Interactive Fly, Drosophila



The Klumpfuss sequence is characterized by four zinc-finger domains of the C2H2 class; part of the N-terminus is negatively charged; the C-terminus, including the zinc-fingers, is positively charged. The N-terminal region contains glutamine-, histidine- and proline-rich stretches, features found in transcriptional activation and repression domains. There are three poly-alanine stretches, two in the N- and one in the C-terminal region. Such stretches are implicated in transcriptional repression. Three putative nuclear localization sites are found in the protein. Klu has a high degree of similarity to the zinc-finger domains of the members of the EGR family. As in WT-1, Klu contains an additional zinc finger1 that is only distantly related to those of the EGR proteins. Besides WT-1, Klu is the only other member of the family with four zinc fingers. There is complete conservation of the amino acids that contact the DNA-binding consensus sequence. So far only two other proteins are described as EGR-like in invertebrates, the Drosophila genes stripe and huckebein. Whereas the zinc fingers of Stripe have the characteristic amino acids for the binding of the DNA consensus sequence, in the zinc fingers of Huckebein, four out of the six amino acids are not conserved. However, the overall homology of the Huckebein finger region shows no greater homology to the EGR sequences than does the finger region of Klumpfuss. In comparison, the Stripe sequence shows a much more marked homology to the vertebrate EGR proteins than do the sequences of either Huckebein or Klu (Klein, 1997).

The Huckebein transcript encodes a Sp1/egr-related zinc finger protein, similar in structure but smaller than Buttonhead, a second Sp1 related protein of Drosophila (Bronner, 1994)

MAB-10/NAB acts with LIN-29/EGR to regulate terminal differentiation and the transition from larva to adult in C. elegans

In Caenorhabditis elegans, a well-defined pathway of heterochronic genes ensures the proper timing of stage-specific developmental events. During the final larval stage, an upregulation of the let-7 microRNA indirectly activates the terminal differentiation factor and central regulator of the larval-to-adult transition, LIN-29, via the downregulation of the let-7 target genes lin-41 and hbl-1. This study identifies a new heterochronic gene, mab-10, and shows that mab-10 encodes a NAB (NGFI-A-binding protein) transcriptional co-factor. MAB-10 acts with LIN-29 to control the expression of genes required to regulate a subset of differentiation events during the larval-to-adult transition, and the NAB-interaction domain of LIN-29 is conserved in Kruppel-family EGR (early growth response) proteins. A similar interaction between Drosophila NAB and the two Drosophila LIN-29 homologs RN and SQZ was reported recently. In mammals, EGR proteins control the differentiation of multiple cell lineages, and EGR-1 acts with NAB proteins to initiate menarche by regulating the transcription of the luteinizing hormone β subunit. Genome-wide association studies of humans and various studies of mouse recently have implicated the mammalian homologs of the C. elegans heterochronic gene lin-28 in regulating cellular differentiation and the timing of menarche. This work suggests that human homologs of multiple C. elegans heterochronic genes might act in an evolutionarily conserved pathway to promote cellular differentiation and the onset of puberty (Harris, 2011).

This study identified mab-10 as a new heterochronic gene that is required for specific aspects of the larval-to-adult transition, specifically molting cycle exit and seam cell exit from the cell cycle. mab-10 encodes the only C. elegans NAB transcriptional co-factor. NAB proteins are thought to physically interact with Kruppel family EGR transcription factors to regulate their activity (Harris, 2011).

Previous work demonstrated that MAB-10 (then known only as the C. elegans NAB protein R166.1) could interact with mammalian EGR proteins in a yeast two-hybrid assay; no corresponding C. elegans EGR protein was identified. This study has demonstrate that MAB-10 interacts with the terminal differentiation factor LIN-29 through an evolutionarily conserved NAB binding domain (R1 domain) and that MAB-10 is required for a subset of LIN-29-dependent activities. This work identifies LIN-29 as a C. elegans EGR-like protein and demonstrates that the C. elegans heterochronic pathway controls the timing of NAB/EGR-mediated differentiation (Harris, 2011).

Several experiments using mammalian tissue culture suggest that NAB proteins negatively regulate EGR activity by binding EGR proteins at specific target genes and preventing EGR-mediated transcription. However, loss of either EGR2 function or NAB function in mice and humans results in hypomyelination, suggesting that EGR and NAB proteins need not act antagonistically in vivo (Harris, 2011).

In C. elegans, MAB-10 and LIN-29 both act to promote terminal differentiation and the onset of adulthood. Furthermore, mab-10 promotes the formation of precocious adult alae in a lin-41 mutant background, suggesting that MAB-10 does not specifically act to control genes required for exit from the molting cycle and seam cell exit from the cell cycle, but more likely acts as a general enhancer of LIN-29 activity (Harris, 2011).

EGR and NAB proteins have been shown to operate in a negative-feedback loop wherein an EGR protein promotes the expression of its NAB co-factor, which then inhibits EGR activity. mab-10 transcription does not depend on LIN-29, despite a dramatic increase of mab-10 transcription during the L4 stage. Thus, mab-10 is not a transcriptional target of LIN-29 (Harris, 2011).

Whereas mab-10 is not a transcriptional target of LIN-29, MAB-10::GFP localization to seam cell nuclei during the L4 stage required LIN-29, indicating that LIN-29 might promote MAB-10 seam cell nuclear localization via a post-transcriptional mechanism or via direct physical interaction (Harris, 2011).

This work demonstrates that MAB-10 and LIN-29 do not operate in a negative-feedback loop. It is proposed that other components of the heterochronic pathway directly regulate mab-10 transcription to temporally regulate MAB-10/LIN-29 activity and that LIN-29 or some factor downstream of LIN-29 controls MAB-10/LIN-29 activity by promoting the accumulation of MAB-10 in seam cell nuclei (Harris, 2011).

By showing that MAB-10 acts with LIN-29 through an evolutionarily conserved EGR R1 domain, LIN-29 and the Drosophila LIN-29 homologs RN and SQZ are identified as EGR-like molecules. It is proposed that NAB proteins and EGR proteins act together in temporal developmental programs to control terminal differentiation. In Drosophila, the LIN-29 homolog SQZ acts with Drosophila NAB to control neuroblast differentiation. In C. elegans, LIN-29 and MAB-10 act together to control the differentiation of a hypodermal stem cell lineage during the transition from larva to adult by regulating the expression of the nuclear hormone receptors nhr-23 and nhr-25 and the cell cycle regulator cki-1. Recently, a study of C. elegans demonstrated that nhr-25 is itself a heterochronic gene and possibly functions with lin-29 to promote aspects of the larval-to-adult transition, including seam cell exit from the cell cycle. Though the mechanism by which nhr-25 regulates seam cell exit from the cell cycle is not known, it is speculated that LIN-29 and NHR-25 might act together to promote cki-1 expression (Harris, 2011).

EGR proteins were originally identified as immediate-early genes and generally have been regarded as differentiation factors. Like mab-10 and lin-29 mutants, Nab and Egr mutant mice are defective in the terminal differentiation of several cell lineages. For example, in Schwann cells, EGR2 promotes the expression of P27, the homolog of C. elegans CKI-1, and acts with NAB proteins to promote terminal differentiation. Mammalian homologs of other C. elegans heterochronic genes also control differentiation. Similar to the role of LIN-28 in C. elegans, mammalian LIN28 and LIN28B promote stem cell identity and prevent differentiation by repressing the let-7 microRNA gene. As in C. elegans, increasing levels of let-7 drive differentiation, and the mouse homolog of LIN-41, LIN41, has been shown to be a let-7 target acting in stem cell niches to prevent premature differentiation (Harris, 2011).

Mammalian LIN-28 controls the timing of the onset of puberty in mice and possibly humans. Mice lacking EGR1 function, like lin-29 mutants of C. elegans, fail to undergo puberty. EGR1 and NAB proteins act with SF1, the homolog of C. elegans NHR-25, in the gonadotrope lineage of the pituitary gland to regulate the expression of luteinizing hormone and the onset of puberty. The molecular mechanism by which mammalian LIN-28 regulates the onset of puberty is not known. This work raises the possibility that homologs of C. elegans heterochronic genes might act in an evolutionarily conserved pathway that controls the terminal differentiation of cell lineages and the onset of adulthood by regulating the activity of NAB and EGR proteins (Harris, 2011).

Corepressors interact with Egr transcription factors

The NGFI-A binding corepressors NAB1 and NAB2 interact with a conserved domain (R1 domain) within the Egr1/NGFI-A and Egr2/Krox20 transactivators, and repress the transcription of Egr target promoters. Using a novel adaptation of the yeast two-hybrid screen, several point mutations have been identified in NAB corepressors that interfere with their ability to bind to the Egr1 R1 domain. Surprisingly, NAB proteins bearing some of these mutations increase Egr1 activity dramatically. The mechanism underlying the unexpected behavior of these mutants was elucidated by the discovery that NAB conserved domain 1 (NCD1) not only binds to Egr proteins but also mediates multimerization of NAB molecules. The activating mutants exert a dominant negative effect on NAB repression by multimerizing with native NAB proteins and preventing binding of endogenous NAB proteins with Egr transactivators. Looking at NAB repression of a native Egr target gene, it can be seen that NAB2 represses Egr2/Krox20-mediated activation of the bFGF/FGF-2 promoter, and that repression is reversed by coexpression of dominant negative NAB2. Because of their specific ability to alleviate NAB repression of Egr target genes, the dominant negative NAB mutants will be useful in elucidating the mechanism and function of NAB corepressors (Svaren, 1998).

The developing vertebrate hindbrain is transiently subdivided along the anterior-posterior axis into metameric units, called rhombomeres (r). These segments constitute units of lineage restriction and display specific gene expression patterns. The transcription factor gene Krox-20 is restricted to r3 and r5, and is required for the development of these rhombomeres. Evidence is presented that Krox-20 transcriptional activity is under the control of a negative feedback mechanism in the hindbrain. This regulatory loop involves two closely related proteins, the Krox-20 co-repressors Nab1 and Nab2, previously identified as antagonists of Krox-20 transcriptional activity in cultured cells. In the mouse hindbrain, Nab1 and Nab2 recapitulate the Krox-20 expression pattern; their expression has been found to be dependent on Krox-20 function. Furthermore, misexpression of Nab1 or Nab2 in zebrafish embryos leads to alterations in the expression patterns of several hindbrain markers, consistent with an inhibition of Krox-20 activity. Taken together, these data indicate that Krox-20 positively regulates the expression of its own antagonists and raise the possibility that this negative feedback regulatory loop may play a role in the control of hindbrain development (Mechta-Grigoriou, 2000).

Targets of Egr genes

The promoter of the rat pgp2/mdr1b gene has a GC-rich region (pgp2GC) that is highly conserved in mdr genes and contains a consensus Sp1 site. Sp1's role in transactivation of the pgp2/mdr1b promoter was tested in Drosophila Schneider cells. The pgp2/mdr1b promoter is strongly activated by co-transfected wild type Sp1 but not mutant Sp1 and mutation of the Sp1 site abrogates Sp1-dependent transactivation. In gel shift assays, the same mutations abolish Sp1-DNA complex formation. Moreover, basal activity of the pgp2/mdr1b Sp1 mutant promoter is dramatically lower. Enforced ectopic overexpression of Sp1 in H35 rat hepatoma cells reveals that cell lines overexpressing Sp1 have increased endogenous pgp2/mdr1b mRNA, demonstrating that Sp1 activates the endogenous pgp2/mdr1b gene. Pgp2GC oligonucleotide also bounds Egr-1 in gel shift assays and Egr-1 competitively displaces bound Sp1. In transient transfections of H35 cells (and human LS180 and HepG2 cells) Egr-1 potently and specifically suppresses pgp2/mdr1b promoter activity; mutations in the Egr-1 site decrease Egr-1 binding and correlate with pgp2/mdr1b up-regulation. Ectopic overexpression of Egr-1 in H35 cells decreases Pgp expression and selectively increases vinblastine sensitivity. In conclusion, Sp1 positively regulates while Egr-1 negatively regulates the rat pgp2/mdr1b gene. Moreover, competitive interactions between Sp1 and Egr-1 in all likelihood determine the constitutive expression of the pgp2/mdr1b gene in H35 cells (Thottassery, 1999).

NGFI-A (also called Egr1, Zif268, or Krox24) and the closely related proteins Krox20, NGFI-C, and Egr3 are zinc-finger transcription factors encoded by immediate-early genes that are induced by a wide variety of extracellular stimuli. NGFI-A has been implicated in cell proliferation, macrophage differentiation, synaptic activation, and long-term potentiation, whereas Krox20 is critical for proper hindbrain segmentation and peripheral nerve myelination. Structure/function analysis of NGFI-A has revealed a 34-aa inhibitory domain that has been hypothesized to be the target of a cellular factor that represses NGFI-A transcriptional activity. Using the yeast two-hybrid system, a cDNA clone was isolated that encodes a protein that interacts with this inhibitory domain and inhibits the ability of NGFI-A to activate transcription. This NGFI-A-binding protein, NAB1, is a 570-aa nuclear protein that bears no obvious sequence homology to known proteins. NAB1 also represses Krox20 activity, but it does not influence Egr3 or NGFI-G, thus providing a mechanism for the differential regulation of this family of immediate-early transcription factors (Russo, 1995).

T cell receptor engagement in the absence of proper accessory signals leads to T cell anergy. E3 ligases are involved in maintaining the anergic state. However, the specific molecules responsible for the induction of anergy have yet to be elucidated. Using microarray analysis early growth response gene 2 (Egr-2) and Egr-3 have been identified as key negative regulators of T cell activation. Overexpression of Egr2 and Egr3 is associated with an increase in the E3 ubiquitin ligase Cbl-b and inhibition of T cell activation. Conversely, T cells from Egr3-/- mice had lower expression of Cbl-b and were resistant to in vivo peptide-induced tolerance. These data support the idea that Egr-2 and Egr-3 are involved in promoting a T cell receptor-induced negative regulatory genetic program (Safford, 2005).

Egr genes and gastrulation

The transcriptional activity of a set of genes, which are all expressed in overlapping spatial and temporal patterns within the Spemann organizer of Xenopus embryos, can be modulated by peptide growth factors. Xegr-1, a zinc finger protein-encoding gene, has been identified as a novel member of this group of genes. The spatial expression characteristics of Xegr-1 during gastrulation are most similar to those of Xbra. Making use of animal cap explants, analysis of the regulatory events that govern induction of Xegr-1 gene activity reveals that, in sharp contrast to transcriptional regulation of Xbra, activation of Ets-serum response factor (SRF) transcription factor complexes is required and sufficient for Xegr-1 gene expression. The Ets-SRF complexes are known to act downstream of the MAP kinase pathway, and in the case of Xegr-1 the complex is shown to function downstream of FGF signaling. The finding that Xegr-1 activation requires Ets-SRF complexes provides the first indication for Ets-SRF complexes binds to serum response elements that are activated during gastrulation. MAP kinase signaling cascades can induce and sustain expression of both Xegr-1 and Xbra. Ectopic Xbra is found to induce Xegr-1 transcription by an indirect mechanism that appears to operate via primary activation of fibroblast growth factor secretion. These findings define a cascade of events that links Xbra activity to the activation of FGF signaling and the subsequent signal-regulated control of Xegr-1 transcription in the context of early mesoderm induction in Xenopus laevis (Panitz, 1998).

Egr genes and the segmentation of the rhombencephalon

The hindbrain is a segmented structure divided into repeating metameric units termed rhombomeres (r). The Hox family, vertebrate homologs of the Drosophila HOM-C homeotic selector genes, are expressed in rhombomere-restricted patterns and are believed to participate in regulating segmental identities. Krox-20, a zinc finger gene, has a highly conserved pattern of expression in r3 and r5 and is functionally required for their maintenance in mouse embryos. Krox-20 has been shown to directly regulate the Hoxb-2 gene (Drosophila homolog: Prosboscipedia) and is involved in regulating multiple Hox genes as a part of its functional role. Hoxa-2 is the only known paralog of Hoxb-2. The patterns of expression of the mouse Hoxa-2 gene were examined with particular focus on r3 and r5 in wild type and Krox-20-/- mutant embryos. There was a clear loss of expression in r3, which indicated that Hoxa-2 is downstream of Krox-20. An r3/r5 enhancer has been identified in the 5' flanking region of the Hoxa-2 gene. Mutation of these Krox-20 sites in the regulatory region specifically abolished r3/r5 activity, but did not affect neural crest and mesodermal components. This indicates that the two Krox-20 sites are required in vivo for enhancer function. Furthermore, ectopic expression of Krox-20 in r4 is able to transactivate Hoxa 2 in this rhombomere. Together these findings suggest that Krox-20 directly participates in the transcriptional regulation of Hoxa-2 during hindbrain segmentation, and is responsible for the upregulation of the r3 and r5 domains of expression of both vertebrate group 2 Hox paralogs. Therefore, the segmental phenotypes in the Krox-20 mutants are likely to reflect the role of Krox-20 in directly regulating multiple Hox genes (Nonchev, 1996).

Retinoic acid application to a recently segmented hindbrain leads to disappearance of posterior rhombomere boundaries. Boundary loss is preceded by changes in segmental expression of Krox-20 and Cek-8 and followed by alterations in Hox gene expression. The characteristic morphology of boundary cells, their expression of follistatin and the periodic accumulation of axons normally associated with boundaries are all lost. In the absence of boundaries, no change in anteroposterior dispersal of precursor cells is detected and, in most cases, there is no substantial cell mixing between former rhombomeric units. This is consistent with the idea that lineage restriction can be maintained by processes other than a mechanical barrier composed of boundary cells. Much of the early organization of the motor nuclei appears normal despite the loss of boundaries and altered Hox expression (Nittenberg, 1997).

The origins of two specifically mammalian structures, the preotic and otic sulci, have been studied. Their formation at the 1/2- and 3-somite stages respectively, divides the hindbrain neuroepithelium into prorhombomeres A, B and C. The preotic sulcus is a deeply recessed structure that forms the rostral boundary of expression of both Hoxb-2 and the first domain of Krox-20. The otic sulcus is a shallow concavity in which the second Krox-20 domain is expressed. DiI labeling followed by whole embryo culture confirms that the later fate of the preotic sulcus is the rhombomere 2/3 boundary, and the fate of the otic sulcus is the cranial part of rhombomere 5. Structurally, the preotic and otic sulci show no specialization with respect to actin, tubulin or proteoglycans, but their maintenance depends on contact with the subjacent mesenchyme. Their formation is inhibited by exposure of embryos to retinoic acid prior to the onset of somitic segmentation, indicating that the molecular events governing prorhombomeric subdivision of the hindbrain are retinoic acid-sensitive. The preotic sulcus may be essential for neuroepithelial cell movement towards and into the rapidly enlarging forebrain; the otic sulcus may simply delineate the caudal boundary of prorhombomere B, an area with a discrete neural crest cell population discontinuous with those populations rostral and caudal to it. Understanding the positional relationships of the preotic and otic sulci to later rhombomeric segments makes them useful landmarks for experimental purposes, but there is no evidence that prorhombomeres are functionally significant as the precursors of rhombomeric segments (Ruberte, 1997).

Rhombomere boundaries were examined, using both morphological and molecular markers, and the fate of specific motor neuron populations were examined using retrograde and anterograde carbocyanine dye tracing. r3 and r5 and their derivatives are completely eliminated in Krox-20(-/-) embryos while overall hindbrain segmentation is maintained. In addition, the disappearance of these territories has important consequences for even-numbered rhombomeres as well (in particular on axonal navigation): (1) a population of r6 motoneurons, presumably normally fated to join the glossopharyngeal nerve, has its axons misrouted toward the facial exit point in r4; (2) the trigeminal motor axons are also misrouted, presumably because of the proximity of the trigeminal and facial exit points. They fasciculate with facial axons outside the neural tube and enter the second branchial arch instead of the first arch. This navigational error could explain the disappearance, at around 17.5 dpc, of the trigeminal motor nucleus in Krox-20(-/-) embryos by inadequate supply of essential, possibly arch-specific survival factors (Schneider-Maunoury, 1997).

The mouse and chicken Hoxb-2 genes are dependent for their expression in rhombdomere 3 and rhombdomere 5 on homologous enhancer elements and on the binding to this enhancer of the r3/r5-specific transcriptional activator Krox-20. Among the three Krox-20 binding sites of the mouse Hoxb-2 enhancer, only the high-affinity site is absolutely necessary for activity. In contrast, an additional cis-acting element, Box1, has been identified as essential for r3/r5 enhancer activity. It is conserved both in sequence and in position respective to the high-affinity Krox-20 binding site within the mouse and chicken enhancers. A short 44 bp sequence spanning the Box1 and Krox-20 sites can act as an r3/r5 enhancer when oligomerized. Box1 may therefore constitute a recognition sequence for another factor cooperating with Krox-20. Taken together, these data demonstrate the conservation of Hox gene regulation and of Krox-20 function during vertebrate evolution (Vesque, 1996).

The morphogenesis of the vertebrate hindbrain involves a transient segmentation process leading to the formation of reiterated organization units called rhombomeres (r). A number of regulatory genes expressed with a rhombomere-specific pattern have been identified, including the gene encoding the transcription factor Krox-20, which is restricted to r3 and r5. In r3 and r5 Krox-20 directly controls the transcription of Hoxa-2 and Hoxb-2. Krox-20 is required for the expression of another Hox gene, Hoxb-3, specifically in r5. The regulatory role of Krox-20 is not restricted to the control of Hox gene expression, since it is also involved in the activation of a receptor tyrosine kinase gene, Sek-1, in r3 and r5 and in the repression of the follistatin gene in r3 but not in r5. In conclusion, at least five regulatory genes belonging to different families are under the direct or indirect control of Krox-20 in r3 and/or r5 and this transcription factor therefore appears as a key regulator of gene expression in the developing hindbrain (Seitanidou, 1997).

The transcription factor genes Hoxa1 and Krox-20 have been shown to play important roles in vertebrate hindbrain segmentation: Hoxa1 is required for maintenance and/or generation of parts of r4 and r5, and Krox-2 plays a similar role for r3 and r5. Evidence is presented for novel functions of these genes, which co-operate in specifying cellular identity in rhombomere (r) 3. Although Hoxa1 has not been observed to be expressed rostrally to the prospective r3/r4 boundary, its inactivation results in (1) the appearance of patches of cells presenting an r2-like molecular identity within r3; (2) early neuronal differentiation in r3, normally characteristic of even-numbered rhombomeres, and (3) abnormal navigation of r3 motor axons, similar to that observed in even-numbered rhombomeres. These phenotypic manifestations become more severe in the context of the additional inactivation of one allele of the Krox-20 gene, demonstrating that Hoxa1 and Krox-20 synergize in a dosage-dependent manner to specify r3 identity and odd- versus even-numbered rhombomere characters. In addition, these data suggest that the control of the development of r3 may not be autonomous but dependent on interactions with Hoxa1-expressing cells. It is thought that a signal originating from r4 or dependent on Hoxa1 function would prevent the proliferation of the early Krox-20-negative cells present in r3. One of the roles of Krox-20 would be to counteract this signal in an autonomous manner and allow for normal proliferation (Helmbacher 1998).

Early in its development, the vertebrate hindbrain is transiently subdivided into a series of compartments called rhombomeres. Genes have been identified whose expression patterns distinguish these cellular compartments. Two of these genes, Hoxa1 and Hoxa2, have been shown to be required for proper patterning of the early mouse hindbrain and the associated neural crest. To determine the extent to which these two genes function together to pattern the hindbrain, mice simultaneously mutant at both loci were generated. The hindbrain patterning defects were analyzed in embryos individually mutant for Hoxa1 and Hoxa2 in greater detail and extended to embryos mutant for both genes. From these data a model is proposed to describe how Hoxa1, Hoxa2, Hoxb1, Krox20 (Egr2) and kreisler function together to pattern the early mouse hindbrain. Critical to the model is the demonstration that Hoxa1 activity is required to set the anterior limit of Hoxb1 expression at the presumptive r3/4 rhombomere boundary. Failure to express Hoxb1 to this boundary in Hoxa1 mutant embryos initiates a cascade of gene misexpressions that result in misspecification of the hindbrain compartments from r2 through r5. Subsequent to misspecification of the hindbrain compartments, ectopic induction of apoptosis appears to be used to regulate the aberrant size of the misspecified rhombomeres (Barrow, 2000).

Hoxa1 and Hoxb1 are coexpressed up to the presumptive r3/4 boundary. Hoxa1 is required to establish Hoxb1 expression in anterior r4. Hoxa1 and Hoxb1 activate the transcription of r4-specific downstream targets including a signal that, in turn, induces Krox 20 expression in cells just anterior to the r3/4 boundary (in cells that are not expressing Hoxa1 or Hoxb1). Krox 20 is repressed, however, in r4 and r5 cells that are expressing Hoxa1 and Hoxb1. Hoxa1 is required for kreisler expression in r5. Without Hoxa1, the anterior limit of Hoxb1 is established in the posterior region of r4. Because of this posterior shift, neither Hoxa1 nor Hoxb1 is expressed in the anterior portion of r4 and Krox 20 is no longer repressed there. Furthermore, the signal downstream of Hoxb1 must be propagated a longer distance causing a delay in the induction of Krox 20 expression in presumptive r3. Due to the absence of Hoxa1, kreisler expression is not activated in r5 (Barrow, 2000).

Without Hoxa1 and Hoxb1 expression, Krox 20 expression is no longer repressed in r4 and r5. In addition, the signal downstream from Hoxa1 and Hoxb1 required to induce Krox 20 expression in r3 is not activated. By E8.5, Hoxa1 expression has completely retreated from the hindbrain. Hoxb1 has also retreated with the exception of the strong autoregulatory expression in r4. Once Hoxa1 and Hoxb1 expression has fully retreated from r5, Krox 20 expression commences at this level. Krox 20 expression also expands into r3. This expansion requires activation of its downstream target(s) Hoxa2 and possibly Hoxb2. Strong kreisler expression in r5 maintains Hoxb1 autoregulated expression at the r4/5 boundary. In Hoxa1 mutants Hoxb1 expression retreats from the caudal hindbrain leaving autoregulated expression in caudal r4. Because kreisler is not activated in r5, autoregulated Hoxb1 expression extends into r5 as well. Krox 20 expansion into r3 although delayed (due to the fewer number of cells that were induced at E8.0) occurs somewhat normally due to the fact that Krox 20 and its downstream target(s) Hoxa2 (and perhaps Hoxb2) are functioning. As a consequence of the larger expression domains of follistatin (r2 and part of r3) and Krox 20 (part of r3 and r4), a regulatory event driven by apoptosis commences in these regions of the neural tube. The hindbrain is similar to that of Hoxa1 single mutants except that Krox 20 expansion into r3 is severely delayed. Hoxa2 is a downstream target of Krox 20 and if absent, cripples the expansion of Krox 20-expressing cells into r3. In double Hoxa1/Hoxa2 mutants Krox 20 is never induced in r3 and thus never expands into r3. As a result, follistatin expression extends to the r3/r4 boundary. Due to enlarged follistatin and Krox 20-expressing domains, apoptosis is activated in the neural tube at this level. Due to the apoptosis at the levels of r2 and r3 in HoxA1 mutants, there is not only a reduction in the number of neural crest cells that will populate the first arch, but also the abnormally large r3 is reduced to almost normal proportions. There is also a reduction in the number of neural crest cells that reach the second arch due to the reduced size of r4 and the fact that the otocyst may act as a barrier to prevent normal migration of the crest. The otocysts do not shift anteriorly to the level of r4; instead, r4 is specified more posteriorly. Double HoxA1/HoxA2 mutants are very similar to Hoxa1 single mutants except that, due to the lack of Hoxa2, the r4 neural crest takes on an r1/r2 identity. In addition, the lack of Hoxa1 causes a reduction in r4 neural crest contributing to the second arch. In HoxA1/HoxA2 double mutants r4 is never specified. Therefore, there is no r4 neural crest to populate the second arch (Barrow, 2000).

Krox-20 and kreisler encode transcription factors involved in the control of hindbrain development and are expressed in rhombomeres (r) 3 and 5, and 5 and 6, respectively. To analyse the regulation of the expression of these genes by positional cues, focusing on the stages just preceding the formation of rhombomeres, ectopic grafts, involving single prospective rhombomeres (pr) or couples of pr, were performed on 4-6 somite avian embryos. Transplantation of pr6 into the pr5 position leads to Krox-20 activation and transplantation of a pr7 into the pr5 position results in kreisler activation. Furthermore, pr6 grafted in the pr5 position develops an r5-like cytoarchitecture. These data establish that rostral transplantation can lead to anteriorization within the hindbrain. However, additional experiments indicate that the competence of the transplanted tissue for such anteriorization appears limited and that transformations corresponding to shifts of a single rhombomere are favored. Caudal transplantation of pr5 into the pr6 position can lead to a down-regulation of Krox-20 expression consistent with posteriorization, suggesting that caudalizing influences are present within the nonsomitic hindbrain after the 4- to 6-somite stage. Finally, combinations of extirpation and grafting experiments suggest that the regulation of kreisler expression in the r6-r7 region may involve anteriorizing influences in addition to posteriorizing signals from the somitic region (Marin, 2000a).

Krox20 and mafB/kreisler are regulatory genes involved in hindbrain segmentation and anteroposterior (AP) patterning. They are expressed in rhombomeres (r) r3/r5 and r5/r6 respectively, as well as in the r5/r6 neural crest. Since several members of the fibroblast growth factor (FGF) family are expressed in the otic/preotic region (r2-r6), their possible involvement in the regulation of Krox20 and mafB/kr was investigated. Application of exogenous FGFs to the neural tube of 4- to 7-somite chick embryos leads to ectopic expression in the neural crest of the somitic hindbrain (r7 and r8) and to the extension of the Krox20- or mafB/kr-positive areas in the neuroepithelium. Application of an inhibitor of FGF signaling leads to severe and specific downregulation of Krox20 and mafB/kr in the hindbrain neuroepithelium and neural crest. These data indicate that FGFs are involved in the control of regional induction and/or maintenance of Krox20 and mafB/kr expression, thus identifying a novel function for these factors in hindbrain development, in addition to their proposed more general role in early neural caudalization (Marin, 2000b).

Inactivation of the Krox20 gene leads to the disappearance of its segmental expression territories in the hindbrain, the rhombomeres (r) 3 and 5. A detailed analysis of the fate of prospective r3 and r5 cells was carried out in Krox20 mutant embryos. Genetic fate mapping indicates that at least some of these cells persist in the absence of a functional Krox20 protein and uncovers the requirement for autoregulatory mechanisms in the expansion and maintenance of Krox20-expressing territories. Analysis of even-numbered rhombomere molecular markers demonstrates that in Krox20-null embryos, r3 cells acquire r2 or r4 identity, and r5 cells acquire r6 identity. Study of embryonic chimaeras between Krox20 homozygous mutant and wild-type cells shows that the mingling properties of r3/r5 mutant cells are changed towards those of even-numbered rhombomere cells. Together, these data demonstrate that Krox20 is essential to the generation of alternating odd- and even-numbered territories in the hindbrain and that it acts by coupling the processes of segment formation, cell segregation and specification of regional identity (Voiculescu, 2001).

In the segmented vertebrate hindbrain, the Hoxa3 and Hoxb3 genes are expressed, respectively, at high relative levels in the rhombomeres (r) 5 and 6, and 5. The single enhancer elements responsible for these activities constitute direct targets of the transcription factor kreisler, which is expressed in r5 and r6. The contribution of the transcription factor Krox20, present in r3 and r5 has been anayzed. Genetic analyses demonstrate that Krox20 is required for activity of the Hoxb3 r5 enhancer, but not of the Hoxa3 r5/6 enhancer. Mutational analysis of the Hoxb3 r5 enhancer, together with ectopic expression experiments, reveals that Krox20 binds to the enhancer and synergizes with kreisler to promote Hoxb3 transcription, restricting enhancer activity to r5, their domain of overlap. These analyses also suggest contributions from an Ets-related factor and from putative factors likely to heterodimerize with kreisler. The integration of multiple independent inputs present in overlapping domains by a single enhancer is likely to constitute a general mechanism for the patterning of subterritories during vertebrate development (Manzanares, 2002).

Neural crest patterning constitutes an important element in the control of the morphogenesis of craniofacial structures. Krox20, a transcription factor gene that plays a critical role in the development of the segmented hindbrain, is expressed in rhombomeres (r) 3 and 5 and in a stream of neural crest cells migrating from r5 toward the third branchial arch. The basis of the specific neural crest expression of Krox20 has been investigated and a cis-acting enhancer element (NCE) located 26 kb upstream of the gene has been identified that is conserved between mouse, man and chick and can recapitulate the Krox20 neural crest pattern in transgenic mice. Functional dissection of the enhancer has revealed the presence of two conserved Krox20 binding sites mediating direct Krox20 autoregulation in the neural crest. In addition, the enhancer included another essential element containing conserved binding sites for high mobility group (HMG) box proteins. This element responds to factors expressed throughout the neural crest. Consistent with this the NCE is strongly activated in vitro by Sox10, a crest-specific HMG box protein, in synergism with Krox20, and the inactivation of Sox10 prevents the maintenance of Krox20 expression in the migrating neural crest. These results suggest that the dependency of the enhancer on both crest- (Sox10) and r5- (Krox20) specific factors limits its activity to the r5-derived neural crest. This organization also suggests a mechanism for the transfer and maintenance of rhombomere-specific gene expression from the hindbrain neuroepithelium to the emerging neural crest and may be of more general significance for neural crest patterning (Ghislain, 2003).

Rostral hindbrain patterning involves the direct activation of a Krox20 transcriptional enhancer by Hox/Pbx and Meis factors

The morphogenesis of the vertebrate hindbrain involves the generation of metameric units called rhombomeres (r), and Krox20 encodes a transcription factor that is expressed in r3 and r5 and plays a major role in this segmentation process. Knowledge of the basis of Krox20 regulation in r3 is rather confusing, especially concerning the involvement of Hox factors. This paper describes a study of one of the Krox20 hindbrain cis-regulatory sequences, element C, which is active in r3-r5 and which is the only initiator element in r3. Element C is shown to contains multiple binding sites for Meis and Hox/Pbx factors; these proteins synergize to activate the enhancer. Mutation of these binding sites showed that Krox20 is under the direct transcriptional control of both Meis (presumably Meis2) and Hox/Pbx factors in r3. Furthermore, the data indicate that element C functions according to multiple modes, in Meis-independent or -dependent manners and with different Hox proteins, in r3 and r5. Finally, it was shown that the Hoxb1 and Krox20 expression domains transiently overlap in prospective r3, and that Hoxb1 binds to element C in vivo, supporting a cell-autonomous involvement of Hox paralogous group 1 proteins in Krox20 regulation. Altogether, these data clarify the molecular mechanisms of an essential step in hindbrain patterning. A model is proposed for the complex regulation of Krox20, involving a novel mode of initiation, positive and negative controls by Hox proteins, and multiple direct and indirect autoregulatory loops (Wassef, 2008).

Krox20 regulation appears to constitute a complex process and this study attempted to amalgamate the observations collected in the present work with previous data to develop a molecular model. The consistent observations in mouse, chick and zebrafish allow combination of data obtained in different vertebrate species. First the regulation in r3 will be envisaged. It is proposed that, in contrast to what was previously thought, at around E8 in the mouse, when Hoxa1/Hoxb1 neural domains reach their maximal rostral extensions, their limits are located within prospective r3. This point is consistent with recent tracing data indicating that derivatives of Hoxa1-expressing cells are found in r3, and is supported by the observation of an overlap between Krox20 and Hoxb1 expression domains in r3. In addition, the existence of another factor (X, unknown) is postulated, whose expression domain extends caudally and will start to overlap with the Hox paralog group (PG) 1 domain around E8. This defines a transversal, narrow stripe of cells where Krox20 is specifically activated under the synergistic transcriptional activities of factor X, Hox PG 1, Pbx and Meis2 proteins, acting through element C. Interestingly, an essential role of Iroquois transcription factors in the activation of krox20 in r3 has been recently uncovered. Factor X might therefore be an Iroquois transcription factors or it might lie downstream to them in the regulatory cascade. A complementary involvement of Hox PG 2 proteins is also likely, although loss-of-function analyses suggest that the major role is played by PG 1 factors. An important feature of this hypothesis is that it provides an explanation for the characteristic initial expression pattern of Krox20, restricted to a very narrow stripe of cells. Krox20 activation will have multiple consequences. (1) It will lead to the progressive retraction of the rostral limit of Hox PG 1 gene expression to the future r3/r4 boundary. This is consistent with the observations that the Hoxb1-positive domain extends within prospective r3 in a Krox20-null mutant and that ectopic Krox20 expression results in Hoxb1 repression. (2) Krox20 initiates several transcriptional autoregulatory loops that are necessary for the maintenance of its own expression. One of them is direct and relies on the binding of Krox20 to element A, whereas the others involve the activation of Hoxa2 and Hoxb2, which will replace Hox PG 1 proteins on element C. These autoregulatory mechanisms are likely to be redundant, as the double mutation of Hoxa2 and Hoxb2 only marginally affects the r3 domain of Krox20 expression. (3) Expression of Krox20 also results in its activation in neighbouring Krox20-negative cells by non-cell autonomous autoregulation, a process thought to participate in the extension of r3. The caudal extension of r3 might also rely on the progression of the front of gene X expression. These processes will give rise to a moving stripe of cells co-expressing Krox20 and Hoxb1 at the caudal edge of developing r3, as was observed in mouse and zebrafish embryos. At some point (around E8.5), these processes of extension of r3 at the expense of adjacent rhombomeres will stop, delimiting the final extensions of r2, r3 and r4 (Wassef, 2008).

In r5, Krox20 is under the control of two initiation enhancer elements, B and C. The severe loss of Krox20 expression in r5 upon mutation of Mafb or vHnf1, and the fact that these factors are likely to act only via element B, suggests that element B is predominant. In r5, element C functions according to a different mode than in r3: although it still requires binding of a Hox protein, Meis factors are not necessary (Wassef, 2008).

Finally, what happens in r4, where element C is active but Krox20 is not expressed? To explain this apparent contradiction, it is proposed that Krox20, in addition to the positive regulatory mechanisms discussed above, is subject to a negative regulation, which may lie downstream of the Hox PG 1 genes and prevent Krox20 expression in r4. The existence of such a negative regulation is consistent with the inactivation of Hoxa1, which results in an extension of the anterior domain of Krox20 into prospective r4, and with the repressive activity of Nlz family members on Krox20 expression (Wassef, 2008).

In conclusion, a particularly interesting feature of this model resides in the initial phase of Krox20 expression in r3. It is proposed that a narrow band of cells is defined by the encounter of two domains extending in opposite directions. In these cells, Krox20 is very transiently activated by Hox PG 1 proteins, which disappear rapidly while Krox20 expression is maintained and propagated by different molecular mechanisms. It is proposed to use the term 'ignition' to refer to the role of Hox PG 1 proteins in this novel type of initiation of gene expression, which may occur in other developmental processes (Wassef, 2008).

Function of Egr genes in the pituitary

Pituitary gonadotropins are critical regulators of gonadal development and function. Expression and secretion of the mature hormones are regulated by gonadotropin-releasing hormone (GnRH), which is itself secreted from the hypothalamus. GnRH stimulation of gonadotropin expression and secretion occurs through the G-protein-linked phospholipase C/inositol triphosphate intracellular signaling pathway, which ultimately leads to protein kinase C (PKC) activation and increased intracellular calcium levels. Transcription factors mediating the effects of GnRH-induced signals on transcription of gonadotropin genes have not yet been identified. Recent studies have identified three key factors involved in luteinizing hormone beta (LHbeta) gonadotropin gene transcription: the nuclear receptor SF-1 (Drosophila homolog: Ftz-f1), the bicoid-related homeoprotein Ptx1 (Pitx1), and the immediate-early Egr-1 gene. GnRH is a potent stimulator of Egr-1, but not Ptx1 or SF-1, expression. Further, Egr-1 activation of the LHbeta promoter is specifically enhanced by PKC, in agreement with a role for Egr-1 in mediating a GnRH effect on transcription. Egr-1 interacts directly with Ptx1 and with SF-1, leading to an enhancement of Ptx1- and SF-1-induced LHbeta transcription. Thus, Egr-1 is a likely transcriptional mediator of GnRH-induced signals for activation of the LHbeta gene (Tremblay, 1999).

The hypothalamic neuropeptide, GnRH, regulates the synthesis and secretion of LH from pituitary gonadotropes. Furthermore, it has been shown that the LH beta-subunit gene is regulated by the transcription factors steroidogenic factor-1 (SF-1) and early growth response protein 1 (Egr1) in vitro and in vivo. The present study investigated the roles played by Egr1 and SF-1 in regulating activity of the equine LH beta-subunit promoter in the gonadotrope cell line, alpha T3-1, and the importance of these factors and cis-acting elements in regulation of the promoter by GnRH. All four members of the Egr family induce activity of the equine promoter. The region responsible for induction by Egr was localized to the proximal 185 bp of the promoter, which contains two Egr response elements. Coexpression of Egr1 and SF-1 leads to a synergistic activation of the equine (e)LH beta promoter. Mutation of any of the Egr or SF-1 response elements attenuate this synergism. Endogenous expression of Egr1 in alpha T3-1 cells is not detectable under basal conditions, but is rapidly induced after GnRH stimulation. Reexamination of the promoter constructs harboring mutant Egr or SF-1 sites indicates that these sites are required for GnRH induction. Mutation of both Egr sites within the eLH beta promoter completely attenuates its induction by GnRH. Thus, GnRH induces expression of Egr1, which subsequently activates the eLH beta promoter. Finally, GnRH not only induces expression of Egr1, but also its corepressor, NGFI-A (Egr1) binding protein (Nab1), which can repress Egr1-induced transcription of the eLH beta promoter (Wolfe, 1999).

Early growth response (Egr) 1-deficient mice exhibit female infertility, reflecting a luteinizing hormone (LH) beta deficiency. Egr-1 activates the LHbeta gene in vitro through synergy with steroidogenic factor-1 (SF-1), a protein required for gonadotrope function. To test if this synergy is essential for stimulation of LHbeta by gonadotropin-releasing hormone (GnRH), the activity of the LHbeta promoter was examined in the gonadotrope cell line LbetaT2. GnRH markedly stimulates the LHbeta promoter (15-fold). Mutation of either Egr-1 or SF-1 elements within the LHbeta promoter attenuates this stimulation, whereas mutation of both promoter elements abrogates GnRH induction of the LHbeta promoter. Furthermore, GnRH stimulates Egr-1 but not SF-1 expression in LbetaT2 cells. Importantly, overexpression of Egr-1 alone is sufficient to enhance LHbeta expression. Although other Egr proteins are expressed in LbetaT2 cells and are capable of interacting with SF-1, GnRH stimulation of Egr-1 is the most robust. The nuclear receptor DAX-1, a repressor of SF-1 activity, reduces Egr-1-SF-1 synergy and diminishes GnRH stimulation of the LHbeta promoter. It is concluded that the synergy between Egr-1 and SF-1 is essential for GnRH stimulation of the LHbeta gene and plays a central role in the dynamic regulation of LHbeta expression (Dorn, 1999).

Egr genes and fertility

Male fertility is complex and depends upon endocrine/paracrine regulatory mechanisms and morphogenetic processes occurring during testicular development, spermatogenesis (mitosis and meiosis) and spermiogenesis (spermatid maturation). The Egr family of zinc-finger transcription factors, whose members include Egr1 (NGFI-A), Egr2 (Krox20), Egr3 and Egr4 (also known as NGFI-C or pAT133), are thought to regulate critical genetic programs involved in cellular growth and differentiation. Egr4 null mice were derived through targeted mutagenesis: they are phenotypically normal with the exception that males, but not females, are infertile. Egr4 is expressed at low levels within male germ cells during meiosis and is critical for germ cell maturation during the early-mid pachytene stage. While most Egr4 null male germ cells undergo apoptosis during early-mid pachytene, some are capable of maturing beyond an apparent Egr4-dependent developmental restriction point. Consequently, a limited degree of spermiogenesis occurs but this is accompanied by markedly abnormal spermatozoon morphology and severe oligozoospermia. Egr4 appears to regulate critical genes involved in early stages of meiosis and has a singularly important role in male murine fertility. These data raise the possibility that Egr4 may contribute to some forms of human idiopathic male infertility (Tourtellotte, 1999).

Egr genes as targets of ETS transcription factors

Continued: Evolutionary Homologs part 2/2

huckebein: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 5 August 97  

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