InteractiveFly: GeneBrief

Olig family: Biological Overview | References


Gene name - Olig family

Synonyms -

Cytological map position - 36D1-36D1

Function - HLH transcription factor

Keywords - CNS, neuron identity, axon pathfinding, motor neurons, brain

Symbol - Oli

FlyBase ID: FBgn0032651

Genetic map position - chr2L:17589425-17591277

Classification - HLH: helix loop helix domain

Cellular location - nuclear



NCBI links: EntrezGene
BIOLOGICAL OVERVIEW

During the development of locomotion circuits it is essential that motoneurons with distinct subtype identities select the correct trajectories and target muscles. In vertebrates, the generation of motoneurons and myelinating glia depends on Olig2, one of the five Olig family bHLH transcription factors. This study investigated the so far unknown function of the single Drosophila homolog Oli. Combining behavioral and genetic approaches, this study demonstrates that oli is not required for gliogenesis, but plays pivotal roles in regulating larval and adult locomotion, and axon pathfinding and targeting of embryonic motoneurons. In the embryonic nervous system, Oli is primarily expressed in postmitotic progeny, and in particular, in distinct ventral motoneuron subtypes. oli mediates axonal trajectory selection of these motoneurons within the ventral nerve cord and targeting to specific muscles. Genetic interaction assays suggest that oli acts as part of a conserved transcription factor ensemble including Lim3, Islet and Hb9. Moreover, oli is expressed in postembryonic leg-innervating motoneuron lineages and required in glutamatergic neurons for walking. Finally, over-expression of vertebrate Olig2 partially rescues the walking defects of oli-deficient flies. Thus, these findings reveal a remarkably conserved role of Drosophila Oli and vertebrate family members in regulating motoneuron development, while the steps that require their function differ in detail (Oyallon, 2012).

The generation of coordinated muscle contractions, enabling animals to perform complex movements, depends on the assembly of functional neuronal motor circuits. Motoneurons lie at the heart of these circuits, receiving sensory input directly or indirectly via interneurons within the central nervous system (CNS) and relaying information to muscles in the periphery. During development neural precursors give rise to progeny that eventually adopt unique motoneuron subtype identities. Their axons each follow distinct trajectories into the periphery to innervate specific target muscles. Understanding of the molecular mechanisms that control the differentiation and respective connectivity of distinct neuronal subtypes is still limited (Oyallon, 2012).

The Olig family of basic Helix-Loop-Helix (bHLH) transcription factors in vertebrates includes the Oligodendrocyte lineage proteins Olig1-3, Bhlhb4 and Bhlhb5 (Bertrand, 2002). All members play pivotal roles in regulating neural development. Olig2 controls the sequential generation of somatic motoneurons and one type of myelinating glia, the oligodendrocytes, from the pMN progenitor domain in the ventral neural tube. Olig2 mediates progenitor domain formation by cross-repressive transcriptional interactions and motoneuron differentiation upstream of the LIM-homeodomain containing transcription factors Lim3 (Lhx3) and Islet1/2 (Isl1/2). Downregulation of Olig2 enables Lim3 and Isl1/2 together with the proneural bHLH transcription factor Neurogenin2 (Neurog2) to activate the expression of Hb9, a homeodomain protein and postmitotic motoneuron determinant. In addition, Olig2 cooperates with the homeodomain protein Nkx2.2 to promote oligodendrocyte formation from uncommitted pMN progenitors. Olig1 mediates gliogenesis redundantly with Olig2, while Olig3 controls interneuron specification within dorsal neural tube progenitor domains. Recent studies uncovered important requirements of Bhlhb4 in retinal bipolar cell maturation, and Bhlhb5 in regulating the specification of retinal amacrine and bipolar cells, area-specific identity acquisition and axon targeting of cortical postmitotic neurons, as well as differentiation and survival of distinct interneuron subtypes in the spinal cord. In Drosophila, genome-wide data base searches identified one single family member, called Olig family (Oli)), and a recent study described Oli expression in the embryonic ventral nerve cord (VNC) (Zhang, 2008). However, despite the central roles of vertebrate Olig family members, the function of their Drosophila counterpart has not been investigated (Oyallon, 2012 and references therein).

In Drosophila, neurons are derived from stem cell-like neuroblasts (NBs). These divide asymmetrically to generate secondary precursor cells, the ganglion mother cells (GMCs), which divide once to produce two postmitotic neurons and/or glia. 15 of 30 embryonic NB lineages give rise to 36 motoneurons in addition to interneurons per abdominal hemisegment. Zfh1 regulates general motoneuron fate acquisition at the postmitotic level. The specification of ventrally projecting motoneuron subtypes is mediated by a combinatorial expression of five transcriptional regulators -- the fly orthologs of Isl, Lim3, Hb9 and Nkx6, as well as the POU protein Drifter (Dfr; Ventral veinless -- FlyBase). Many of these determinants are highly conserved, raising the question as to whether Oli functions as part of this genetic network that shapes motoneuron diversity. Although related molecules in vertebrates and invertebrates appear to mediate late aspects of glial function, factors that regulate early steps of gliogenesis and are molecularly and functionally conserved have so far not been identified. Olig2 is essential for oligodendrocyte development in vertebrates, and a recent study also implicated the C. elegans homolog Hlh-17 in regulating gliogenesis). Thus, Oli is also a potential candidate that could control early glial development in Drosophila (Oyallon, 2012).

This study provides insights into the so far unexplored function of the Oli bHLH transcription factor in the Drosophila nervous system. Oli is not required in glia; however, taking advantage of the well-defined embryonic motoneuron lineages and axonal projections, this study demonstrates that oli controls trajectory selection and muscle targeting of ventral motoneuron subtypes. Moreover, Oli is expressed in postembryonic lineages, which include glutamatergic leg-innervating motoneurons. Loss-of-function experiments revealed that oli is required for larval and adult locomotion. Chick Olig2 can partially rescue these defects in adults, highlighting at least one evolutionarily conserved role of Olig transcription factors in flies and vertebrates (Oyallon, 2012).

Oli protein is mainly expressed in postmitotic neurons, as well as in some GMCs during embryonic development. This is consistent with in situ hybridization labeling detecting high levels of oli mRNA in postmitotic progeny, in addition to transient expression in MP2 and 7.1 NBs (Zhang, 2008). Oli is also expressed in postmitotic progeny of postembryonic lineages. By contrast, vertebrate Olig2 is required in progenitors to promote commitment to a general motoneuron identity (Mizuguchi, 2001). Also Olig1 and 3 largely function in progenitors (Lu, 2000; Muller, 2005; Takebayashi, 2000; Zechner, 2007; Zhou, 2000)). Interestingly, Bhlhb4 and Bhlhb5 are expressed and required in postmitotic progeny of the retina, brain and spinal cord (Bramblett, 2004; Feng, 2006; Joshi, 2008; Liu, 2007). Thus, with respect to its primarily postmitotic expression, Drosophila Oli resembles more that of Bhlhb4 and Bhlhb5 than Olig1–3 in vertebrates (Oyallon, 2012).

The dynamic expression of Drosophila Oli is not consistent with that of a member of the temporal series of transcriptional regulators (Brody, 2000; Isshiki, 2001). With the latter, neurons largely maintain the determinant they expressed at the time of their birth. By contrast, Oli is widely expressed in newly born progeny, but subsequently levels decrease, and only some subtypes show high expression during late stages. Vertebrate Olig2 acts as a transcriptional repressor in homomeric and heteromeric complexes, and expression is downregulated in differentiating motoneurons to enable the activation of postmitotic determinants such as Hb9 by Lim3, Isl1/2 and Neurog2 (Lee, 2005; Ma, 2008). Strikingly in flies, Oli expression decreases in RP and lateral ISNb motoneurons during embryogenesis and prolonged high expression of Oli elicits muscle innervation defects, supporting the notion that Oli downregulation is critical for its function in some neurons. Oli could thus act in a dual mode to regulate the differentiation of neuronal subtypes. The first one may rely on downregulation and be a feature shared with vertebrate Olig2, the second one may require persistent activity, and possibly be a feature more in common with Bhlhb4 and Bhlhb5 family members (Oyallon, 2012).

The findings indicate that Drosophila Oli, unlike vertebrate Olig2, does not act as a general early somatic motoneuron determinant. It rather contributes to shaping ventral motoneuron subtype development as part of a postmitotic transcriptional regulatory network in concert with Drosophila Lim3, Isl, Hb9 and Dfr (Drifter/Vvl). This notion is supported by findings that (1) Oli is co-expressed in specific combinations with these determinants in differentiated ISNb and TN motoneuron subtypes; (2) similar to other ventral determinants, oli mutant embryos display distinct axonal pathfinding and muscle targeting defects; (3) oli does not act upstream of hb9, isl, lim3 or Dfr; and (4) oli and hb9 genetically interact, as loss of both enhances phenotypes in ISNb axons. Because of the proximity of oli, isl and lim3 genetic loci, it has so far not been possible to further extend these interaction assays. Some defects observed in oli mutants, such as failure to innervate the clefts of muscles 12/13 or aberrant contacts between ISNb and TN motoneurons are qualitatively similar to those observed in isl, lim3, hb9 and dfr, while the phenotype of isl-τ-myc-positive neurons abnormally exiting the VNC via the SN branch appears characteristic for oli. Moreover, the connectivity phenotypes observed in oli gain-of-function experiments were not reminiscent of trajectories of other motoneuron subtypes. This suggests that although Oli is a member of the combinatorial code, unlike for instance Dfr, it does not act as a simple switch between fates. It may rather act in concert or partially redundantly with these other determinants in regulating the stepwise process of axon guidance to ensure robustness of trajectory selection (Oyallon, 2012).

Individual transcription factors within an ensemble may regulate different biological properties to tightly coordinate the differentiation and synaptic connectivity of a given neuron subtype. As Oli does not act upstream of Isl, Lim3, Hb9 and Dfr, it may control the expression of other yet to be identified transcription factors, or - similar to dfr, Nkx6 and eve in Drosophila and Bhlhb5 in mice - axon guidance determinant or - as reported for Neurog2 - cytoskeletal regulators. Examining Fasciclin 3, N-Cadherin, PlexinA, and Frazzled, no obvious altered expression was observed in the absence of oli). Thus, future studies using approaches such as microarrays will be required to identify oli downstream targets that control subtype-specific axonal connectivity (Oyallon, 2012).

While the role of oli in controlling neuronal development linked to locomotion appears conserved in Drosophila and vertebrates, conservation does not extend to glia. Oli is neither expressed in glia during embryonic or postembryonic development, nor is it essential for basic glial formation in the embryonic VNC or required in glia for locomotion. This also applied to other parts of the nervous system, such as the 3rd instar larval visual system endowed with large glial diversity. hlh-17, the C. elegans Oli homolog, is expressed in cephalic sheath glia in the brain, and interestingly in some motoneurons in the larval CNS. However, as analysis of hlh-17 mutants could not pinpoint any requirement in glial generation and differentiation possibly due to redundancy with related factors, the precise role of the worm homolog remains elusive. Although ensheathing glia can be found in both invertebrates and vertebrates, myelinating glia have so far only been identified in vertebrates. This raises the possibility that the glial requirement of vertebrate Olig family members could be secondary, and Olig2 may have been recruited to collaborate with additional transcriptional regulators to promote the formation of myelinating glia. Indeed, Olig2 promotes motoneuron development together with Neurog2, and subsequently collaborates with Nkx2.2 to enable the generation of oligodendrocyte precursors and differentiating offspring from newly formed, uncommitted pMN progenitors (Wu, 2006). Interestingly in cell-based assays, Oli can physically interact with the Nkx2.2 homolog Ventral nervous system defective (Vnd) (Zhang, 2008). Together with the current observation that Oli is not essential for glial development, this suggests that the potential of these determinants to interact is evolutionarily conserved, while the steps depending on them diverged in flies and vertebrates (Oyallon, 2012).

The locomotion defects in oli mutant larvae are likely the consequence of embryonic wiring defects, whereas the adult phenotypes may be due to an additional or even sole postembryonic requirement. Unlike the so far identified widely expressed determinants Chinmo, Broad Complex or Castor in the postembryonic VNC, Oli expression is restricted to distinct lineages. That these include motoneurons is supported by observations that Oli is detected in postembryonic lineages 20-22 and 15, and expression overlaps with that of OK371-Gal4. Moreover, locomotion defects can be partially rescued by over-expressing oli in glutamatergic neurons with this driver. This initial characterization raises many new questions regarding the specific postembryonic role of Oli. Because of the expression in lineage 15, future experiments will need to specifically test, whether oli contributes to consolidating motoneuron subtype identity by regulating dendritic arbor-formation or leg muscle innervation with single cell resolution. The wider expression of Oli and the partial rescue with OK371-Gal4 further suggest a requirement of oli in lineages that are part of locomotion-mediating neural circuits beyond motoneurons. Because of the expression pattern and the severe walking defects of adult oli escapers, these observations open the door for future functional studies to unravel the mechanisms that shape neural circuits underlying adult locomotion (Oyallon, 2012).

The Drosophila homeodomain transcription factor, Vnd, associates with a variety of co-factors, is extensively phosphorylated and forms multiple complexes in embryos.

Vnd is a dual transcriptional regulator that is essential for Drosophila dorsal-ventral patterning. Yet, understanding of the biochemical basis for its regulatory activity is limited. Consistent with Vnd's ability to repress target expression in embryos, endogenously expressed Vnd physically associates with the co-repressor, Groucho, in Drosophila Kc167 cells. Vnd exists as a single complex in Kc167 cells, in contrast with embryonic Vnd, which forms multiple high-molecular-weight complexes. Unlike its vertebrate homolog, Nkx2.2, full-length Vnd can bind its target in electrophoretic mobility shift assay, suggesting that co-factor availability may influence Vnd's weak regulatory activity in transient transfections. This study identifies the high mobility group 1-type protein, D1, and the helix-loop-helix protein, Olig, as Vnd-interacting proteins using co-immunoprecipitation assays. Furthermore, it was demonstrated that both D1 and Olig are co-expressed with Vnd during Drosophila embryogenesis, consistent with a biological basis for this interaction. It is also suggested that the phosphorylation state of Vnd influences its ability to interact with co-factors, because Vnd is extensively phosphorylated in embryos and can be phosphorylated by activated mitogen-activated protein kinase in vitro. These results highlight the complexities of Vnd-mediated regulation (Zhang, 2008).


REFERENCES

Search PubMed for articles about Drosophila Oli

Bertrand, N., Castro, D. S. and Guillemot, F. (2002). Proneural genes and the specification of neural cell types. Nat Rev Neurosci 3: 517-530. PubMed ID: 12094208

Bramblett, D. E., Pennesi, M. E., Wu, S. M. and Tsai, M. J. (2004). The transcription factor Bhlhb4 is required for rod bipolar cell maturation. Neuron 43: 779-793. PubMed ID: 15363390

Brody, T. and Odenwald, W. F. (2000). Programmed transformations in neuroblast gene expression during Drosophila CNS lineage development. Dev Biol 226: 34-44. PubMed ID: 10993672

Feng, L., Xie, X., Joshi, P. S., Yang, Z., Shibasaki, K., Chow, R. L. and Gan, L. (2006). Requirement for Bhlhb5 in the specification of amacrine and cone bipolar subtypes in mouse retina. Development 133: 4815-4825. PubMed ID: 17092954

Isshiki, T., Pearson, B., Holbrook, S. and Doe, C. Q. (2001). Drosophila neuroblasts sequentially express transcription factors which specify the temporal identity of their neuronal progeny. Cell 106: 511-521. PubMed ID: 11525736

Joshi, P. S., Molyneaux, B. J., Feng, L., Xie, X., Macklis, J. D. and Gan, L. (2008). Bhlhb5 regulates the postmitotic acquisition of area identities in layers II-V of the developing neocortex. Neuron 60: 258-272. PubMed ID: 18957218

Lee, S. K., Lee, B., Ruiz, E. C. and Pfaff, S. L. (2005). Olig2 and Ngn2 function in opposition to modulate gene expression in motor neuron progenitor cells. Genes Dev 19: 282-294. PubMed ID: 15655114

Liu, B., Liu, Z., Chen, T., Li, H., Qiang, B., Yuan, J., Peng, X. and Qiu, M. (2007). Selective expression of Bhlhb5 in subsets of early-born interneurons and late-born association neurons in the spinal cord. Dev Dyn 236: 829-835. PubMed ID: 17219401

Lu, Q. R., Yuk, D., Alberta, J. A., Zhu, Z., Pawlitzky, I., Chan, J., McMahon, A. P., Stiles, C. D. and Rowitch, D. H. (2000). Sonic hedgehog--regulated oligodendrocyte lineage genes encoding bHLH proteins in the mammalian central nervous system. Neuron 25: 317-329. PubMed ID: 10719888

Ma, Y. C., Song, M. R., Park, J. P., Henry Ho, H. Y., Hu, L., Kurtev, M. V., Zieg, J., Ma, Q., Pfaff, S. L. and Greenberg, M. E. (2008). Regulation of motor neuron specification by phosphorylation of neurogenin 2. Neuron 58: 65-77. PubMed ID: 18400164

Mizuguchi, R., Sugimori, M., Takebayashi, H., Kosako, H., Nagao, M., Yoshida, S., Nabeshima, Y., Shimamura, K. and Nakafuku, M. (2001). Combinatorial roles of olig2 and neurogenin2 in the coordinated induction of pan-neuronal and subtype-specific properties of motoneurons. Neuron 31: 757-771. PubMed ID: 11567615

Muller, T., Anlag, K., Wildner, H., Britsch, S., Treier, M. and Birchmeier, C. (2005). The bHLH factor Olig3 coordinates the specification of dorsal neurons in the spinal cord. Genes Dev 19: 733-743. PubMed ID: 15769945

Oyallon, J., Apitz, H., Miguel-Aliaga, I., Timofeev, K., Ferreira, L. and Salecker, I. (2012). Regulation of locomotion and motoneuron trajectory selection and targeting by the Drosophila homolog of Olig family transcription factors. Dev Biol 369: 261-276. PubMed ID: 22796650

Takebayashi, H., Ohtsuki, T., Uchida, T., Kawamoto, S., Okubo, K., Ikenaka, K., Takeichi, M., Chisaka, O. and Nabeshima, Y. (2002). Non-overlapping expression of Olig3 and Olig2 in the embryonic neural tube. Mech Dev 113: 169-174. PubMed ID: 11960707

Wu, S., Wu, Y. and Capecchi, M. R. (2006). Motoneurons and oligodendrocytes are sequentially generated from neural stem cells but do not appear to share common lineage-restricted progenitors in vivo. Development 133: 581-590. PubMed ID: 16407399

Zechner, D., Muller, T., Wende, H., Walther, I., Taketo, M. M., Crenshaw, E. B., 3rd, Treier, M., Birchmeier, W. and Birchmeier, C. (2007). Bmp and Wnt/beta-catenin signals control expression of the transcription factor Olig3 and the specification of spinal cord neurons. Dev Biol 303: 181-190. PubMed ID: 17150208

Zhang, H., Syu, L. J., Modica, V., Yu, Z., Von Ohlen, T. and Mellerick, D. M. (2008). The Drosophila homeodomain transcription factor, Vnd, associates with a variety of co-factors, is extensively phosphorylated and forms multiple complexes in embryos. FEBS J 275: 5062-5073. PubMed ID: 18795949

Zhou, Q. and Anderson, D. J. (2002). The bHLH transcription factors OLIG2 and OLIG1 couple neuronal and glial subtype specification. Cell 109: 61-73. PubMed ID: 11955447


Biological Overview

date revised: 4 March 2013

Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.

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