of ventral dominance (as proposed in the neuroectoderm of the ventral nerve cord where the more ventral gene represses the gene expressed more dorsally) but rather support the model that in the brain cross-repression between DV factors is crucial for stabilizing these borders (Seibert, 2010)

However, despite the ability of Msh and Ind to repress vnd, neither factor seems to be sufficient to define the dorsal border of vnd expression in trito- and deutocerebrum, as has been shown for Ind in the ventral nerve cord (from stage 9 onwards). Instead of reinforcing this border through repressive interaction, vnd expression in the brain could also be limited by a (too) low concentration or absence of an activator, like Dorsal (as has been speculated for the trunk), or be regulated by BMP signalling in a dosage-dependent fashion. Neuromere-specific differences are also observed regarding limitation of ind and msh expression domains along the DV axis. Vnd establishes the ventral border of ind expression in the trunk and tritocerebrum, but not in the deutocerebrum or protocerebrum (where the expression domains of ind and vnd do not abut). ind expression was found to be limited dorsally by repression through Msh in the trito- and deutocerebrum, but not in the protocerebrum (where msh is not expressed before stage 11) or trunk, although evidence is available that Msh might act in rendering the dorsal border of ind expression more precisely in the ventral nerve cord. Taking into account that ind expression does not expand into the complete dorsal neuroectoderm of trito- and deutocerebrum in msh mutants, this may also indicate an involvement of the nuclear Dorsal gradient, possibly in concert with graded activity of EGFR (as was shown for the trunk neuroectoderm), or BMP (which can repress ind in the trunk neuroectoderm), in establishing a rough dorsal border of ind expression that is further defined and stabilized via repression by Msh. Whereas Vnd is initially responsible for keeping msh expression confined to the dorsal neuroectoderm in trito- and deutocerebrum, it is only indirectly involved in defining the ventral border of msh expression in the trunk neuroectoderm. Later in development Ind helps to maintain repression of msh in trito- and deutocerebrum (together with Nkx6), which is in contrast to the trunk where Ind directly establishes the ventral limit of msh expression from the beginning (Seibert, 2010)

DV neuroectodermal and corresponding stem cell domains in the Drosophila brain become established and maintained through cross-repressive regulation, and it has been speculated that such genetic interactions are more common in the fly brain. This study has presented further examples supporting this hypothesis. Notably, this is a feature that bears similarity to DV patterning in the neural tube of vertebrates where cross-repressive interactions of homeodomain proteins are common and indeed crucial for the establishment of discrete DV progenitor domains (Seibert, 2010)

All interactions between DV genes in the brain identified so far are based on the interplay of transcriptional repressors. Likewise, this study shows that Vnd does not act as a direct activator to positively regulate ind, but according to a double-negative mechanism, it suppresses the ind-repressor Msh. It has been shown previously, that interactions of the AP patterning gene ems with the DV genes (vnd, ind, msh, and Nkx6) are indispensable for proper development of the trito- and deutocerebrum. This study demonstrates that the segmentation gene en is significantly involved in regionalization of DV gene expression domains, thus representing a further example of an AP patterning gene integrating into the DV gene regulatory network that patterns the brain. This study has shown that En acts differently on the respective DV genes, but no evidence is available that, vice versa, DV genes control en, as has been observed for expression of ems. DV genes, as well as En and Ems, all contain an Eh1 repressor domain and are able to interact with the co-repressor Groucho (Gro), and thus are capable of mediating repression on target genes (including each other). But how could it be possible that all DV genes interact with the same co-factor to stabilize expression domains by conferring repression onto genes expressed in neighboring domains? In the first place, the DV genes display spatio-temporal differences in their respective expression. In addition, conformational changes of the protein seem to be necessary to enable binding of Gro which has been at least shown for Nkx6. It has been observed that Vnd can be phosphorylated by activated MAPK and is present in different isoforms in the developing embryo, which most likely leads to a change in its binding partners. Another critical point could be inactivation of the co-repressor, in case of Gro also through phosphorylation by activated MAPK, or modification of target genes so that binding of the repressor complex is impaired. Still, that the DV genes are able to interact with Groucho, does not exclude that their repressor activity is Gro-independent, since also other repressor domains have been reported for these genes, as well as activator domains, at least for Vnd, Ind, and Nkx6. Whether the DV gene products function as repressors or activators seems to depend on co-factor availability as well as on the respective target gene, since not only the presence of a transcriptional binding site, but also its accessibility is limiting in this context (Seibert, 2010)


GENE STRUCTURE

cDNA clone length - 3.5 kb

Bases in 5' UTR - 341

Exons - two

Bases in 3' UTR - 394


PROTEIN STRUCTURE

Amino Acids 723

Structural Domains

VND has both a homeodomain and a PEST-domain, affording the protein a short half life due to rapid turnover through degradation. The region before the homeobox contains both an arginine rich region and an acidic region. The C terminal 30 amino acids are rich in histidine, proline, and glycine (Kim, 1989). Following the homeodomain is an NK2 box (Jimenez, 1995).

This study describes the NMR determination of the three-dimensional structure of a 77 amino acid residue protein, which consists of the 60 residue NK-2 homeodomain from Drosophila melanogaster and adjacent amino acid residues. The NK-2 homeodomain protein is part of a 723 amino acid residue protein that is expressed early in embryonic development in part of the central nervous system. NK-2 was characterized using both a natural abundance and a uniformly 15N enriched sample by two-dimensional and three-dimensional NMR experiments. The average root-mean-square deviation for 30 structures for residues 8 to 53 is 0.40 A for the backbone heavy-atoms and 0.72 A for the backbone and side-chain heavy-atoms. These structures were obtained from 986 NOE-derived upper and lower bound restraints. The three-dimensional structure contains three helices that consist of homeodomain amino acid residues 10 to 22, 28 to 38 and 42 to 52, as well as a turn between helix II and III, characteristic of homeodomains. Residues 53 to 60 of the DNA recognition helix are not fully ordered in the absence of DNA. In the free state, this segment adopts a flexible but helix-like structure between residues 53 and 56 and is disordered from residues 57 to 60, although the helix elongates by eight residues upon binding to DNA. Also discussed are the roles of variable residues 52, 54 and 56 in determining the structure and flexibility of the recognition helix, as well as the stability of the NK-2 homeodomain as manifested by its thermal denaturation (Tsao, 1995).

The Engrailed homeoprotein is a dominantly acting, so-called 'active' transcriptional repressor, both in cultured cells and in vivo. When retargeted via a homeodomain swap to the endogenous fushi tarazu gene (ftz), Engrailed actively represses ftz, resulting in a ftz mutant phenocopy. Functional regions of Engrailed have been mapped using this in vivo repression assay. In addition to a region containing an active repression domain identified in cell culture assays, there are two evolutionarily conserved regions that contribute to activity. The one that does not flank the HD is particularly crucial to repression activity in vivo. This domain is present not only in all engrailed-class homeoproteins but also in all known members of several other classes, including goosecoid, Nk1, Nk2 (vnd) and muscle segment homeobox. The repressive domain is located in the eh1 region, known as 'region three', found several hundred amino acids N-terminal to the homeodomain. The consensus sequence, arrived at by comparing Engrailed, Msh, Gsc, Nk1 and NK2 proteins from a variety of species, consists of a 23 amino acid homologous motif found in all these proteins. Thus Engrailed's active repression function in vivo is dependent on a highly conserved interaction that was established early in the evolution of the homeobox gene superfamily. Using rescue transgenes it has been shown that the widely conserved in vivo repression domain is required for the normal function of Engrailed in the embryo (Smith, 1996).


ventral nervous system defective: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 20 January 99

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