In C. elegans, VA and VB motor neurons arise as lineal sisters but synapse with different interneurons to regulate locomotion. VA-specific inputs are defined by the UNC-4 homeoprotein and its transcriptional corepressor, UNC-37/Groucho, which function in the VAs to block the creation of chemical synapses and gap junctions with interneurons normally reserved for VBs. To reveal downstream genes that control this choice, a cell-specific microarray strategy was used that has identified unc-4-regulated transcripts. One of these genes, ceh-12, a member of the HB9 family of homeoproteins, is normally restricted to VBs. Expression of CEH-12/HB9 in VA motor neurons in unc-4 mutants imposes VB-type inputs. Thus, this work reveals a developmental switch in which motor neuron input is defined by differential expression of transcription factors that select alternative presynaptic partners. The conservation of UNC-4, HB9, and Groucho expression in the vertebrate motor circuit argues that similar mechanisms may regulate synaptic specificity in the spinal cord (Von Stetina, 2007).
Transcription factor cascades define the structure of the vertebrate motor circuit by regulating the differentiation of specific neurons that contribute to this network. A striking feature of these pathways is the frequent use of negative gene regulation to produce distinct fates between neurons generated from adjacent progenitor domains. This study shows that a similar mechanism of repression involving conserved transcriptional components distinguishes the fates of C. elegans motor neurons born as sisters from a common mother cell. These results also offer a strikingly new finding, an explicit link between this biological strategy and the choice of presynaptic partners, a developmental decision of critical importance to motor neuron function. A model is presented of transcriptional regulation of synaptic specificity in C. elegans and the possibility that related schemes may also define wiring in the vertebrate spinal cord is discussed (Von Stetina, 2007).
C. elegans mutants in the unc-4 homeodomain gene display a strong backward movement defect that results from the miswiring of VA class motor neurons with inputs normally reserved for VB motor neurons. Intriguingly, other aspects of VA cell fate (i.e., axon trajectory and process placement) are unchanged, suggesting that UNC-4 functions to control only the synaptic fate of this cell type. This study shows that this change in synaptic specificity depends in part on misexpression of the VB-specific transcription factor, CEH-12/HB9, in VA motor neurons. Normally, UNC-4 functions with UNC-37/Groucho to block ceh-12/HB9 expression in the VAs. Because HB9 is also believed to function as a transcriptional repressor in other organisms, it is proposed that ectopic CEH-12/HB9 in unc-4 and unc-37 mutants triggers miswiring by turning off genes that specify VA inputs. It is possible that ectopic CEH-12/HB9 also activates VB genes that drive the creation of VB-type inputs. These results provide strong genetic evidence for at least one additional pathway downstream from UNC-4 that functions in parallel to CEH-12/HB9. The relative contributions of these pathways to VA input specificity are biased along the anterior-posterior (A/P) axis with ectopic CEH-12 selectively driving the creation of VB inputs to posterior VA motor neurons in unc-4 mutants and the presumptive parallel pathway imposing VB inputs to anterior VAs. Finally, a third set of VB genes, glr-4, del-1, and acr-5 are negatively regulated by unc-4 but have no detectable role in the VA miswiring defect. These cell surface proteins and ion channel components could be indicative of physiologically important differences in the excitability or signaling capacity of VA versus VB motor neurons. In the future, it will be interesting to determine if ectopic ceh-12 expression contributes to the observed depletion of synaptic vesicles in unc-4 mutant neurons (Von Stetina, 2007).
Although ceh-12 is required for the imposition of VB-type inputs to posterior VA motor neurons in unc-4 mutants, inputs to most VB motor neurons apparently do not depend on ceh-12 activity. Two lines of evidence support this conclusion: (1) ceh-12 knock-out mutants do not show an obvious forward movement defect as would be expected if VB motor neurons were miswired; (2) the elimination of ceh-12 activity in these mutants does not perturb the creation of gap junctions between most VBs and AVB command interneurons. These data are consistent with the proposal that ceh-12 functions in parallel to a redundant pathway in VB motor neurons that is sufficient to retain VB-type inputs (Von Stetina, 2007).
This work describes the use of a GFP-tagged UNC-7S marker protein for visualizing gap junctions between specific neuron pairs in the C. elegans motor circuit. This assay has provided an unprecedented opportunity to score gap junction specificity in the light microscope in multiple animals and in a variety of different mutant backgrounds. These experiments indicate that the innexin, UNC-7S, is expressed in AVB command interneurons for assembly into gap junctions with B-class motor neurons. Genetic and physiological data suggest that these gap junctions are likely to be heterotypic, and also include the innexin UNC-9. The ectopic gap junctions between AVB and A-class motor neurons that appear in unc-4 mutants may have a similar subunit composition, since unc-9 is the most abundant innexin transcript expressed in A-class motor neurons. It follows that UNC-9 is also a likely candidate for assembly into gap junctions between VA and AVA command interneurons in wild-type animals. Gap junctions with AVB tend to be located on the motor neuron soma, whereas gap junctions with AVA are more often distributed along the length of the motor neuron partner. Thus, unc-4 may orchestrate the assembly of UNC-9 into gap junctions at particular locations within A-class motor neurons and with selected presynaptic partners. Although gap junctions have been previously thought to provide a largely developmental role in the generation of neural networks in higher vertebrates, recent evidence suggests that these 'electrical' synapses are also important for neural function in adult nervous systems. This view is consistent with ultrastructural and immunochemical data showing that gap junctions are widely distributed in the mature mammalian brain and spinal cord. Since the mechanisms that control the specificity of gap junction assembly in the vertebrate CNS are unknown, the discovery of downstream genes that regulate gap junction placement in C. elegans could provide targets for molecular studies in more complex nervous systems. Moreover, the joint regulation by unc-4 (or ceh-12) of the specificity of chemical and electrical synapse formation is indicative of a common nexus for pathways controlling the assembly of both types of synapses (Von Stetina, 2007).
These findings indicate that ceh-12 conspires with at least one additional pathway in VA motor neurons to control input specificity. unc-4 regulation of ceh-12 is restricted to VA motor neurons in the posterior region of the ventral nerve cord. Because anterior VA motor neurons are also miswired in unc-4 mutants, it is proposed that the presumptive downstream pathway functioning in parallel to ceh-12 may be selectively derepressed in anterior VAs. Other unc-4-regulated genes should be represented in the microarray profile of unc-37 mutant VA motor neurons. One plausible candidate in this data set that could function in parallel to ceh-12 is cog-1, the C. elegans homolog of the homeodomain transcription factor, Nkx6. In Drosophila, dHB9 and Nkx6 act together in ventrally projecting motor neurons to repress dorsal motor neuron traits. COG-1 regulates a similar decision in the C. elegans nervous system by preventing ASER sensory neurons from adopting characteristics normally reserved for ASEL. Potential COG-1 interactions with CEH-12 are suggested by the observation that cog-1::GFP is also expressed in VA and VB motor neurons. cog-1 and other candidate unc-4 target genes in the microarray data set that function in parallel to ceh-12 may be revealed by RNA interference (RNAi) tests currently underway to detect genes that enhance ceh-12-dependent suppression of the Unc-4 phenotype (i.e., improved backward locomotion). Conversely, RNAi of transcripts that are depleted in the unc-37 microarray data set and therefore potentially repressed by ectopic ceh-12 should result in an Unc-4 like movement defect if these genes are required for specifying VA-type inputs (Von Stetina, 2007).
The results showing that ceh-12 preserves VB motor neuron fate by repressing VAB-7/Eve, parallels earlier observations that HB9 regulates motor neuron differentiation in flies, birds, and mammals. In Drosophila, dHB9 is expressed in a subset of ventrally projecting motor neurons where it represses the dorsal motor neuron determinant, Eve, and blocks the adoption of a dorsal axon trajectory. Eve, in turn, opposes ventral fates in dorsal motor neurons by reciprocally repressing dHB9 in a Groucho-dependent mechanism. Interestingly, HB9 is also restricted to ventrally projecting motor neurons in the vertebrate spinal cord where it acts to prevent expression of markers for interneurons arising from the adjacent V2 progenitor domain. In this case, ectopic expression of HB9 in V2 neuroblasts is sufficient to drive expression of motor neuron markers as well as impose motor neuron-like morphological characteristics (i.e., ventral axonal projections). This dual function of HB9 to block as well as activate expression of motor neuron-specific traits is similar to the finding that CEH-12 inhibits VA motor neuron differentiation while simultaneously promoting a specific VB trait. Together, these observations suggest that the key role of HB9 function in motor neuron differentiation is evolutionarily ancient. In this regard, it is noted that the UNC-4 homolog, UNCX4.1, is strongly expressed in the V3 neural progenitor domain immediately adjacent to the MN region in which HB9 resides. It will be interesting to determine if UNCX4.1 functions in the V3 domain to block HB9 expression (Von Stetina, 2007).
Characterization of a sea urchin (P. lividus) homeobox gene PIHbox 9 has shown that the homeodomain of PIHbox9 is 95% identical to the homeodomain of the human HB9 gene, indicating that the two genes are highly related. Temporal expression analysis during sea urchin embryogenesis showed an absence of transcripts at early cleavage stages. At late gastrula stage, transcripts were barely detectable and reached the highest abundance at prism/early pluteus stages. By whole mount in situ hybridization, a highly restricted expression was observed in a few cells of the ectoderm-endoderm boundary of embryos at the prism stage. At pluteus stages, expression of PIHbox 9 was confined around the anus (Bellomonte, 1998).
The HB9 homeobox gene has been cloned from several vertebrates and is implicated in motor neuron differentiation. In the chick, a related gene, MNR2, acts upstream of HB9 in this process. An amphioxus homolog of these genes is described; it diverged before the gene duplication yielding HB9 and MNR2. AmphiMnx RNA is detected in two irregular punctate stripes along the developing neural tube, comparable to the distribution of 'dorsal compartment' motor neurons, and also in dorsal endoderm and posterior mesoderm. A new homeobox class, Mnx, is proposed to include AmphiMnx, HB9, MNR2 and their Drosophila and echinoderm orthologs; it is suggested that vertebrate HB9 is renamed Mnx1 and MNR2 be renamed Mnx2 (Ferrier, 2001).
While the role of the notochord and floor plate in patterning the dorsal-ventral (D/V) axis of the neural tube is clearly established, relatively little is known about the earliest stages of D/V regionalization. In an effort to examine more closely the initial, preneural plate stages of regionalization along the prospective D/V neural axis, a series of explant experiments were performed employing xHB9, a novel marker of the motor neuron region in Xenopus. Using tissue recombinants and Keller explants it has been shown that direct mesodermal contact is both necessary and sufficient for the initial induction of xHB9 in the motor neuron region. Presumptive neural plate explants removed as early as midgastrulation and cultured in isolation are already specified to express xHB9 but do so in an inappropriate spatial pattern, while identical explants are specified to express the floor plate marker vhh-1 with correct spatial patterning. These data suggest that, in addition to floor plate signaling, continued interactions with the underlying mesoderm through neural tube stages are essential for proper spatial patterning of the motor neuron region (Saha, 1997).
The homeobox gene Hb9, like its close relative MNR2, is expressed selectively by motor neurons (MNs) in the developing vertebrate CNS. In embryonic chick spinal cord, the ectopic expression of MNR2 or Hb9 is sufficient to trigger MN differentiation and to repress the differentiation of an adjacent population of V2 interneurons. Genetic evidence is provided that Hb9 has an essential role in MN differentiation. In mice lacking Hb9 function, MNs are generated on schedule and in normal numbers but transiently acquire molecular features of V2 interneurons. The aberrant specification of MN identity is associated with defects in the migration of MNs, the emergence of the subtype identities of MNs, and the projection of motor axons. These findings show that HB9 has an essential function in consolidating the identity of postmitotic MNs (Arber, 1999).
A homeobox gene, HB9, has been isolated from the tarsometatarsal skin of 13-day-old chick embryos using a degenerate RT-PCR-based screening method. In situ hybridization analysis has revealed that, during development of chick embryonic skin, the HB9 gene is expressed in epidermal basal cells of the placodes, but not in those of interplacodes, and in the dermal cells under the placodes at 9 days before addition of an intermediate layer by proliferation of the basal cells in the placodes. With the onset of epidermal stratification, the direction of the basal cell mitosis changes, with the axis becoming vertical to the epidermal surface. Placodes and interplacodes form outer and inner scales, respectively, after they have elongated distally. During scale ridge elongation at 12-15 days, HB9 is strongly expressed in the epidermis of the outer scale face, where the cell proliferation is more active than in the epidermis of the inner scale face; hence, stratification of the outer scale face is more prominent than that of the inner scale face. After 16 days, when mitotic activity in the epidermal basal cells decreases and the thickness of the epidermis is maintained at a constant level, the HB9 expression decreases with the onset of epidermal keratinization. These results suggest that HB9 may be involved in the proliferation of the epidermal basal cells that accompany epidermal stratification (Kosaka, 2000a).
In situ hybridization and immunohistochemical analysis of HB9 homeobox gene mRNA and protein, respectively, were performed during chick feather development. HB9 mRNA is highly expressed in epidermal basal cells and dermal cells of the placodes and feather buds, but not in those of the interplacodes and interbud regions. HB9 protein is predominantly expressed in dermal cells of the symmetric short buds and decreases after the asymmetric bud stage when the feather bud had becomes elongated along the anterior-posterior (A-P) and proximal-distal (P-D) axis. These results suggest that HB9 gene is regulated in a spatiotemporal manner during feather development, and may be involved in early feather bud morphogenesis (Kosaka, 2000b).
Sonic hedgehog signaling controls the differentiation of motor neurons in the ventral neural tube, but the intervening steps are poorly understood. A differential screen of a cDNA library derived from a single Shh-induced motor neuron has identified a novel homeobox gene, MNR2, expressed by motor neuron progenitors and transiently by postmitotic motor neurons. The ectopic expression of MNR2 in neural cells initiates a program of somatic motor neuron differentiation characterized by the expression of homeodomain proteins, by neurotransmitter phenotype, and by axonal trajectory. These results suggest that the Shh-mediated induction of a single transcription factor, MNR2, is sufficient to direct somatic motor neuron differentiation (Tanabe, 1998).
Sonic hedgehog (Shh) specifies the identity of both motor neurons (MNs) and interneurons with morphogen-like activity. Evidence is presented that the homeodomain factor HB9 is critical for distinguishing MN and interneuron identity in the mouse. Presumptive MN progenitors and postmitotic MNs express HB9, whereas interneurons never express this factor. This pattern resembles a composite of the avian homologs MNR2 and HB9. In mice lacking Hb9, the genetic profile of MNs is significantly altered, particularly by upregulation of Chx10, a gene normally restricted to a class of ventral interneurons. This aberrant gene expression is accompanied by topological disorganization of motor columns, loss of the phrenic and abducens nerves, and intercostal nerve pathfinding defects. Thus, MNs actively suppress interneuron genetic programs to establish their identity (Thaler, 1999).
In the developing spinal cord, motor neurons acquire columnar subtype identities that can be recognized by distinct profiles of homeodomain transcription factor expression. The mechanisms that direct the differentiation of motor neuron columnar subtype from an apparently uniform group of motor neuron progenitors remain poorly defined. In the chick embryo, the Mnx class homeodomain protein MNR2 is expressed selectively by motor neuron progenitors, and has been implicated in the specification of motor neuron fate. MNR2 expression persists in postmitotic motor neurons that populate the median motor column (MMC), whereas its expression is rapidly extinguished from lateral motor column (LMC) neurons and from preganglionic autonomic neurons of the Column of Terni (CT). The extinction of expression of MNR2, and the related Mnx protein HB9, from postmitotic motor neurons appears to be required for the generation of CT neurons but not for LMC generation. In addition, MNR2 and HB9 are likely to mediate the suppression of CT neuron generation that is induced by the LIM HD protein Lim3. Finally, MNR2 appears to regulate motor neuron identity by acting as a transcriptional repressor, providing further evidence for the key role of transcriptional repression in motor neuron specification (William, 2003).
Several lines of evidence suggest that MNR2, and its relative HB9, function as transcriptional repressors during the process of motor neuron specification: (1) the N-terminal domain of MNR2 essential for its activity in motor neuron specification can function as a potent transcriptional repressor in cell-based reporter assays; (2) the HD of MNR2, when fused to a known co-repressor recruitment domain, the E1a C-terminal domain, can mimic the activity of the wild-type MNR2 protein, both in motor neuron specification and in repression of CT subtype identity. These findings are complemented by genetic studies of HB9 function in mouse, in which HB9 has been shown to repress its own expression and to repress expression of V2 interneuron determinants in motor neurons (William, 2003).
The precise mechanism of MNR2- and HB9-mediated transcriptional repression remains unclear. MNR2, like many other HD proteins, possesses a well conserved eh1 motif that, in other contexts, can recruit Groucho class co-repressors. However, elimination of the eh1 motif in MNR2 does not abolish its ability to induce motor neuron generation. Moreover, fusion of the HD of MNR2 to a potent Groucho recruitment domain results in poor motor neuron-inducing activity in vivo. Thus, the repressor functions of MNR2, and by inference of HB9, may not simply reflect the recruitment of Groucho class co-repressors. The data show that the MNR2 HD-E1a C-terminal repressor domain fusion protein mimics the activity of the wild-type MNR2 protein, raising the possibility that MNR2 repressor activity involves the recruitment of Ctbp class co-repressors. However, additional experiments are necessary to resolve whether the repressor functions of MNR2 normally involve the recruitment of Ctbp class co-repressors. In addition, studies on co-repressor function in Drosophila raise the possibility of cooperative interactions between eh1 Groucho recruitment and Ctbp recruitment domains present within the same transcription factor (William, 2003).
Regardless of the precise co-repressors recruited by MNR2, the evidence supports the view that MNR2 function in vivo is likely to reflect its role as a transcriptional repressor. These findings therefore add to the emerging view that the logic of motor neuron fate specification is grounded in transcriptional repression. Many of the progenitor transcription factors involved in motor neuron specification at steps upstream of MNR2, e.g. Nkx6.1, Nkx6.2 and Olig2, also function as transcriptional repressors. Unlike the Nkx6 and Mnx proteins, Olig2 does not possess a clear eh1 motif, further supporting the idea that the transcriptional repressors that function in motor neuron specification recruit distinct classes of co-repressor protein. Finally, the similarities in sequence and activities of Mnx class HD proteins, and genetic studies of HB9 in mouse and Drosophila indicate that all Mnx class proteins may function as transcriptional repressors. Since HB9 expression in spinal cord is restricted largely to postmitotic motor neurons, these observations imply that the key role of transcriptional repression in motor neuron fate specification extends from progenitor cells into postmitotic neurons (William, 2003).
Spinal motor neurons (MNs) and V2 interneurons (V2-INs) are specified by two related LIM-complexes, MN-hexamer and V2-tetramer, respectively. This study shows how multiple parallel and complementary feedback loops are integrated to assign these two cell fates accurately. While MN-hexamer response elements (REs) are specific to MN-hexamer, V2-tetramer-REs can bind both LIM-complexes. In embryonic MNs, however, two factors cooperatively suppress the aberrant activation of V2-tetramer-REs. First, LMO4 blocks V2-tetramer assembly. Second, MN-hexamer induces a repressor, Hb9, which binds V2-tetramer-REs and suppresses their activation. V2-INs use a similar approach; V2-tetramer induces a repressor, Chx10, which binds MN-hexamer-REs and blocks their activation. Thus, this study uncovers a regulatory network to segregate related cell fates, which involves reciprocal feedforward gene regulatory loops (Lee, 2008).
A SELEX study revealed that the Lhx3-binding sites deviate between HxRE and TeRE in sequence. As HxRE is recognized by MN-hexamer but not by V2-tetramer, the conformation of Lhx3 in MN-hexamer and V2-tetramer is likely different. This may involve an allosteric structural change in the DNA binding domain of Lhx3 in MN-hexamer, induced by the Isl1:Lhx3 interaction. As MN-hexamer is assembled only in MNs, HxRE-containing genes would be stimulated specifically in MNs but not in V2-INs. In contrast, TeRE is activated by both V2-tetramer and MN-hexamer in vitro, suggesting that TeRE-containing V2 genes could be inappropriately induced in MNs. However, TeRE is a V2-specific response element in the developing embryos due to a collaborative action of Hb9 and LMO4 to silence TeRE in MNs. Thus, these studies demonstrate that DNA-REs for specific transcription complexes are sufficient to confer gene expressions to proper cell types in developing embryos (Lee, 2008).
Although Lhx4 and their cofactor NLI (Ldb, CLIM, Chip) dimerization is dispensable for the DNA-binding activity of V2-tetramer and MN-hexamer, it is essential for their robust transactivation. Thus, reiterated TeRE1/2s and HxRE1/2s are necessary for functional TeREs and HxREs. Indeed, the MN-specific enhancer of Hb9 has two functional HxRE1/2s spaced ~150 nt apart. Similarly, three evolutionarily conserved TeRE1/2 sequences were found in the Chx10-TeRE region. Three possible advantages can be proposed for the NLI dimerization in V2-tetramer and MN-hexamer. First, the requirement for multiple repeats of TeRE1/2 and HxRE1/2 may impose higher stringency for functional target gene selection. Second, NLI dimerization bridges two NLI-interacting transcription factors bound to their DNA-binding sites separated by a relatively long spacer region in Drosophila (Heitzler, 2003; Morcillo, 1997). This raises the possibility that V2-tetramer and MN-hexamer may integrate the transcriptional activity of multiple TeRE1/2s or HxRE1/2s located within a single target gene or across multiple target genes. For instance, both Chx10 and Lin-52 have additional TeRE1/2s within their gene. Thus, it will be interesting to test whether the Chx10-TeRE region is required to regulate both Chx10 and Lin-52 by V2-tetramer and whether the multiple TeRE1/2s throughout these two genes enable V2-tetramer to temporally coordinate expression of these genes during development. Third, NLI dimerization may potentiate the transcriptional activity of LIM-complexes by stabilizing the LIM-complexes and/or facilitating recruitment of transcriptional coactivators and chromatin remodeling complexes. Indeed, single-stranded DNA-binding proteins have been found to interact with NLI and augment the transactivation of NLI-containing complexes (Lee, 2008).
DNA-REs affect the protein-protein interaction properties of their cognate transcription factors. Sox2 and Pou factor family members Oct1 or Oct4 dimerize onto different DNA-REs in distinct conformational arrangements, offering one molecular explanation for the wide spectrum of developmental functions for Sox/Pou factors. Thus, it will be interesting to interrogate the role of TeRE and HxRE on the spatial alignment of DNA-protein complex of Lhx3/TeRE1/2 and of Isl1:Lhx3/HxRE1/2 (Lee, 2008).
As p2 and pMN cells are exposed to relatively similar concentration of Shh, deregulation of the transcriptional events downstream of Shh often results in V2-MN fate conversion or hybrid phenotypes. The initial segregation of V2 and MN pathways appears to involve transcriptional crossrepression of progenitor factors Irx3 and Olig2 in neuroepithelial cells. However, additional mechanisms are likely to be needed, as both differentiating V2 and MN cells express Lhx3/4, which are necessary for their cell fates. The results reveal an efficient feedforward gene regulatory circuitry in which a cell-type specific LIM-complex triggers expression of a transcriptional repressor, which in turn binds and represses the DNA-RE of another related LIM-complex, thereby blocking the unwanted choice of alternative fates. This likely contributes to establishing a precise cell identity once a specific LIM-complex is assembled and activates the downstream target genes in neural precursors (Lee, 2008).
First, MN-hexamer upregulates Hb9, which in turn refines MN-gene expression by silencing V2-genes. Hb9 selectively binds TeREs, which prevents undesirable recruitment of MN-hexamer (and any V2-tetramer) to TeREs and represses their inappropriate activation in embryonic MNs. Indeed, V2 genes are upregulated in MNs lacking Hb9. Hb9, a transcriptional repressor, may suppress TeRE-containing V2 genes by recruiting corepressors to their TeREs. Mnr2, an Hb9 paralog, is also known to recruit Ctbp-like corepressor. Thus, TeRE-containing V2 genes are likely to be activated by V2-tetramer in V2-INs, while they are simultaneously silenced by Hb9 in MNs (Lee, 2008).
Second, V2-tetramer directly binds Chx10-TeRE and upregulates Chx10 in chick spinal cord, suggesting that Chx10 is a direct target gene of V2-tetramer. Although Chx10 regulates retinal development, neither its function in the developing spinal cord nor its in vivo target genes are known. Interestingly, Chx10 binds Hb9-HxRE through its homeodomain and represses both the basal and MN-hexamer-induced levels of transcriptional activity mediated by HxRE, consistent with a previous report that Chx10 primarily functions as a transcriptional repressor. In V2 cells, Chx10 could be necessary to completely shut off any leaky expression of HxRE-containing MN genes or to actively block erroneous activation of MN genes by other transcription factors shared between V2-INs and MNs via recruiting corepressors to HxREs. Overall, repression of HxRE by Chx10 is likely to contribute to further refining V2 identity by suppressing unwanted MN-gene expression in V2 cells. Analysis of V2 specification in Chx10 mutant embryos should help examine this possibility genetically (Lee, 2008).
Overall, these studies reveal a sequential regulatory cascade of gene expression that operates to ensure the high fidelity in gene regulation required to specify two closely related, but distinct neural subtypes during vertebrate CNS development. This cascade resembles feedforward loops described in other organisms such as E. Coli, yeast, C. elegans and Drosophila. In particular, this study highlights the key role for DNA-REs in feedfoward gene regulatory loop and combinatorial transcription code, which have been underappreciated previously. Both HxRE and TeRE function as binary switches in the developing spinal cord; i.e., TeRE is off-switch in MNs and on-switch in V2-INs, while HxRE is on-switch in MNs and off-switch in V2-INs. Importantly, these strategies should reinforce the distinct gene expression outcomes in MNs and V2-INs, as expression of HxRE- and TeRE-containing genes would be precisely coregulated to opposite directions depending on the cell context. Thus, cell-type-specific DNA-RE alone is capable of decoding all the cell-fate-specifying genetic programs installed in each cell type, sensing both transcriptional activation and repression machineries. This model also predicts that a set of genes with TeRE or HxRE would be synonymously regulated during cell fate specification. Thus, the defined consensus TeRE and HxRE sequences could be useful in bioinformatics approaches to find a group of genes, which are specifically expressed in V2-INs and MNs and direct V2 and MN differentiations and maturations. Together, these findings provide a prototypic gene regulatory network for cell-type specification in development, which involves feedforward gene regulatory loops (Lee, 2008).
Hb9-MNe of Hb9 gene consists of functional HxRE and E-box elements that recruit proneural basic helix-loop-helix (bHLH) factors. MN-hexamer transcriptionally synergizes with proneural bHLH factors Ngn2 and NeuroM to fully activate Hb9 gene and subsequently specify MNs in the developing spinal cord and P19 cells. This synergistic interaction of MN-hexamer and Ngn2/NeuroM requires DNA bindings of these transcription factors in proximity. Thus, full activation of Chx10-TeRE in the neural tube may also need other transcription factors bound elsewhere in Chx10. It will be interesting to test whether V2-tetramer indeed cooperates with other transcription factors involved in V2 specification, such as Mash1, GATA2, FoxN4, or SCL, to promote V2-IN fate (Lee, 2008).
LMO4 disrupts the assembly of V2-tetramer in newborn MNs by displacing Lhx3 from NLI and suppresses V2-IN development in chick embryos. Among LMOs, LMO4 is most highly expressed in differentiating MNs in chick and mouse embryos and it binds NLI with a 2-fold higher affinity than LMO2. Thus, LMO4 is a good candidate to regulate the formation of LIM-complexes in MNs. Under the condition of 1:1 interactions, the affinities of Lhx3 and Isl1 for NLI binding are comparable, suggesting that LMO4 inhibits similarly the formation of V2-tetramer and MN-hexamer through competition for NLI binding. However, the data indicate that LMO4 functions as a selective competitor to disrupt V2-tetramer over MN-hexamer assembly. Interestingly, the binding of NLI and Isl1 without Lhx3 is also sensitive to LMO4, suggesting that the resistance of NLI:Isl1-Lhx3 binding to LMO4 is not simply due to the differences in the binding affinities between NLI:Lhx3 interaction and NLI:Isl1 interaction. Rather, the differences in the complex architecture of MN-hexamer and V2-tetramer may contribute to the distinct sensitivity of the two complexes to LMO4. Relative to V2-tetramer, MN-hexamer is a higher-order multiprotein complex. Thus, it could be more stable through multiple protein-protein interactions and less sensitive to a competitor such as LMO4. In MNs, LMO4 should increase the population of MN-hexamer, as MN-hexamer is formed at the expense of V2-tetramer. Consistently, deletion of LMO4 results in progressive increase of V2-INs. Although V2-MN hybrid cells are consistently found in LMO4 mutants, the phenotype is relatively subtle in LMO4 single mutant and greatly enhanced in LMO4:Hb9 compound mutants. Thus, LMO4 may provide a fine-tuning mechanism to control the stoichiometry of LIM-complexes in the developing spinal cord by increasing MN-hexamer concentration in MNs (Lee, 2008).
The results demonstrate that Hb9 and LMO4 cooperate to silence V2 genes. Why is the cooperative action of Hb9 and LMO4 necessary to inhibit V2 genes in MNs? LMO4 seems to function as a modulator, rather than an active MN fate selector, to promote MN-hexamer formation over other possible LIM-complexes in MNs. Likewise, although Hb9 is dominant over MN-hexamer for TeRE binding and thus blocks the access of MN-hexamer to TeRE-containing V2 genes, Hb9 may have intrinsically modest affinity to TeRE (weaker than V2-tetramer). Thus, Hb9 alone could be inefficient in blocking binding of V2-tetramer to TeRE, and may not completely shut down V2-gene expression in MNs, unless LMO4 helps Hb9 to bind TeRE more readily by destabilizing V2-tetramer, Hb9's competitor to bind TeRE. The loss of LMO4 and Hb9 in LMO4:Hb9-DKO likely permits both V2-tetramer and MN-hexamer to bind TeREs and upregulate V2 genes. As a consequence, both TeRE-containing V2 genes and HxRE-containing MN genes are activated in MNs, thereby resulting in MN-V2 hybrid cells. Thus, the functional cooperation between Hb9 and LMO4 is expected to be a critical component in the overall strategy to suppress expression of V2 genes in MNs. Together, these findings underscore the importance of actively suppressing alternative fate choice to generate correct neuronal subtype (Lee, 2008).
The data demonstrate that transcriptionally active endogenous MN-hexamer is assembled in MMCm-MNs. Interestingly, LMO4 is maintained mainly in MMCm-MNs among motor columns. Thus, the HxRE may mediate not only MN specification but also postmitotic MN diversification to MMCm cells and LMO4 may also antagonize the unwanted formation of V2-tetramer in MMCm cells. V2-INs also undergo further diversification to excitatory Chx10+ V2a-INs and inhibitory GATA2/3+ V2b-INs. Analogous to MN development, Lhx3 is maintained in V2a-INs, but extinguished in V2b-INs. Thus, TeRE activity could be maintained only in V2a subtype in which V2-tetramer is assembled. In comparison, V2b-INs express GATA2/3, SCL, and LMO4, which could assemble a transactivating complex similar to a hematopoietic complex containing NLI, GATA1, SCL, and LMO2. This raises the possibility that LMO4 might control the V2 subtype segregation by acting as an activator in V2b and a repressor in V2a (Lee, 2008).
In summary, these studies have established that subtle differences in DNA-REs can direct segregation of lineage-specific transcription pathways in the developing nervous system by concertedly mobilizing the action of transcriptional activators and repressors. This regulatory network likely represents a prototypic genetic mechanism for segregating related but distinct cell fates during the nervous system development. Importantly, this knowledge should provide a rational strategy to direct stem/progenitor cells into MNs in vitro (Lee, 2008).
The underlying transcriptional mechanisms that establish the proper spatial and temporal pattern of gene expression required for specifying neuronal fate are poorly defined. This study characterizes how the Hb9 gene is expressed in developing motoneurons in order to understand how transcription is directed to specific cells within the developing CNS. Non-specific general-activator proteins such as E2F and Sp1 are capable of driving widespread low level transcription of Hb9 in many cell types throughout the neural tube; however, their activity is modulated by specific repressor and activator complexes. The general-activators of Hb9 are suppressed from triggering inappropriate transcription by repressor proteins Irx3 and Nkx2.2. High level motoneuron expression is achieved by assembling an enhancesome on a compact evolutionarily-conserved segment of Hb9 located from -7096 to -6896. The ensemble of LIM-HD and bHLH proteins that interact with this enhancer change as motoneuron development progresses, facilitating both the activation and maintenance of Hb9 expression in developing and mature motoneurons. These findings provide direct support for the derepression model of gene regulation and cell fate specification in the neural tube, as well as establishing a role for enhancers in targeting gene expression to a single neuronal subtype in the spinal cord (Lee, 2004).
Developing motoneurons sequentially express several bHLH proteins, including Ngn2 in the progenitor cells followed by NeuroM in the early postmitotic motoneurons and NeuroD in the more mature cells. Ngn2 and NeuroM have been shown to contribute to the activation of Hb9 during the initial stages of motoneuron development, but it remained unclear whether NeuroD in the mature cells could also stimulate Hb9 expression. To compare the activity of these transcription factors, P19 cells were transfected with expression constructs encoding bHLH proteins together with a luciferase reporter containing seven E box elements. Under these conditions Ngn2 activated the reporter much more than either NeuroM or NeuroD. Despite this inherent difference in transactivation, Ngn2, NeuroM, and NeuroD each synergized in a similar way with the LIM factors Isl1 and Lhx3 to trigger Hb9 expression. Likewise, each bHLH factor dimerizes with E47 and binds to the M50 and M100 E box elements in a sequence-specific manner, and exhibits a similar ability to promote motoneuron differentiation from transfected P19 embryonic carcinoma cells when expressed with Isl1 and Lhx3. Taken together, these findings suggest that the initial activation of Hb9 expression is dependent on Ngn2 and NeuroM as motoneurons become postmitotic, and that NeuroD contributes to the maintenance of Hb9 expression in mature motoneurons (Lee, 2004).
Nkx2.2, Nkx6.1, Pax6 and Irx3 control progenitor cell fate by repressing transcription. Since the deletion analysis of Hb9 indicated that repressor proteins might interact with the 2.5 kb distal segment from -8129 to -5575, tests were performed to see whether constructs with this DNA segment were repressed by Nkx2.2, Nkx6.1, Pax6 and/or Irx3 using 293 cell transfections. The Hb9 promoter was repressed ~50-500 fold by Nkx2.2 and Irx3, whereas Pax6 and Nkx6.1 were significantly less active. These findings suggest that progenitor cell factors such as Nkx2.2 and Irx3 expressed by non-motoneuron cells suppress the expression of Hb9 (Lee, 2004).
Genetic studies have shown that Hb9 feeds back negatively to modulate its own expression. Whether Hb9 could suppress the activity of its enhancer when LIM and bHLH factors synergize to activate transcription was tested. The native Hb9 protein and the EnR-Hb9 repressor (Hb9 homeodomain linked to eh1 engrailed repressor domain) both inhibited transcription under these conditions, whereas the Hb9-HD and a fusion of Hb9 to the VP16 activation domain (VP16-Hb9) lacked this activity. Thus, in developing motoneurons where Hb9 transcription is synergistically activated, co-repressors such as those recruited by the engrailed fusion (EnR) appear to be involved in negative feedback regulation. Consistent with these findings, Hb9 protein binds in a sequence-specific manner to the ATTA motifs in the enhancer (Lee, 2004).
A novel human homeobox gene, HB9, was isolated from a cDNA library prepared from in vitro stimulated human tonsil B lymphocytes and from a human genomic library. The HB9 gene is composed of 3 exons spread over 6 kilobases of DNA. An open reading frame of 1206 nucleotides is in frame with a diverged homeodomain. The predicted HB9 protein has a molecular mass of 41 kilodaltons and is enriched for alanine, glycine, and leucine. The HB9 homeodomain is most similar to that of the Drosophila melanogaster homeobox gene proboscipedia. Northern blot analysis of poly(A) RNA purified from the human B cell line RPMI 8226 and from activated T cells has revealed a major mRNA transcript of 2.2 kilobases. Similar analysis of poly(A) RNA from a variety of adult tissues has demonstrated HB9 transcripts in pancreas, small intestine, and colon. Reverse transcriptase-polymerase chain reaction was used to examine HB9 RNA transcripts in hematopoietic cell lines. HB9 RNA transcripts were most prevalent in several human B cell lines and K562 cells. In addition, transcripts were detected in RNA prepared from tonsil B cells and in situ hybridization studies have localized them in the germinal center region of adult tonsil. These findings suggest the involvement of HB9 in regulating gene transcription in lymphoid and pancreatic tissues (Harrison, 1994).
In most mammals the pancreas develops from the foregut endoderm as ventral and dorsal buds. These buds fuse and develop into a complex organ composed of endocrine, exocrine and ductal components. This developmental process depends upon an integrated network of transcription factors. Gene targeting experiments have revealed critical roles for Pdx1, Isl1, Pax4, Pax6 and Nkx2-2. The homeobox gene HLXB9 (encoding HB9) is prominently expressed in adult human pancreas, although its role in pancreas development and function is unknown. To facilitate its study, the mouse HLXB9 ortholog, Hlxb9, was isolated. During mouse development, the dorsal and ventral pancreatic buds and mature beta-cells in the islets of Langerhans express Hlxb9. In mice homologous for a null mutation of Hlxb9, the dorsal lobe of the pancreas fails to develop. The remnant Hlxb9-/- pancreas has small islets of Langerhans with reduced numbers of insulin-producing beta-cells. Hlxb9-/- beta-cells express low levels of the glucose transporter Glut2 and homeodomain factor Nkx 6-1. Thus, Hlxb9 is key to normal pancreas development and function (Harrison, 1999).
The initial stages of pancreatic development occur early during mammalian embryogenesis, but the genes governing this process remain largely unknown. The homeodomain protein Pdx1 is expressed in the developing pancreatic anlagen from the approximately 10-somite stage, and mutations in the gene Pdx1 prevent the development of the pancreas. The initial stages of pancreatic development, however, still occur in Pdx1-deficient mice. Hlxb9 is a homeobox gene that in humans has been linked to dominant inherited sacral agenesis and Hb9 is expressed at early stages of mouse pancreatic development and later in differentiated beta-cells. Hlxb9 has an essential function in the initial stages of pancreatic development. In the absence of Hlxb9 expression, the dorsal region of the gut epithelium fails to initiate a pancreatic differentiation program. In contrast, the ventral pancreatic endoderm develops but exhibits a later and more subtle perturbation in beta-cell differentiation and in islet cell organization. Thus, Hlxb9 is required dorsally for specifying the gut epithelium to a pancreatic fate and ventrally for ensuring proper endocrine cell differentiation (Li, 1999).
The homebox gene Hlxb9, encoding Hb9, exhibits a dual expression profile during pancreatic development. The early expression in the dorsal and ventral pancreatic epithelium is transient and spans from embryonic day 8 to 9 and 10, whereas the later expression is confined to differentiating beta-cells as they appear. Hlxb9 is critically required for the initiation of the dorsal, but not the ventral, pancreatic program. This study demonstrates the requirement for a stringent temporal regulation of Hlxb9 expression during early stages of pancreatic development. In transgenic mice, where Hlxb9 expression (under control of the Ipf1/Pdx1 promoter) is extended beyond e9-e10, the development of the pancreas is drastically perturbed. Morphological analyses show that the growth and morphogenesis of the pancreatic epithelium is impaired. Moreover, differentiation of pancreatic endocrine and exocrine cells is diminished; instead, the pancreatic epithelium with its adjacent mesenchyme adopts an intestinal-like differentiation program. Together, these data point to a need for a tight temporal regulation of Hlxb9 expression. Thus, a total loss of Hlxb9 expression results in a block of the initiation of the dorsal pancreatic program, while a temporally extended expression of Hlxb9 results in a complete impairment of pancreatic development (Li, 2001).
The vertebrate endocrine pancreas has the crucial function of maintaining blood sugar homeostasis. This role is dependent upon the development and maintenance of pancreatic islets comprising appropriate ratios of hormone-producing cells. In all vertebrate models studied, an initial precursor population of Pdx1-expressing endoderm cells gives rise to separate endocrine and exocrine cell lineages. Within the endocrine progenitor pool a variety of transcription factors influence cell fate decisions, such that hormone-producing differentiated cell types ultimately arise, including the insulin-producing beta cells and the antagonistically acting glucagon-producing alpha cells. In previous work, it was established that the development of all pancreatic lineages requires retinoic acid (RA) signaling. Zebrafish was used to uncover genes that function downstream of RA signaling, and this study identified mnx1 (hb9) as an RA-regulated endoderm transcription factor-encoding gene. By combining manipulation of gene function, cell transplantation approaches and transgenic reporter analysis it was established that Mnx1 functions downstream of RA within the endoderm to control cell fate decisions in the endocrine pancreas progenitor lineage. It was confirmed that Mnx1-deficient zebrafish lack beta cells, and, importantly, the novel observation was made that they concomitantly gain alpha cells. In Mnx1-deficient embryos, precursor cells that are normally destined to differentiate as beta cells instead take on an alpha cell fate. These findings suggest that Mnx1 functions to promote beta and suppress alpha cell fates (Dalgin, 2011).
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