Xcoe2 is a recently identified HLH transcription factor of the Xenopus primary neurogenesis pathway, which is necessary downstream of Neurogenin to stabilize neuroblast determination. The embryonic expression pattern of Zcoe2, its zebrafish homolog, is reported. As observed for Xcoe2, Zcoe2 is strongly expressed in a subset of the neurogenin1- (ngn1-) positive primary neuroblasts of the spinal cord. In the anterior neural plate, in contrast, Zcoe2 is expressed earlier and more widely than ngn1. This pattern is strongly maintained in the presumptive mesencephalon and rhombomeres 1-4 until the 2-3-somite stage. This expression of Zcoe2 in the brain anlage calls for a re-analysis in zebrafish of the functional relationship demonstrated in Xenopus between Coe2 and Neurogenin factors. At later stages, Zcoe2 is expressed in early forming neurons of the anterior brain and is a marker of the olfactory placodes (Bally-Cuif, 1998).

Primary neurogenesis in Xenopus is a model for studying the control of neural cell fate decisions. The specification of primary neurons appears to be driven by transcription factors containing a basic region and a helix-loop-helix (HLH) motif: expression of Xenopus neurogenin-related-1 (X-ngnr-1) defines the three prospective domains of primary neurogenesis, and expression of XNeuroD coincides with neuronal differentiation. However, the transition between neuronal competence and stable commitment to a neuronal fate remains poorly characterised. Drosophila Collier and rodent early B-cell factor/olfactory-1 are both members of a family of HLH transcription factors containing a previously unknown type of DNA-binding domain. An orthologous gene from Xenopus, Xcoe2, has been isolated and found to be expressed in precursors of primary neurons. Xcoe2 is transcribed after X-ngnr-1 and before XNeuroD. Overexpression of a dominant-negative mutant of XCoe2 prevents neuronal differentiation. Conversely, overexpressed wild-type Xcoe2 could promote ectopic differentiation of neurons, in both the neural plate and the epidermis. In contrast to studies with X-ngnr-1 or XNeuroD, the supernumerary neurons induced by Xcoe2 appear in a 'salt-and-pepper' pattern, resulting from the activation of X-Delta1 expression and feedback regulation by lateral inhibition. XCoe2 may play a pivotal role in the transcriptional cascade that specifies primary neurons in Xenopus embryos: by maintaining Delta-Notch signaling, XCoe2 stabilizes the higher neural potential of selected progenitor cells that express X-ngnr-1, ensuring the transition between neural competence and irreversible commitment to a neural fate; and it promotes neuronal differentiation by activating XNeuroD expression, directly or indirectly (Dubois, 1998).

Both gain-and loss-of-function analyses indicate that proneural basic/helix-loop-helix (bHLH) proteins direct not only general aspects of neuronal differentiation but also specific aspects of neuronal identity within neural progenitors. In order to better understand the function of this family of transcription factors, hormone-inducible fusion constructs were used to assay temporal patterns of downstream target regulation in response to proneural bHLH overexpression. In these studies, two distantly related Xenopus proneural bHLH genes, Xash1 and XNgnr1, were compared. Both Xash1 and XNgnr1 induce expression of the general neuronal differentiation marker, N-tubulin, with a similar time course in animal cap progenitor populations. In contrast, these genes each induce distinct patterns of early downstream target expression. Both genes induce expression of the HLH-containing gene, Xcoe2, at early time points, but only XNgnr1 induces early expression of the bHLH genes, Xath3 and XNeuroD. Structure:function analyses indicate that the distinct pattern of XNgnr1-induced downstream target activation is linked to the XNgnr1 HLH domain, demonstrating a novel role for this domain in mediating the differential function of individual members of the proneural bHLH gene family (Talikka, 2002).

Early B-cell factor (EBF) is a tissue-specific and differentiation stage-specific DNA-binding protein that participates in the regulation of the pre-B and B lymphocyte-specific mb-1 gene (MB-1 is a non-immunoglobulin component of the B-cell antigen receptor complex that couples the B cell antigen receptor to multiple src family protein tyrosine kinases). Partial amino acid sequences obtained from purified EBF were used to isolate cDNA clones, which by multiple criteria encode EBF. The recombinant polypeptide forms sequence-specific complexes with the EBF-binding site in the mb-1 promoter. The cDNA hybridizes to multiple transcripts in pre-B and B-cell lines, but transcripts are not detected at significant levels in plasmacytoma, T-cell, and nonlymphoid cell lines. Expression of recombinant EBF in transfected nonlymphoid cells strongly activates transcription from reporter plasmids containing functional EBF-binding sites. Analysis of DNA binding by deletion mutants of EBF hs identified an amino-terminal cysteine-rich DNA-binding domain lacking obvious sequence similarity to known transcription factors. DNA-binding assays with cotranslated wild-type and truncated forms of EBF indicate that the protein interacts with its site as a homodimer. Deletions delineate a carboxy-terminal dimerization region containing two repeats of 15 amino acids that show similarity with the dimerization domains of basic-helix-loop-helix proteins. Together, these data suggest that EBF represents a novel regulator of B lymphocyte-specific gene expression (Hagman, 1993).

Early-B-cell factor (EBF) is a nuclear protein that recognizes a functionally important sequence in the promoter of the mb-1 gene. Like the mb-1 gene, which encodes an immunoglobulin-associated protein, EBF is specifically expressed in the early stages of B-lymphocyte differentiation. EBF was purified by sequence-specific DNA affinity chromatography and its biochemical properties and DNA-binding specificity were examined. EBF generates protein-DNA complexes with the mb-1 promoter. EBF DNA-binding activity can be reconstituted from polypeptides with molecular masses of 62 to 65 kDa. It has been suggested that native EBF has a molecular mass of 140 kDa, if one assumes a globular shape for the protein. Thus, EBF appears to be a dimer with subunits of 62 to 65 kDa. To characterize the DNA-binding specificity of purified EBF, two sets of experiments were performed. (1) Various mutant EBF-binding sites were examined for interaction with purified EBF in an electrophoretic mobility shift assay. (2) Oligonucleotides containing pairs of randomized bases were used in a binding-site selection and amplification experiments to determine a preferred sequence for DNA binding by EBF. Taken together, the results indicate that EBF recognizes variations on the palindromic sequence 5'-ATTCCCNNGGGAAT, with an optimal spacer of 2 bp between the half-sites (Travis, 1993).

The structural requirements for DNA binding, homodimerization and transcriptional activation by EBF have been examined. A carboxyl-terminal region, containing a repeat of alpha-helices related to the helix-loop-helix motif, is important for dimerization of EBF in solution and can confer dimerization on a heterologous DNA binding protein. The amino-terminal DNA binding domain by itself is monomeric, but can mediate assembly of dimers on optimized and correctly spaced half-sites. Mutational analysis of the DNA binding domain of EBF indicates that a novel zinc coordination motif consisting of H-X3-C-X2-C-X5-C is important for DNA recognition. Deletion analysis and transfer of regions of EBF onto a heterologous DNA binding domain identify a serine/threonine-rich transcriptional activation domain. Moreover, the DNA binding domain of EBF can mediate transcriptional activation from optimized binding sites. Thus, EBF contains both a complex DNA binding domain that allows for dimerization and transcriptional activation, and additional dimerization and activation domains (Hagman, 1995).

Two novel mouse genes, Ebf2 and Ebf3, have been identified that show high similarity to Drosophila collier and the rodent Ebf/Olf-1 genes. The strong conservation of the protein regions corresponding to the DNA binding and dimerization domains previously defined in Ebf strongly suggests that Ebf2 and Ebf3 also constitute DNA sequence-specific transcription factors. Determination of the chromosomal locations of the two genes indicates that the different members of this novel mouse multigene family are not clustered. A detailed analysis of the expression of each of the three Ebf genes in the developing central nervous system reveals partially overlapping patterns with two salient features: (1) in the region extending from the midbrain to the spinal cord, the expression of the three genes correlates with neuronal maturation, with a general activation in early post-mitotic cells, followed by specific patterns of extinction also consistent with the neurogenic gradient. (2) In the forebrain area, although the patterns of Ebf gene expression also reflect neuronal maturation, in addition they appear to be region specific. These data suggest that Ebf genes may be involved in the control of neuronal differentiation in the CNS and in enforcing regional diversity in populations of post-mitotic forebrain neurons (Garel, 1997).

The Olf-1/EBF helix-loop-helix (HLH) transcription factor has been implicated in olfactory gene regulation and in B-cell development. Using homology screening methods, two additional Olf-1/EBF-like cDNAs were identified from a mouse embryonic cDNA library. The Olf-1/EBF-like (O/E) proteins O/E-1, O/E-2, and O/E-3 define members of a family of transcription factors that share structural similarities and biochemical activities. Although these O/E genes are expressed within olfactory epithelium in an identical pattern, they exhibit different patterns of expression in the developing nervous system. Although O/E-1 mRNA is present in several tissues in addition to olfactory neurons and developing B-cells, O/E-2 and O/E-3 are expressed at high levels only in olfactory tissue. In O/E-1 knock-out animals, the presence of two additional O/E family members in olfactory neurons may provide redundancy and allow normal olfactory neurodevelopment. The identification of members of the O/E family of HLH transcription factors and their embryonic expression patterns suggest that the O/E proteins may have a more general function in neuronal development (Wang, 1997).

Facial branchiomotor (fbm) neurons undergo a complex migration in the segmented mouse hindbrain. They are born in the basal plate of rhombomere (r) 4, migrate caudally through r5, and then dorsally and radially in r6. To study how migrating cells adapt to their changing environment and control their pathway, this stereotyped migration was analyzed in wild-type and mutant backgrounds. During their migration, fbm neurons regulate the expression of genes encoding the cell membrane proteins TAG-1, Ret and cadherin 8. Specific combinations of these markers are associated with each migratory phase in r4, r5 and r6. In Krox20 and kreisler mutant mouse embryos, both of which lack r5, fbm neurons migrate dorsally into the anteriorly positioned r6 and adopt an r6-specific expression pattern. In embryos deficient for Ebf1, a gene normally expressed in fbm neurons, part of the fbm neurons migrates dorsally within r5. Accordingly, fbm neurons prematurely express a combination of markers characteristic of an r6 location. These data suggest that fbm neurons adapt to their changing environment by switching on and off specific genes, and that Ebf1 is involved in the control of these responses. In addition, they establish a close correlation between the expression pattern of fbm neurons and their migratory behaviour, suggesting that modifications in gene expression participate in the selection of the local migratory pathway (Garel, 2000).

ebf1, ebf2 and ebf3 are all expressed in both immature olfactory neuronal precursors and mature neurons in the adult olfactory epithelium, and also in the developing nervous system during mouse embryogenesis. Expression of all three ebf genes in early post-mitotic neurons along the rostro-caudal axis from the midbrain to the spinal cord and at specific sites in the embryonic forebrain suggests a role in the control of neuronal maturation in the CNS. The absence of any anomaly of the olfactory epithelium, or global defect in the CNS in ebf1-/- mice further raises the possibility of complementation or redundancy between the different EBF in tissues where several ebf genes are expressed. This hypothesis was substantiated by the observation that ebf1-/- embryos show specific defects in the embryonic striatum, where ebf1 is the only ebf gene to be expressed; ebf1 expression is detected throughout embryonic development in differentiating cells of the subventricular zone (SVZ) and mantle cells in the lateral ganglionic eminence (LGE). In ebf1 minus embryos, post-mitotic cells that leave the SVZ appear to be unable to downregulate genes whose expression is normally restricted to this zone, such as Ephrin A-4 and SCIP/Oct-6 or to activate mantle-specific genes such as cellular retinoic acid binding protein I (CRABP I) and cadherin 8. These early molecular defects are followed by a dramatic reduction in the size of the postnatal striatum, due to massive cell death. Impaired striatal cell differentiation in turn leads to defects in navigation and fasciculation of thalamocortical fibers travelling through the striatum, possibly due to change in expression of surface components such as Ephrin A-4 or Cadherin 8. This axonal navigation phenotype of thalamic neurons is reminiscent of the non-cell autonomous defects in motor neuron axonal pathfinding observed in unc-3 mutant worms, suggesting that at least some specific aspects of coe gene function in neuronal differentiation have been conserved throughout evolution. ebf1 is also the only ebf gene expressed in facial branchiomotor (fbm) neurons. In the mouse, fbm neurons undergo a complex and stereotyped migration in the hindbrain, from rhombomere r4, their site of birth, to rhombomere 6, to form the facial motor (nVII) nucleus. In ebf1 mutant embryos, the migration of fbm neurons is abnormal, a phenotype suggestive of a misinterpretation of their rhombomeric environment. Supporting this conclusion, analysis with molecular markers has revealed no general defect in neuronal differentiation (Dubois, 2001 and references therein).

As observed in the striatum, differentiating ebf1 minus fbm neurons do not downregulate the expression of early genes, leading to a discrepancy between their location and their pattern of gene expression. The observation of ebf1 mutant neuronal phenotypes at sites where ebf1 is the only expressed member of the ebf family suggests that ebf genes may play similar roles in other neuronal populations, and that the absence of one family member may be compensated by expression of another. It does not rule out, however, the possible importance of the expression in the same tissue of several EBF proteins, all of which can bind DNA as heterodimers. It is worth noting that ebf3 and ebf2, in contrast to ebf1, are expressed in the progenitors of fbm neurons, but not in mature or migrating neurons whereas in the spinal cord, ebf2 is activated and down-regulated earlier than ebf1 and ebf3. These sequential patterns of expression raise the questions both of specific neuronal functions and epistatic relations between the different ebf genes. Targeted disruption of ebf2 and ebf3, in progress in several laboratories, should help to clarify the respective roles of each EBF protein in various developmental situations (Dubois, 2001 and references therein).

Olf/Ebf transcription factors have been implicated in numerous developmental processes, ranging from B-cell development to neuronal differentiation. Mice are described that carry a targeted deletion within the Ebf2 (O/E3) gene. In Ebf2-null mutants, because of defective migration of gonadotropin releasing hormone-synthesizing neurons, formation of the neuroendocrine axis (which is essential for pubertal development) is impaired, leading to secondary hypogonadism. In addition, Ebf2-/- peripheral nerves feature defective axon sorting, hypomyelination, segmental dysmyelination and axonal damage, accompanied by a sharp decrease in motor nerve conduction velocity. Ebf2-null mice reveal a novel genetic cause of hypogonadotropic hypogonadism and peripheral neuropathy in the mouse, disclosing an important role for Ebf2 in neuronal migration and nerve development (Corradi, 2003).

The mammalian Olf1/EBF (O/E) family of repeated helix-loop-helix (rHLH) transcription factors has been implicated in olfactory system gene regulation, nervous system development and B-cell differentiation. Ebf (O/E1) mutant animals show defects in B-cell lineage and brain regions where it is the only O/E family member expressed, but the olfactory epithelium appears unaffected and olfactory marker expression is grossly normal in these animals. In order to further study the mammalian O/E proteins, murine O/E2 and O/E3 genes were disrupted and tau-lacZ and tau-GFP reporter genes were placed under the control of the respective endogenous O/E promoters. Mice mutant for each of these genes display reduced viability and other gene-specific phenotypes. Interestingly, both O/E2 and O/E3 knockout mice as well as O/E2/O/E3 double heterozygous animals share a common phenotype: olfactory neurons (ORN) fail to project to dorsal olfactory bulb. It is suggested that a decreased dose of O/E protein may alter expression of O/E target genes and underlie the ORN projection defect (Wang, 2004).

Collier homologs in C. elegans

The expression of specialized signal transduction components in mammalian olfactory neurons is thought to be regulated by the O/E (Olf-1/EBF) family of transcription factors. The O/E proteins are expressed in cells of the olfactory neuronal lineage throughout development and are also expressed transiently in neurons in the developing nervous system during embryogenesis. A C. elegans homolog of the mammalian O/E proteins has been identified that displays greater than 80% similarity over 350 amino acids. Like its mammalian homologs, CeO/E is expressed in certain chemosensory neurons (ASI amphid neurons) throughout development and is also expressed transiently in developing motor neurons when these cells undergo axonal outgrowth. In L1 larvae, 16 cells of 22 motor neurons express unc-3; by late L1 and early L2, when 57 new motor neurons are added to the ventral cord, up to 41 ventral motor neurons express the gene. CeO/E is the product of the unc-3 gene, mutations in which cause defects in the axonal outgrowth of motor neurons, as well as defects in dauer formation, a process requiring chemosensory inputs. CeO/E is expressed in two cells outside the ventral cord. These are identified as ASI amphid neurons, chemosensory neurons implicated in a number of sensory processes, including dauer formation. The dauer program is an alternative developmental stage that is activated under conditions of crowding, low food, and unfavorable temperatures. It is possible that unc-3 is required for the proper function and/or development of the ASI neurons. The continual expression of CeO/E in the ASI amphid neurons long after synaptic connections are complete is similar to the pattern seen in mammalian olfactory neurons where expression in these cells remains elevated throughout development. The presence of O/E-binding sites proximal to olfactory-specific genes suggests that O/E proteins regulate the odorant signal transduction cascade. The observations presented in this paper suggest that the O/E family of transcription factors play a central and evolutionarily conserved role in the expression of proteins essential for axonal pathfinding and/or neuronal differentiation in both sensory and motor neurons (Prasad, 1998).

collier is expressed in specific subsets of post-mitotic neurons of both the central nervous system (CNS) and peripheral nervous system (PNS) (Crozatier, 1996). Although the significance of this expression remain to be analysed, neurogenesis may emerge as one unifying theme of ebf/coe developmental functions in both vertebrates and invertebrates. Genetic evidence for multiple roles of coe genes in neural development came with the identification of C. elegans unc-3. unc-3 is expressed, starting in late stage embryos, in a fraction of motor neurons that form in the ventral nerve cord. In newly hatched first stage (L1) larvae, these correspond to the cholinergic DA and DB neurons, and exclude the GABAergic DD neurons. unc-3 mutant worms move abnormally, a behavioral phenotype that correlates with defects in motor neuron axonal pathfinding. Together, these observations suggest that Unc-3 might regulate expression of genes involved in growth cone pioneering along the ventral nerve cord, or in fasciculation. The existence of axonal projection defects in D class motor neurons that do not express Unc-3 further suggested a non-cell autonomous effect, possibly due to changes of surface components of Unc-3 expressing neurons. unc-3 mutants are also defective in the process of dauer formation (entry into dormancy), which requires inputs from the ASI amphid neurons. Contrary to motor neurons, the sensory neurons that express unc-3 throughout development do not display abnormal axonal projections in unc-3 mutants, suggesting that their activity rather than their circuitry is defective. unc3 might therefore regulate expression in these chemosensory neurons of components essential for transducing environmental cues (Dubois, 2001 and references therein).

Collier/OLF/EBF-Dependent Transcriptional Dynamics Control Pharyngeal Muscle Specification from Primed Cardiopharyngeal Progenitors

In vertebrates, pluripotent pharyngeal mesoderm progenitors produce the cardiac precursors of the second heart field as well as the branchiomeric head muscles and associated stem cells. However, the mechanisms underlying the transition from multipotent progenitors to distinct muscle precursors remain obscured by the complexity of vertebrate embryos. Using Ciona intestinalis as a simple chordate model, this study shows that bipotent cardiopharyngeal progenitors are primed to activate both heart and pharyngeal muscle transcriptional programs, which progressively become restricted to corresponding precursors. The transcription factor COE (Collier/OLF/EBF) orchestrates the transition to pharyngeal muscle fate both by promoting an MRF-associated myogenic program in myoblasts and by maintaining an undifferentiated state in their sister cells through Notch-mediated lateral inhibition. The latter are stem cell-like muscle precursors that form most of the juvenile pharyngeal muscles. The implications of these findings for the development and evolution of the chordate cardiopharyngeal mesoderm are discussed (Razy-Krajka, 2014).

Collier homologs and brain patterning

Several genetic factors have been proven to contribute to the specification of the metencephalic-mesencephalic territory, a process that sets the developmental foundation for prospective morphogenesis of the cerebellum and mesencephalon. However, evidence stemming from genetic and developmental studies performed in man and various model organisms suggests the contribution of many additional factors in determining the fine subdivision and differentiation of these central nervous system regions. In man, the cerebellar ataxias/aplasias represent a large and heterogeneous family of genetic disorders. Mmot1 is a new gene, encoding a DNA-binding protein strikingly similar to the helix-loop-helix factor Ebf/Olf1. Throughout midgestation embryogenesis, Mmot1 is expressed at high levels in the metencephalon, mesencephalon, and sensory neurons of the nasal cavity. In vitro DNA binding data suggest some functional equivalence of Mmot1 and Ebf/Olf1, possibly accounting for the reported lack of olfactory or neural defects in Ebf-/- knockout mutants. The isolation of Mmot1 and of an additional homolog in the mouse genome defines a novel, phylogenetically conserved mammalian family of transcription factor genes with potential relevance to studies of neural development and aberration (Malgaretti, 1997).

Because modifications in the expression pattern of guidance and adhesion molecules were observed in the LGE of Ebf1 -/- embryos, an examination was carried out to see if fiber tracts passing through the striatum are affected. The internal capsule is a major fiber tract that travels through the striatum and is composed of cortical axons projecting subcortically and axons originating from various CNS structures projecting towards the cortex. fiber bundles running in the caudal part of the internal capsule fasciculate before turning medially and entering the pallidum. In Ebf1 -/- animals, the fibers of the internal capsule exhibit an abnormal fasciculation: the bundles of fibers are generally larger than in wild-type animals and their distribution appear less regular. In the caudal part, where the internal capsule normally forms a single large bundle, a disorganization is also observed. This fasciculation defect is already visible at E15.5, before any other morphological alteration of the striatum is detected. Finally, the posterior branch of the anterior commissure, which runs underneath the striatum, is also affected: a large majority of the fibers fail to cross the midline. To further characterize the phenotype affecting telencephalic fiber tracts, the thalamocortical fibers, which are an important component of the internal capsule, were traced by placing DiI crystals in the dorsolateral thalamus of E15.5 and E18.5 embryos. In E15.5 Ebf1 -/- embryos, DiI-labelled fibers present a normal navigation and fasciculation before entering the ganglionic eminences. In the LGE, these fibers spread out in the mantle and enter the ventralmost part, whereas in wild-type embryos these fibers are restricted to the dorsal part. At E18.5, the characteristic fan-shaped distribution of the thalamocortical fibers in the striatal mantle is completely disorganized in the homozygous mutant. However, after exiting the striatum, DiI-labelled fibers refasciculate and reach the cortex. In conclusion, these results indicate that thalamocortical fibers are specifically affected as they travel through the striatum primordium, and this phenotype is detected before any other morphological defect can be observed in the LGE (Garel, 1999).

Neurogenesis in both vertebrates and invertebrates is tightly controlled in time and space involving both positive and negative regulators. The bHLH factor Her5 acts as a prepattern gene to prevent neurogenesis in the anlage of the midbrain/hindbrain boundary in the zebrafish neural plate. This involves selective suppression of both neurogenin1 (ngn1) and coe2 mRNA expression in a process that is independent of Notch signalling, and where inhibition of either ngn1 or coe2 expression is sufficient to prevent neuronal differentiation across the midbrain-hindbrain boundary. A ngn1 transgene faithfully responds to Her5 and deletion analysis of the transgene identifies an E-box in a ngn1 upstream enhancer to be required for repression by Her5. Together these data demonstrate a role for Her5 as a prepattern factor in the spatial definition of proneural domains in the zebrafish neural plate, in a manner similar to its Drosophila homolog Hairy (Geling, 2004).

Both Ngn1 and Coe2 functions are necessary for the progression of neurogenesis and for the early events of neuronal differentiation in the midbrain-hindbrain domain. Blocking Coe2 activity downregulates ngn1 expression throughout the neural plate, suggesting a requirement for Coe2 in all primary neurons. The absence of ngn1 function prevents deltaB expression in the anterior proneural clusters, including the presumptive motorneurons of rhombomeres 2 and 4, and the first anterior neuronal cluster (ventrocaudal cluster, vcc), and is also necessary for neuronal differentiation of vcc derivatives, which comprise at least the first differentiating populations of the reticulospinal nMLF neurons. This, together with previous reports, indicates a strict requirement for Ngn1 in spinal sensory neurons and the MH area of the embryonic zebrafish CNS. By contrast, Ngn1 is not essential for motorneuron and interneuron development in the trunk and spinal cord, and for epiphysial neurons. Differential requirements for Ngn in CNS neuronal differentiation were also observed in other vertebrates, a typical example being the complementary requirements for Ngn2 and Mash1 in the mouse embryonic neural tube. Other bHLH factors, such as Achaete-scute or Olig, may play redundant or prominent roles in neurogenic areas that differentiate normally in ngn1-deficient embryos (Geling, 2004 and references therein).

These results point to synergistic roles of Ngn1 and Coe2 in MH neurogenesis, possibly reflecting the positive cross-regulation of their expression, and a parallel activity of these factors rather than their action in a linear cascade. It is possible that the crossregulation of ngn1 and coe2 expression helps stabilize the committed state of neuronal progenitors, as described for Xenopus Xcoe2. Together, these results led to a model for the spatial control of MH neurogenesis. In this process, ngn1 and coe2 expression are crucial elements that permit neurogenesis throughout the MH, which is initially identified as a single territory competent to form neurons. At the MHB, ngn1 and coe2 expression are the targets of Her5 inhibition. This inhibition prevents the specification of a proneural cluster in this location and permits the generation of the 'intervening zone' (Geling, 2004).

Ebf1/Olf-1, the mammalian homolog of Drosophila collier, belongs to a small multigene family encoding closely related helix-loop-helix transcription factors, which have been proposed to play a role in neuronal differentiation. Ebf1 controls cell differentiation in the murine embryonic striatum, where it is the only gene of the family to be expressed. The striatum is a large structure located in the ventral telencephalon. During embryogenesis, the telencephalic vesicles differentiate dorsally into the cortex and ventrally into two bulges, the lateral (LGE) and medial (MGE) ganglionic eminences. The LGE will give rise to the striatum and the MGE will form the pallidum, two structures that are part of the basal ganglia and are involved in motion control. In both the cortex and the ganglionic eminences, two distinct proliferative zones contribute to the generation of neuronal precursors: a ventricular zone (VZ) organized as a pseudostratified neuroepithelium and a VZ-derived subventricular zone (SVZ), where dividing cells are scattered and do not form an epithelial structure. After their exit from the cell cycle in the VZ or in the SVZ, cells migrate laterally into the mantle, where they undergo terminal differentiation. Two histochemical compartments designated 'patch' (striosome) and 'matrix' can be distinguished in the postnatal striatum. The patch and matrix compartments differ in their expression of neurotransmitters, neuropeptides and receptors, as well as in their input from the cortex and output to the pallidum and substantia nigra. In addition, the peak of generation of patch neurons occurs earlier than that of the matrix neurons. Ebf1 targeted disruption affects postmitotic cells that leave the subventricular zone (SVZ) en route to the mantle: they appear to be unable to downregulate genes normally restricted to the SVZ or to activate some mantle-specific genes. These downstream genes encode a variety of regulatory proteins including transcription factors and proteins involved in retinoid signaling as well as adhesion/guidance molecules. These early defects in the SVZ/mantle transition are followed by an increase in cell death, a dramatic reduction in size of the postnatal striatum and defects in navigation and fasciculation of thalamocortical fibers travelling through the striatum. These data show that Ebf1 plays an essential role in the acquisition of mantle cell molecular identity in the developing striatum and provide information on the genetic hierarchies that govern neuronal differentiation in the ventral telencephalon (Garel, 1999).

Ebf gene function is required for coupling neuronal differentiation and cell cycle exit

Helix-loop-helix transcription factors of the Ebf/Olf1 family have been implicated in the control of neurogenesis in the central nervous system in both Xenopus laevis and the mouse, but their precise roles have remained unclear. Two family members have been characterized in the chick, and a functional analysis was performed by gain- and loss-of-function experiments. This study reveals several specific roles for Ebf genes in the spinal cord and hindbrain regions of higher vertebrates, and enables their precise positioning along the neurogenic cascade. During neurogenesis, cell cycle exit appears to be tightly coupled to migration to the mantle layer and to neuronal differentiation. Antagonizing Ebf gene activity allows the uncoupling of these processes. Ebf gene function is necessary to initiate neuronal differentiation and migration toward the mantle layer in neuroepithelial progenitors, but it is not required for cell cycle exit. Ebf genes therefore appear to be master controllers of neuronal differentiation and migration, coupling them to cell cycle exit and earlier steps of neurogenesis. Mutual activation between proneural and Ebf genes suggests that besides their involvement in the engagement of differentiation, Ebf genes may also participate in the stabilization of the committed state. Finally, gain-of-function data raise the possibility that, in addition to these general roles, Ebf genes may be involved in neuronal subtype specification in particular regions of the CNS (Garcia-Dominguez, 2003).

Analysis of Ebf1 and Ebf3 mRNAs in the chick neural tube indicated that their accumulation is coincidental with the onset of neurogenesis and that they are detected within the entire mantle layer. This is in agreement with the expression pattern of the mouse orthologs, and shows that these genes are expressed at a high level in early post-mitotic neurons and that their expression is maintained during neuronal differentiation. Low level, scattered expression has also been observed in the neuroepithelium for mouse Ebf2 and Ebf3, presumably corresponding to cells en route to the mantle layer. Forced expression of both Ngn2, a proneural gene, and NeuroM, an early neuronal differentiation regulator, promotes Ebf1 and Ebf3 expression, indicating that the latter genes are downstream of the former in the neurogenic cascade, consistent with Ebf gene expression pattern. Expression of a dominant-negative molecule, which presumably antagonizes all Ebf activities, does not affect cell cycle exit, but prevents neuroepithelial precursor migration towards the mantle layer and expression of differentiation markers. Furthermore, the dominant-negative Ebf is also able to prevent neuronal differentiation and migration induced by the forced expression of Ngn2, but it does not affect the endogenous expression of this latter gene. Together, these observations suggest that Ebf genes play an essential role in cell engagement into neuronal differentiation and migration towards the mantle layer, coupling these processes to cell cycle exit. In agreement with a role of Ebf genes in the control of neuronal differentiation and migration, misexpression of Ebf1 in neuroepithelial progenitors promotes these processes, which indicates that Ebf genes are both necessary and sufficient. However, surprisingly, forced expression of Ebf1 also leads to exit from the cell cycle. This is correlated with a transient reinforcement of Ngn1 and Ngn2, and NeuroM expression. Induction of the complete neurogenic program by Ebf1 is largely dependent on bHLH proteins, presumably including proneural gene products, as shown by Ebf1 inhibition by the bHLH antagonist Id2. At this stage, the possibility that forced high level expression of Ebf1 in neuroepithelial progenitors may lead to non-physiological proneural gene activation, subsequently resulting in the activation of the complete program cannot be excluded. An alternative explanation, involving a second function of Ebf genes, can nevertheless be envisaged. Ebf1 misexpression also leads to changes in the balance of neuronal subtypes. This suggests the existence of a third level of intervention of Ebf genes in the neurogenic cascade. Ebf genes are considered to be downstream from proneural genes and cell cycle exit, but are absolutely required for neuronal differentiation and migration towards the mantle layer (Garcia-Dominguez, 2003).

Collier homologs and axon guidance

Axon pathfinding relies on the ability of the growth cone to detect and interpret guidance cues and to modulate cytoskeletal changes in response to these signals. The murine POU domain transcription factor Brn-3.2 regulates pathfinding in retinal ganglion cell (RGC) axons at multiple points along their pathways and the establishment of topographic order in the superior colliculus. Using representational difference analysis, Brn-3.2 gene targets likely to act on axon guidance have been identified at the levels of transcription, cell-cell interaction, and signal transduction, including the actin-binding LIM domain protein abLIM. Evidence is presented that abLIM plays a crucial role in RGC axon pathfinding, sharing functional similarity with its C. elegans homolog, UNC-115. These findings provide insights into a Brn-3.2-directed hierarchical program linking signaling events to cytoskeletal changes required for axon pathfinding (Erkman, 2000).

To understand the molecular mechanisms by which Brn-3.2 exerts its effects on RGC axon guidance, candidate target genes have been identified using a modification of representational difference analysis. Thus far, screening has yielded three potentially novel genes, and seven with matching sequences in mouse EST and human genomic and cDNA databases, the structure and function of which have not been reported. A number of genes were obtained that share a very low degree of homology with known genes and cannot be classified at this time. In addition, five candidate target genes have been identified that represent previously characterized molecules, including the transcription factors Irx6, EBF/Olf-1, EBF/Olf-2, and a mouse homolog of rat Neuritin. Irx6 is a homeodomain transcription factor with homology to the Drosophila genes of the iroquois complex, Olf-1 and Olf-2, belonging to the early B cell factor (EBF) family of HLH transcription factors, and Neuritin, a GPI-anchored neuronal cell surface protein, are all well characterized. One RDA product that did not present homology to published sequences was ultimately identified by analysis of cDNA clones as part of the 3'UTR of the mouse homolog of the human actin binding zinc finger protein abLIM. The amino acid sequence of the LIM domain containing all four LIM motifs specific for the retinal isoform is highly conserved and shows 97% identity to the human sequence. In situ hybridization analysis of m-abLIM in E15.5 mice shows, in addition to its expression in the inner layer of the retina, expression in other neuronal structures including peripheral sensory ganglia, spinal cord, SC, and nonneural tissues such as the thymus (Erkman, 2000).

If these genes are regulated by Brn-3.2, their mRNA levels should decrease in Brn-3.2-/- retina, and their spatial and temporal patterns of expression should support such an assumption. Indeed, comparison of mRNA levels in E15.5 wild-type and Brn-3.2-/- retinas reveals a dramatic decrease in the levels of Irx6, Olf-1, m-abLIM, and Neuritin, and a modest effect on Olf-2 mRNA levels, indicating that even relatively small differences can be detected by the modified RDA protocol. Temporal expression patterns of Brn-3.2, Irx6, Olf-2, and m-abLIM were determined using adjacent sections of retina at different developmental stages. Expression of Brn-3.2 mRNA, first detectable in the retina at E11.5, precedes initial detectable expression of Irx6 and Olf-2 around E12.5, and m-abLIM around E13.5. Thus, these genes may represent components of a molecular cascade regulated by Brn-3.2 (Erkman, 2000).

The prevailing model to explain the formation of topographic projections in the nervous system stipulates that this process is governed by information located within the projecting and targeted structures. In mammals, different thalamic nuclei establish highly ordered projections with specific neocortical domains and the mechanisms controlling the initial topography of these projections remain to be characterized. To address this issue, Ebf1-/- embryos were examined in which a subset of thalamic axons does not reach the neocortex. Ebf1 (also known as Olf-1, O/E-1, COE1) encodes a HLH transcription factor. The projections that do form between thalamic nuclei and neocortical domains have a shifted topography, in the absence of regionalization defects in the thalamus or neocortex. This shift is first detected inside the basal ganglia, a structure on the path of thalamic axons, and one that develops abnormally in Ebf1-/- embryos. A similar shift in the topography of thalamocortical axons inside the basal ganglia and neocortex was observed in Dlx1/2-/- embryos, which also have an abnormal basal ganglia development. Furthermore, Dlx1 and Dlx2 are not expressed in the dorsal thalamus or in cortical projection neurons. Thus, this study shows that: (1) different thalamic nuclei do not establish projections independently of each other; (2) a shift in thalamocortical topography can occur in the absence of major regionalization defects in the dorsal thalamus and neocortex, and (3) the basal ganglia may contain decision points for thalamic axons' pathfinding and topographic organization. These observations suggest that the topography of thalamocortical projections is not strictly determined by cues located within the neocortex and may be regulated by the relative positioning of thalamic axons inside the basal ganglia (Garel, 2002).

If defects in structures located on the path of thalamic axons can shift the topography of thalamocortical axons, a similar phenotype should be found in other mutant mice that have basal ganglia defects. Thus Dlx1/2 mutants, where differentiation of the basal ganglia and ventral thalamus is abnormal, were examined. Furthermore, Dlx1 and Dlx2 are not expressed in the dorsal thalamus or in cortical projection neurons. In Dlx1/2 mutants, the formation of the internal capsule is perturbed, probably because of a block in basal ganglia differentiation and, as in Ebf1 mutants, the topography of thalamocortical projections is shifted in the neocortex and internal capsule. Thus, the phenotype of Dlx1/2-/- mice supports the possibility that affecting structures on the path of thalamic axons can shift the topography of thalamocortical projections. It could be argued that the reduction of cortical interneurons in Dlx1/2 mutants might contribute to this phenotype. However, this is unlikely because Nkx2.1 mutants, which also have a major deficit in neocortical interneurons, have normal thalamocortical projections. Thus, the combined study of Ebf1 and Dlx1/2 mutants shows that the topography of thalamocortical axons can be systematically shifted in the absence of apparent abnormalities in the neocortex and dorsal thalamus, and suggests that this shift is due to defects in structures located on the path of the axons (Garel, 2002).

Collier homologs and the B-cell lineage

Early B-cell factor (EBF) is a tissue-specific and differentiation stage-specific DNA-binding protein that participates in the regulation of the pre-B and B lymphocyte-specific mb-1 gene (MB-1 is a non-immunoglobulin component of the B-cell antigen receptor complex that couples the B cell antigen receptor to multiple src family protein tyrosine kinases). Partial amino acid sequences obtained from purified EBF were used to isolate cDNA clones, which by multiple criteria encode EBF. The recombinant polypeptide forms sequence-specific complexes with the EBF-binding site in the mb-1 promoter. The cDNA hybridizes to multiple transcripts in pre-B and B-cell lines, but transcripts are not detected at significant levels in plasmacytoma, T-cell, and nonlymphoid cell lines. Expression of recombinant EBF in transfected nonlymphoid cells strongly activates transcription from reporter plasmids containing functional EBF-binding sites. Analysis of DNA binding by deletion mutants of EBF hs identified an amino-terminal cysteine-rich DNA-binding domain lacking obvious sequence similarity to known transcription factors. DNA-binding assays with cotranslated wild-type and truncated forms of EBF indicate that the protein interacts with its site as a homodimer. Deletions delineate a carboxy-terminal dimerization region containing two repeats of 15 amino acids that show similarity with the dimerization domains of basic-helix-loop-helix proteins. Together, these data suggest that EBF represents a novel regulator of B lymphocyte-specific gene expression (Hagman, 1993).

Murine B lymphocytes, adipocytes, and olfactory neurons all contain a DNA-binding protein that participates in the regulation of genes encoding tissue-specific components of signal transduction. Purification and cloning of this protein from murine B lymphocytes, termed early B-cell factor (EBF), and independent cloning of a protein from olfactory neuronal cells, termed Olf-1, reveals a virtually complete amino acid sequence identity between these proteins. As a first step towards identifying a human genetic disorder or mouse mutation for which EBF could be a candidate gene, the corresponding locus has been chromosomally mapped in both species. EBF homologous sequences have been found on human Chromosome (Chr) 5, band q34, and on proximal mouse Chr 11, in an evolutionarily conserved region (Milatovich 1994).

Early B-cell factor (EBF) is a cell type-specific transcription factor that is expressed at all antigen-independent stages of B-lymphocyte differentiation and participates in the regulation of the mb-1 gene. EBF is necessary for the generation of immunoglobulin-expressing B cells. EBF-deficient mice lack B cells and evince rearranged immunoglobulin D and JH gene segments; however, these EBF null mutants contain B220+CD43+ progenitor cells that express germline mu and IL-7 receptor transcripts. Various non-lymphoid tissues that express EBF are apparently normal in homozygous mutant mice, including olfactory neurons in which EBF has been identified as Olf-1. Together, these data suggest that EBF plays a specific and important role in the transcriptional control of B-cell differentiation at a stage before Ig (immunoglobulin) gene rearrangement but after commitment of cells to the B-lymphoid lineage (Lin, 1995).

Early B cell factor (EBF) and E47 (Drosophila homolog: Daughterless) participate in the transcriptional control of early B lymphocyte differentiation. To identify genetic targets for these transcription factors, cDNAs encoding EBF or a covalent homodimer of E47 were stabily transfected, either individually or together, into immature hematopoietic Ba/F3 cells, lacking both factors. In combination, EBF and E47 induce efficient expression of the endogenous immunoglobulin surrogate light chain genes, lambda5 and VpreB, whereas other pre-B cell-specific genes remain silent. Multiple functionally important EBF and E47 binding sites were identified in the lambda5 promoter/enhancer region, indicating that lambda5 is a direct genetic target for these transcription factors. Taken together, these data suggest that EBF and E47 synergize to activate expression of a subset of genes that define an early stage of the B cell lineage (Sigvardsson, 1997).

The pre-B cell-specific expression of the lambda5 gene is regulated at the level of transcription. The 5' region of the lambda5 gene has been shown to contain an enhancer that activates heterologous promoters. This enhancer, B(lambda5), also acts as a lineage- and tissue-restricted enhancer on its own promoter. The enhancer core, b(lambda5), carries approximately 50% of the total enhancer activity. The transcription factor early B cell factor (EBF) binds to a DNA motif in the lambda5 core enhancer that is crucial for enhancer activity, suggesting that lambda5 is a second target gene of EBF (Martensson, 1997).

A conserved sequence element situated between the decamer and TATA box in V kappa II and V kappa V promoters has a high homology to the binding site for early B cell factor (EBF). The kappa promoter element binds EBF specifically. Concomitant binding of EBF and Oct proteins to a wild-type kappa promoter template is observed at low efficiency; such dual occupancy is dependent on an intact amino terminus of the Oct protein. When the two binding sites are separated by a 10-bp spacer, this dependency disappears. A single kappa promoter EBF site, together with a TATA box and an Ig heavy chain enhancer, show marginal transcriptional stimulatory activity. In contrast, the EBF site acts synergistically with a decamer element in EBF-negative plasmacytoma cells, but not in B cells of an earlier differentiation stage. In these cells, a distinct protein is observed that interacts with the EBF binding motif, while overexpression of EBF down-regulates the expression of a reporter construct containing Ig control elements (Sigvardsson, 1996).

The immunoglobulin heavy chain intron enhancer contains two potential binding sites for early B-cell factor (EBF). To investigate the functional properties of these, EBF was expressed in the EBF non-expressing S194 plasmacytoma cell line and found to down-regulate the activity of a co-transfected immunoglobulin heavy chain intron enhancer reporter construct. The expression of an unrelated reporter construct remains unaltered. Dividing the immunoglobulin heavy chain intron enhancer into two subregions shows that the EBF mediated down-regulation of expression is mediated by at least two independent sites. These data indicate a role for EBF in the regulation of immunoglobulin gene expression (Akerblad, 1997).

Early differentiation of B lymphocytes requires the function of multiple transcription factors that regulate the specification and commitment of the lineage. Loss- and gain-of-function experiments have provided important insight into the transcriptional control of B lymphopoiesis, whereby E2A was suggested to act upstream of EBF1 and Pax5 downstream of EBF1. However, this simple hierarchy cannot account for all observations, and understanding of a presumed regulatory network, in which transcription factors and signaling pathways operate, is limited. This study shows that the expression of the Ebf1 gene involves two promoters that are differentially regulated and generate distinct protein isoforms. Interleukin-7 signaling, E2A, and EBF1 activate the distal Ebf1 promoter, whereas Pax5, together with Ets1 and Pu.1, regulates the stronger proximal promoter. In the absence of Pax5, the function of the proximal Ebf1 promoter and accumulation of EBF1 protein are impaired and the replication timing and subcellular localization of the Ebf1 locus are altered. Taken together, these data suggest that the regulation of Ebf1 via distinct promoters allows for the generation of several feedback loops and the coordination of multiple determinants of B lymphopoiesis in a regulatory network (Roessler, 2006).

Alternative lineage restriction and B cell fate commitment require the transcription factor Pax5, but the function of early B cell factor (EBF) in these processes remains mostly unexplored. This study shows that in the absence of EBF, 'expandable' and clonal lymphoid progenitor cells retain considerable myeloid potential. Conversely, ectopic expression of EBF in multipotential progenitor cells directs B cell generation at the expense of myeloid cell fates. EBF induces Pax5 and antagonizes expression of genes encoding the transcription factors C/EBPalpha, PU.1 and Id2. Notably, sustained expression of EBF in Pax5-/- hematopoietic progenitor cells is sufficient to block their myeloid and T lineage potential in vivo. Furthermore, in Pax5-/- pro-B cells, higher EBF expression represses alternative lineage genes. Thus, EBF can restrict alternative lineage 'choice' and promote commitment to the B cell fate independently of Pax5 (Pongubala, 2008).

Somatic hypermutation introduces point mutations into immunoglobulin genes in germinal centre B cells during an immune response. The reaction is initiated by cytosine deamination by the activation-induced deaminase (AID) and completed by error-prone processing of the resulting uracils by mismatch and base excision repair factors. Somatic hypermutation represents a threat to genome integrity and it is not known how the B cell genome is protected from the mutagenic effects of somatic hypermutation nor how often these protective mechanisms fail. This study shows, by extensive sequencing of murine B cell genes, that the genome is protected by two distinct mechanisms: selective targeting of AID and gene-specific, high-fidelity repair of AID-generated uracils. Numerous genes linked to B cell tumorigenesis, including Myc, Pim1, Pax5, Ocab (also called Pou2af1), H2afx, Rhoh and Ebf1, are deaminated by AID but escape acquisition of most mutations through the combined action of mismatch and base excision repair. However, approximately 25% of expressed genes analysed were not fully protected by either mechanism and accumulated mutations in germinal centre B cells. These results demonstrate that AID acts broadly on the genome, with the ultimate distribution of mutations determined by a balance between high-fidelity and error-prone DNA repair (Liu, 2008).

Critical role for Ebf1 and Ebf2 in the adipogenic transcriptional cascade

The Ebf (O/E) family of helix-loop-helix transcription factors plays a significant role in B lymphocyte and neuronal development. The three primary members of this family, Ebf1, 2, and 3, are all expressed in adipocytes, and Ebf1 promotes adipogenesis when overexpressed in NIH 3T3 fibroblasts. These three proteins have adipogenic potential in multiple cellular models and peroxisome proliferator-activated receptor gamma (PPARgamma) is required for this effect, at least in part due to direct activation of the PPARgamma1 promoter by Ebf1. Ebf1 also directly binds to and activates the C/EBPalpha promoter, which exerts positive feedback on C/EBPdelta expression. Despite this, C/EBPalpha is dispensable for the adipogenic action of Ebf proteins. Ebf1 itself is induced by C/EBPbeta and delta, which bind and activate its promoter. Reduction of Ebf1 and Ebf2 proteins by specific short hairpin RNA blocks differentiation of 3T3-L1 cells, suggesting a critical role for these factors and the absence of functional redundancy between members of this family. Altogether, these data place Ebf1 within the known transcriptional cascade of adipogenesis and suggest critical roles for Ebf1 and Ebf2 (Jimenez, 2006).

Interaction of CCR4-NOT with EBF1 regulates gene-specific transcription and mRNA stability in B lymphopoiesis

Transcription factor EBF1 (early B-cell factor 1; see Drosophila Collier/Knot) regulates early B-cell differentiation by poising or activating lineage-specific genes and repressing genes associated with alternative cell fates. SILAC (stable isotope labeling by amino acids in cell culture)-based mass spectrometry of was used to identify proteins associated with endogenous EBF1 in pro-B cells. This analysis identified most components of the multifunctional CCR4-NOT complex, which regulates transcription and mRNA degradation. CNOT3 interacts with EBF1, and histidine 240 in EBF1 was identified as a critical residue for this interaction. Complementation of Ebf1-/- progenitors with EBF1H240A revealed a partial block of pro-B-cell differentiation and altered expression of specific EBF1 target genes that show either reduced transcription or increased mRNA stability. Most deregulated EBF1 target genes show normal occupancy by EBF1H240A, but genes were also detected with altered occupancy, suggesting that the CCR4-NOT complex affects multiple activities of EBF1. Mice with conditional Cnot3 inactivation recapitulate the block of early B-cell differentiation, which was found to be associated with an impaired autoregulation of Ebf1 and reduced expression of pre-B-cell receptor components. Thus, the interaction of the CCR4-NOT complex with EBF1 diversifies the function of EBF1 in a context-dependent manner and may coordinate transcriptional and post-transcriptional gene regulation (Yang, 2016).

knot/collier: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.