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Neuronal temporal identity in post-embryonic Drosophila brain
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Neuronal temporal identity in post-embryonic Drosophila brain Hung-Hsiang Yu and Tzumin Lee Department of Neurobiology, University of Massachusetts Medical School, Worcester, MA 01605, USA
Understanding how a vast number of neuron types derive from a limited number of neural progenitors remains a major challenge in developmental neurobiology. In the post-embryonic Drosophila brain, specific neuron types derive from specific progenitors at specific times. This suggests involvement of time-dependent cell fate determinants acting as ‘temporal codes’ along with lineage cues to specify neuronal cell fates. Interestingly, such temporal codes might be provided not only by several regulators acting in sequence, but also by the differential protein levels of the BTB-zinc finger nuclear protein Chinmo. Identifying temporal codes and determining their origins should allow us to elucidate how neuronal diversification occurs through protracted neurogenesis. Introduction Neurons acquire characteristic morphologies and exhibit discrete electrophysiological properties. Many neuronal traits are prefigured when the neuron is born according to its spatial and temporal origin [1,2]. However, despite much understanding about spatial patterning of neural diversification [3], our knowledge of neuron type specification based on time or order of birth within a lineage remains limited. One of our greatest challenges is determining not only when particular neurons are born but also from which lineages they arise in complex brains. In Drosophila, breakthroughs were first made through detailed characterization of the embryonic neurogenesis of the ventral nervous cord (VNC), where a fixed number of neural progenitors persist in each hemi-segment and can be individually identified based on when and where they delaminate into the embryo from the ventral neuroectoderm. DiI labeling of single neural progenitors has revealed that every VNC progenitor yields a specific reproducible set of progeny [4–6]. One of the VNC progenitors produces five identifiable distinct motor neurons in an invariant sequence before making interneurons [7]. Using this as a model lineage, Doe and coworkers [7] have substantiated that a set of transcription factors are serially expressed with one cell cycle per expression window in the progenitor to specify birth-order-dependent cell fates in sequentially derived post-mitotic neurons [7–9]. Hunchback (Hb), Kruppel (Kr), POU domain transcription factors (Pdm), and Castor (Cas) are transiently and sequentially expressed in the respective order, and post-mitotic cells Corresponding author: Lee, T. ([email protected]). Available online 6 September 2007. www.sciencedirect.com
born in different expression windows inherit different transcription factors and acquire different cell fates. Intriguingly, these transcription factors are broadly expressed in mutually exclusive layers in the late embryonic VNC, with Hb restricted to the deepest layer neurons and Cas located in the most superficial ones [8,10]. In general, early-derived post-mitotic neurons occupy deeper layers than their later-born siblings [6,8]. Thus, this broad layered pattern of expression suggests that many or all of the VNC neural progenitors generate their diverse progeny in an invariant sequence, and implicates the orderly expression of Hb, Kr, Pdm, and Cas as a common mechanism governing neuronal temporal cell fates during early embryonic neurogenesis of Drosophila. There are multitudes of neuronal lineages that contribute to building the nervous system, among which the production of various types of neurons occurs with distinctive tempos. Whether and how the transcription factors implicated in early embryonic neurogenesis play roles in other neuronal lineages remains to be determined. Nevertheless, four transcription factors appear insufficient to encode all the temporal identities in most neuronal lineages. Encouragingly, lineage and single-cell analysis by MARCM (mosaic analysis with a repressible cell marker), a genetic mosaic system permitting positive labeling [11], has recently expanded the field. Use of a lineage-specific GAL4 driver in MARCM permits the labeling of single neurons born at specific times in a particular lineage (see Box 1). Holometabolous insects experience two neurogenic periods. An initial brief period during embryogenesis produces neurons that are involved in larval behavior, and a second prolonged phase during larval growth results in a much larger set of neurons that govern adult behavior [12]. The application of MARCM to characterize larval-born neurons consistently revealed the derivation of neurons from specific progenitors at specific developmental times [13–17]. This suggests the broad involvement of time-dependent cell fate determinants that act as ‘temporal codes’ in the combinatorial specification of neuronal cell fates. Unbiased genetic mosaic screens with MARCM provide information on mutations in genes involved in a wide array of cellular activities (see below, section on Protein concentrations of Chinmo as novel temporal code) and thus can allow identification of such temporal codes. For example, a recent MARCM-based screen provided for the discovery of Chinmo (pronounced as kee-mo), a novel BTB (broad complex, tramtrack, bric-a-brac)-zinc finger nuclear protein required for proper specification of multiple neuronal temporal cell fates [18]. We believe the study of neuronal
0166-2236/$ – see front matter . Published by Elsevier Ltd. doi:10.1016/j.tins.2007.07.003
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Box 1. MARCM technology and application Mosaic analysis with a repressible cell marker (MARCM) is a genetic mosaic system used to label homozygous mutant cells in otherwise heterozygous tissues (Figure I); this can facilitate phenotypic analysis in the complicated nervous system. Initially, cells contain a yeast transcriptional repressor, GAL80, to inhibit the transcriptional activity of GAL4, which, in turn, suppresses the expression of a reporter gene, UAS-mCD8::GFP. Following the induction of flip recombinase (FLP) by heat shock to mediate FLP recognition target (FRT)-based mitotic recombination, the GAL80 transgene is segregated into one of the
daughter cells, thus allowing UAS-mCD8::GFP expression in the other daughter cell. A mutation of a gene of interest (x) can be placed distal to the site of mitotic recombination and in trans with the GAL80 transgene when phenotypic analysis is desired. Homozygous mutant cells (x/x) are visualized by the expression of UAS-mCD8::GFP. When different GAL4 lines (A and B) and different heat shock windows (1 and 2) are used, distinct lineage (round neuron versus square neuron) and temporal (solid green neuron versus lined green neuron) information can be revealed in MARCM analyses, respectively.
Figure I. MARCM technology and application.
temporal identity in diverse Drosophila post-embryonic neuronal lineages will bring many new opportunities for uncovering the mechanisms that govern neuronal diversification through protracted neurogenesis. Here we selectively review neuronal diversification during Drosophila post-embryonic development. First, we discuss two specific Drosophila post-embryonic neuronal lineages in which the derivation of multiple neuron types occurs with distinct tempos. Second, we elaborate the mechanism of Chinmo as a single nuclear protein capable of specifying multiple neuronal temporal cell fates, in contrast to temporal cues provided by sequential action of multiple transcription factors during early embryonic neurogenesis of Drosophila. Finally, we further discuss how studying neuronal temporal identity in diverse lineages of Drosophila and at various developmental stages can synergistically www.sciencedirect.com
enhance our understanding of birth time- or birth order-governed neuron type specification. We ask the reader to refer to the excellent reviews [19,20] by Doe and coworkers for additional details on neuronal temporal cell fate specification in the Drosophila embryonic VNC; this cell fate specification is an effective model system that has been proven very informative in the elucidation of neuronal temporal identity and in its underlying molecular mechanisms. Post-embryonic mushroom body neurons The mature mushroom bodies (MB), which are crucial for Drosophila olfactory learning and memory [21], each derive from four indistinguishable lineages [22]. Each lineage provides neurons that are serially deposited in pairs by a neural progenitor, called a neuroblast (Nb). The
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production of neuron pairs involves the generation of an intermediate neural precursor, called a ganglion mother cell (GMC), from each asymmetric division of the Nb and the subsequent mitotic division of the GMC to form the neurons [13,23]. Intriguingly, MB Nbs continuously deposit post-mitotic neurons through embryonic, larval and pupal stages, providing for the most extended of Drosophila neuronal lineages [13,24]. The prominent structural features of the fully developed MBs provided for early speculations about the types of neurons they contain. Each mature MB exhibits five axon lobes [25] (Figure 1), and distinct sets of these lobes are revealed when the neurite projection patterns of subsets of MB neurons are labeled using various subtype-specific GAL4 drivers in conjunction with cell markers like GFP [22,26]. The medial g lobe can be selectively labeled, whereas the dorsal a0 and a lobes are consistently colabeled with the medial b0 and b lobes, respectively. This indicates the presence of three major types of MB neurons, called g, a0 /b0 , and a/b neurons, that can be distinguished not only by subtype-specific GAL4s but also based on axon trajectories (Figure 1). Furthermore, some MB GAL4s appear to label different subsets of a/b neurons indicating the presence of subtypes of MB a/b neurons [18,27]. The pioneer a/b (pa/b) neurons, named according to the order of birth (see the next paragraph), extend their medial projections along the upper surface of the b lobe while their dorsal processes form a tight fascicle in the periphery of the a lobe [16] (Figure 1). The core a/b (ca/b) neurons, which are the last-born MB neurons (see the next paragraph),
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send neurites through the centers of the a and b lobes, whereas typical a/b (ta/b) neurons fill in the majority of the a and b lobes. Although the developing MBs continuously grow while serially derived MB neurons sequentially project their neurites into the MB lobes, the adult sets of axon lobes form at about the same time during early pupal stages following a drastic remodeling of the larval MBs [13,28]. That the adult sets of axon lobes form at about the same time makes it impossible to determine the temporal origins of these distinct sets of MB lobes or neurons without a birth time-dependent labeling system. Further, additional neuron types potentially exist, which can be identified after detailed characterization of single neuron projections. Use of a pan-MB GAL4 driver in MARCM permits selective labeling of single MB neurons that are born at different specific times depending on when mitotic recombination was induced. Targeting single neurons through all of the MB lineages has not only substantiated previous speculation about MB neuronal diversity but also demonstrated that these distinct neuron types derive from common progenitors in an invariant order [13]. MB progenitors continuously make g neurons through embryonic and midlarval stages and then switch over to production of a0 /b0 neurons until puparium formation, after which they yield the various subtypes of a/b neurons in the following order: pa/b ! ta/b ! ca/b (Figure 1). The g to a0 /b0 transition takes place at the mid-third instar stage [13], whereas the brief production of pa/b neurons (14 per MB Nb lineage) coincides with puparium formation [16] (Figure 1). Next,
Figure 1. Sequential production of g, a0 /b0 and a/b MB neurons in the Drosophila MB lineage. (a) The MBs are located in the upper medial part of an adult fly brain. Different types of MB neurons possessing distinctive axon and dendrite projection patterns are shown in drawings of single neurons [(i) g, (ii) a0 /b0 , (iii) pioneer (p) a/b, (iv) typical (t) a/b and core (c) a/b neurons]. (b) A neuroblast (Nb) of a MB lineage sequentially deposits different types of MB neurons (g, a0 /b0 and a/b) during different developmental periods (from left to right: late embryo, first-instar larvae, second-instar larvae, third-instar larvae, pupa and eclosion of an adult fly). Abbreviations: ALH, after larval hatching; d, days. www.sciencedirect.com
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production of a large number of ta/b neurons (80 per MB Nb lineage) commences followed by the deposition of the ca/b neurons (26 per MB Nb lineage) towards the end of development (ChihFei Kao, Suewei Lin and Tzumin Lee, unpublished observation). Because these protracted lineages are so easily accessible with mosaic analysis, they should serve as a great model system for elucidating the underlying mechanisms of birth order- or birth time-dependent neuronal cell fate specification. Antennal lobe projection neurons The antennal lobe (AL) projection neurons (PNs) serve as relays for olfactory information from olfactory receptor neurons to the MBs and the lateral horns (LHs), and recent studies that utilized analogous lineage and single-cell analysis of PNs revealed orderly derivation of distinct PNs in the adPN lineage [14] (Figure 2). Like most other non-MB Nbs that experience two neurogenic periods [24], the adPN Nb does not resume self-renewing asymmetric divisions until the second instar stage and completes its neurogenesis before pupal formation [14]. In this three-day window (as opposed to nine days for post-embryonic MB neurogenesis), one adPN Nb makes 75 post-mitotic neurons that together innervate 15 of the 40+ identifiable AL glomeruli [29]. Each adPN targets dendrites only to one
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glomerulus [14], and PNs with distinct glomerular targets acquire different characteristic axon arborization patterns in the LHs [30,31] (Figure 2). The distinct PNs are latent and morphologically similar before pupal formation, and although they do not acquire their stereotypical projection patterns until early metamorphosis [32] they derive from the adPN Nb in an invariant sequence [14]. Taking these three things together, one adPN Nb sequentially makes 15 types of monoglomerular PNs, which, like MB development, is representative of another example of birth order- or birth time-dependent neuronal diversification (Figure 2). Compared with the MB neuronal lineages, the adPN lineage yields many more neuron types in a much shorter period of time. Further, the adPN Nb makes distinct PNs in a fast but steady tempo (Figure 2) with each type of PN consisting of about five post-mitotic neurons (Sen-Lin Lai and Tzumin Lee, unpublished observation). By contrast, MB Nbs alter their competence status (i.e. switch to produce different types of MB neurons) at an irregular pace and more in a stage-specific manner (Figure 1). The discovery of these lineage differences presents exciting opportunities for comparative analyses. We hope that elucidating how post-mitotic neurons acquire different cell fates based on their temporal origins in such diverse
Figure 2. Orderly production of distinct PNs in the Drosophila anterodorsal PN (adPN) lineage. (a) The positions of antennal lobe (AL, labeled in purple), MB and lateral horn (LH) are illustrated in the adult fly brain. Different types of PNs possess their own axon and dendrite projection patterns, and three types of PNs (DL1, D and VA1lm) are shown in the drawings. (b) A neuroblast (Nb) of an adPN lineage rests at the post-embryonic stage, resumes its proliferation at the second-instar larval stage, and stops depositing PNs around the puparium formation. About fifteen different types of PNs are produced within a relatively short period in the adPN lineage as compared to fewer types of MB neurons produced in the longer MB lineage (shown in Figure 1). www.sciencedirect.com
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neuronal lineages sheds light on the different modalities providing for temporal identity specification in the developing nervous system. Protein concentrations of Chinmo as novel temporal codes MARCM allows one to label single neurons and at the same time learn the developmental history (e.g. lineage and temporal origins) of the labeled cells (see Box 1). If desired, MARCM permits manipulation of gene functions (by loss of heterozygosity or induction of dominant transgenes) selectively in the labeled neurons [33,34]. In fact, MARCM analysis of lethal mutations in the MB lineages has allowed us to identify genes, including usp [35], Dscam [15], babo [36], dSmad2 [36], cul3 [37], ph [38] and chinmo [18], that control various aspects of neural development. Among them, the Chinmo BTB-zinc finger nuclear protein was recovered as a master regulator for MB neuronal temporal identity at the larval stages. Interestingly, levels of Chinmo exist in a gradient through larval MB neurogenesis (high to low through time) and the levels of Chinmo in newly derived post-mitotic neurons dictate their temporal cell fates [18] (Figure 3). Briefly, early larval-born MB neurons contain
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more Chinmo than their later-derived siblings during the initial development of individual cells; and a reduction or an increase in the expression of Chinmo can accelerate or delay neuronal temporal cell fate transitions. Taken together, these observations suggest concentration of Chinmo might act as a novel temporal code in the combinatorial specification of neuronal cell fates. As revealed by immunostaining, Chinmo broadly exists and gradually decreases through larval development of the Drosophila CNS [18]. It has the potential for being a universal determinant of neuronal temporal identity among larval-derived post-mitotic neurons in the way that Hb, Kr, Pdm and Cas confer temporal identity in diverse embryonic neuronal lineages. However, distinct neuron types arise in different temporal patterns in different post-embryonic neuronal lineages, as illustrated above. In the adPN lineage, although the first-larval-born adPN does require Chinmo for proper temporal cell fate specification [18], it remains unclear whether Chinmo also exits in a gradient in the developing adPN lineage and whether a similar gradient alone is sufficient to specify all the distinct adPN neurons. It is possible that distinct, perhaps multiple, gradients could exist in the lineages that use
Figure 3. A model for formation of the Chinmo gradient and an illustration of the precocious shift in neuronal temporal identity upon loss of chinmo. (a) There is no detectable gradient in the transcription of chinmo as reflected by abundant chinmo mRNA in precursor cells. However, a gradual reduction in the Chinmo expression level is observed among sequentially deposited post-mitotic neurons, suggesting a post-transcriptional control over the production of Chinmo. An unidentified negative (or positive) post-transcriptional regulator with a gradually increasing (or decreasing) influence might govern the production of the Chinmo gradient in post-mitotic neurons. (b) A hypothetical neuronal lineage is used to illustrate how the Chinmo gradient controls the production of sequentially derived neurons (A to D from early-born to lateborn). When chinmo is knocked out of early-born neurons in single-cell MARCM analyses (green circle cells), they adopt the fate of late-born neurons. Abbreviation: wt, wild type. www.sciencedirect.com
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different temporal strategies in their orderly derivation of multiple neuron types. Alternatively, an analogous gradient could have different consequences in different lineages. Furthermore, multiple mechanisms might act independently and/or in concert to simultaneously and/or sequentially govern neuronal temporal identity. For example, a non-Chinmo mechanism is apparently required for proper specification of distinct subtypes of MB a/b neurons and gradients of Chinmo might operate collaboratively with additional mechanisms to increase neuronal diversity in the adPN lineage. Given that levels of Chinmo dictate neuronal temporal identity, the elucidation of how the high to low gradient of Chinmo is established through development of a neuronal lineage is an important task for our understanding the origins of neuronal temporal identity. Several interesting observations have been made [18]. First, locating endogenous Chinmo proteins versus chinmo transcripts revealed that the gradient expression of chinmo is only evident at the protein level (Figure 3). This implies involvement of post-transcriptional regulation in the establishment of gradients of the Chinmo protein. Second, transgenic chinmo with the endogenous 50 UTR faithfully recapitulated the high to low Chinmo gradient in both wild-type and chinmo mutant MB Nb clones. By contrast, chinmo transcripts lacking the 50 UTR were constitutively translated, resulting in high Chinmo expression throughout MB lineage development. These observations indicate that a 50 UTR-dependent translational control underlies the initial specification of MB neuronal temporal cell fates. Although several Caenorhabditis elegans heterochronic mutants carry mutations in miRNA genes [39–41], it is uncommon for miRNAs to suppress gene expression through 50 UTR [42]. In fact, no putative miRNA target sequences could be identified in the chinmo 50 UTR and abolition of miRNA or siRNA processing did not affect MB neuronal temporal cell fate specification (ChihFei Kao and Tzumin Lee, unpublished results). Given that 50 UTR-dependent translational control often involves RNA-binding proteins [43,44], it is more likely that such neuronal temporal cell fates governed by a Chinmo gradient are originally specified by differential expression of some RNAbinding proteins (Figure 3). Identifying additional temporal codes and elucidating how they are derived promise to unravel how distinct lineages generate multiple neuron types in different temporal patterns. New possibilities and further challenges Single-cell analysis of diverse Drosophila neuronal lineages has clearly revealed the broad involvement of birth order- or birth time-dependent cell fate determinants in the derivation of multiple neuron types from a common progenitor. This lays a solid ground for elucidating various modalities used in the Drosophila nervous system for temporal cell fate specification. However, identifying temporal codes and determining their origins remain challenging especially in non-MB neuronal lineages. For instance, in lineages that quickly produce different types of cells, it is impossible to assure any temporal cell fate transformation in a given mutant single-cell clone, because in current MARCM one cannot determine exactly which www.sciencedirect.com
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neuronal precursor has undergone mitotic recombination to give rise to a particular MARCM-labeled neuron. Newer, more sophisticated MARCM techniques that incorporate finer temporal control over the induction of MARCM clones or methodology for identifying when mitotic recombination has occurred are needed for detecting subtle neuronal temporal identity phenotypes in fast-tempo neuronal lineages. In addition, most temporal codes likely exist transiently during the initial specification of neuronal cell fates. To locate them and determine how they are generated, we must better characterize neuronal precursors. Though some progress has been made along this line [45], it remains extremely challenging to identify specific neuron progenitors and their immediate derivatives throughout development of the Drosophila brain (except the MB lineages). Systematic efforts should be made to study the development of the Drosophila brain at the level of individual cells so that we can elucidate the diverse strategies used in the Drosophila nervous system for temporal cell fate specification. We hope that the lessons learned from the highly complex Drosophila brain will facilitate the study of vastly more complex brains, ultimately leading us to a better understanding of how multitudes of neuron types derive from a limited number of neural progenitors in higher organisms. Acknowledgements We would like to thank C.T. Zugates and members in the Lee laboratory for helpful discussions regarding the manuscript. The work of the authors is supported by the National Institutes of Health and March of Dimes Birth Defects Foundation.
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Neuronal temporal identity in post-embryonic Drosophila brain Hung-Hsiang Yu and Tzumin Lee Department of Neurobiology, University of Massachusetts Medical School, Worcester, MA 01605, USA
Understanding how a vast number of neuron types derive from a limited number of neural progenitors remains a major challenge in developmental neurobiology. In the post-embryonic Drosophila brain, specific neuron types derive from specific progenitors at specific times. This suggests involvement of time-dependent cell fate determinants acting as ‘temporal codes’ along with lineage cues to specify neuronal cell fates. Interestingly, such temporal codes might be provided not only by several regulators acting in sequence, but also by the differential protein levels of the BTB-zinc finger nuclear protein Chinmo. Identifying temporal codes and determining their origins should allow us to elucidate how neuronal diversification occurs through protracted neurogenesis. Introduction Neurons acquire characteristic morphologies and exhibit discrete electrophysiological properties. Many neuronal traits are prefigured when the neuron is born according to its spatial and temporal origin [1,2]. However, despite much understanding about spatial patterning of neural diversification [3], our knowledge of neuron type specification based on time or order of birth within a lineage remains limited. One of our greatest challenges is determining not only when particular neurons are born but also from which lineages they arise in complex brains. In Drosophila, breakthroughs were first made through detailed characterization of the embryonic neurogenesis of the ventral nervous cord (VNC), where a fixed number of neural progenitors persist in each hemi-segment and can be individually identified based on when and where they delaminate into the embryo from the ventral neuroectoderm. DiI labeling of single neural progenitors has revealed that every VNC progenitor yields a specific reproducible set of progeny [4–6]. One of the VNC progenitors produces five identifiable distinct motor neurons in an invariant sequence before making interneurons [7]. Using this as a model lineage, Doe and coworkers [7] have substantiated that a set of transcription factors are serially expressed with one cell cycle per expression window in the progenitor to specify birth-order-dependent cell fates in sequentially derived post-mitotic neurons [7–9]. Hunchback (Hb), Kruppel (Kr), POU domain transcription factors (Pdm), and Castor (Cas) are transiently and sequentially expressed in the respective order, and post-mitotic cells Corresponding author: Lee, T. ([email protected]). Available online 6 September 2007. www.sciencedirect.com
born in different expression windows inherit different transcription factors and acquire different cell fates. Intriguingly, these transcription factors are broadly expressed in mutually exclusive layers in the late embryonic VNC, with Hb restricted to the deepest layer neurons and Cas located in the most superficial ones [8,10]. In general, early-derived post-mitotic neurons occupy deeper layers than their later-born siblings [6,8]. Thus, this broad layered pattern of expression suggests that many or all of the VNC neural progenitors generate their diverse progeny in an invariant sequence, and implicates the orderly expression of Hb, Kr, Pdm, and Cas as a common mechanism governing neuronal temporal cell fates during early embryonic neurogenesis of Drosophila. There are multitudes of neuronal lineages that contribute to building the nervous system, among which the production of various types of neurons occurs with distinctive tempos. Whether and how the transcription factors implicated in early embryonic neurogenesis play roles in other neuronal lineages remains to be determined. Nevertheless, four transcription factors appear insufficient to encode all the temporal identities in most neuronal lineages. Encouragingly, lineage and single-cell analysis by MARCM (mosaic analysis with a repressible cell marker), a genetic mosaic system permitting positive labeling [11], has recently expanded the field. Use of a lineage-specific GAL4 driver in MARCM permits the labeling of single neurons born at specific times in a particular lineage (see Box 1). Holometabolous insects experience two neurogenic periods. An initial brief period during embryogenesis produces neurons that are involved in larval behavior, and a second prolonged phase during larval growth results in a much larger set of neurons that govern adult behavior [12]. The application of MARCM to characterize larval-born neurons consistently revealed the derivation of neurons from specific progenitors at specific developmental times [13–17]. This suggests the broad involvement of time-dependent cell fate determinants that act as ‘temporal codes’ in the combinatorial specification of neuronal cell fates. Unbiased genetic mosaic screens with MARCM provide information on mutations in genes involved in a wide array of cellular activities (see below, section on Protein concentrations of Chinmo as novel temporal code) and thus can allow identification of such temporal codes. For example, a recent MARCM-based screen provided for the discovery of Chinmo (pronounced as kee-mo), a novel BTB (broad complex, tramtrack, bric-a-brac)-zinc finger nuclear protein required for proper specification of multiple neuronal temporal cell fates [18]. We believe the study of neuronal
0166-2236/$ – see front matter . Published by Elsevier Ltd. doi:10.1016/j.tins.2007.07.003
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Box 1. MARCM technology and application Mosaic analysis with a repressible cell marker (MARCM) is a genetic mosaic system used to label homozygous mutant cells in otherwise heterozygous tissues (Figure I); this can facilitate phenotypic analysis in the complicated nervous system. Initially, cells contain a yeast transcriptional repressor, GAL80, to inhibit the transcriptional activity of GAL4, which, in turn, suppresses the expression of a reporter gene, UAS-mCD8::GFP. Following the induction of flip recombinase (FLP) by heat shock to mediate FLP recognition target (FRT)-based mitotic recombination, the GAL80 transgene is segregated into one of the
daughter cells, thus allowing UAS-mCD8::GFP expression in the other daughter cell. A mutation of a gene of interest (x) can be placed distal to the site of mitotic recombination and in trans with the GAL80 transgene when phenotypic analysis is desired. Homozygous mutant cells (x/x) are visualized by the expression of UAS-mCD8::GFP. When different GAL4 lines (A and B) and different heat shock windows (1 and 2) are used, distinct lineage (round neuron versus square neuron) and temporal (solid green neuron versus lined green neuron) information can be revealed in MARCM analyses, respectively.
Figure I. MARCM technology and application.
temporal identity in diverse Drosophila post-embryonic neuronal lineages will bring many new opportunities for uncovering the mechanisms that govern neuronal diversification through protracted neurogenesis. Here we selectively review neuronal diversification during Drosophila post-embryonic development. First, we discuss two specific Drosophila post-embryonic neuronal lineages in which the derivation of multiple neuron types occurs with distinct tempos. Second, we elaborate the mechanism of Chinmo as a single nuclear protein capable of specifying multiple neuronal temporal cell fates, in contrast to temporal cues provided by sequential action of multiple transcription factors during early embryonic neurogenesis of Drosophila. Finally, we further discuss how studying neuronal temporal identity in diverse lineages of Drosophila and at various developmental stages can synergistically www.sciencedirect.com
enhance our understanding of birth time- or birth order-governed neuron type specification. We ask the reader to refer to the excellent reviews [19,20] by Doe and coworkers for additional details on neuronal temporal cell fate specification in the Drosophila embryonic VNC; this cell fate specification is an effective model system that has been proven very informative in the elucidation of neuronal temporal identity and in its underlying molecular mechanisms. Post-embryonic mushroom body neurons The mature mushroom bodies (MB), which are crucial for Drosophila olfactory learning and memory [21], each derive from four indistinguishable lineages [22]. Each lineage provides neurons that are serially deposited in pairs by a neural progenitor, called a neuroblast (Nb). The
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production of neuron pairs involves the generation of an intermediate neural precursor, called a ganglion mother cell (GMC), from each asymmetric division of the Nb and the subsequent mitotic division of the GMC to form the neurons [13,23]. Intriguingly, MB Nbs continuously deposit post-mitotic neurons through embryonic, larval and pupal stages, providing for the most extended of Drosophila neuronal lineages [13,24]. The prominent structural features of the fully developed MBs provided for early speculations about the types of neurons they contain. Each mature MB exhibits five axon lobes [25] (Figure 1), and distinct sets of these lobes are revealed when the neurite projection patterns of subsets of MB neurons are labeled using various subtype-specific GAL4 drivers in conjunction with cell markers like GFP [22,26]. The medial g lobe can be selectively labeled, whereas the dorsal a0 and a lobes are consistently colabeled with the medial b0 and b lobes, respectively. This indicates the presence of three major types of MB neurons, called g, a0 /b0 , and a/b neurons, that can be distinguished not only by subtype-specific GAL4s but also based on axon trajectories (Figure 1). Furthermore, some MB GAL4s appear to label different subsets of a/b neurons indicating the presence of subtypes of MB a/b neurons [18,27]. The pioneer a/b (pa/b) neurons, named according to the order of birth (see the next paragraph), extend their medial projections along the upper surface of the b lobe while their dorsal processes form a tight fascicle in the periphery of the a lobe [16] (Figure 1). The core a/b (ca/b) neurons, which are the last-born MB neurons (see the next paragraph),
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send neurites through the centers of the a and b lobes, whereas typical a/b (ta/b) neurons fill in the majority of the a and b lobes. Although the developing MBs continuously grow while serially derived MB neurons sequentially project their neurites into the MB lobes, the adult sets of axon lobes form at about the same time during early pupal stages following a drastic remodeling of the larval MBs [13,28]. That the adult sets of axon lobes form at about the same time makes it impossible to determine the temporal origins of these distinct sets of MB lobes or neurons without a birth time-dependent labeling system. Further, additional neuron types potentially exist, which can be identified after detailed characterization of single neuron projections. Use of a pan-MB GAL4 driver in MARCM permits selective labeling of single MB neurons that are born at different specific times depending on when mitotic recombination was induced. Targeting single neurons through all of the MB lineages has not only substantiated previous speculation about MB neuronal diversity but also demonstrated that these distinct neuron types derive from common progenitors in an invariant order [13]. MB progenitors continuously make g neurons through embryonic and midlarval stages and then switch over to production of a0 /b0 neurons until puparium formation, after which they yield the various subtypes of a/b neurons in the following order: pa/b ! ta/b ! ca/b (Figure 1). The g to a0 /b0 transition takes place at the mid-third instar stage [13], whereas the brief production of pa/b neurons (14 per MB Nb lineage) coincides with puparium formation [16] (Figure 1). Next,
Figure 1. Sequential production of g, a0 /b0 and a/b MB neurons in the Drosophila MB lineage. (a) The MBs are located in the upper medial part of an adult fly brain. Different types of MB neurons possessing distinctive axon and dendrite projection patterns are shown in drawings of single neurons [(i) g, (ii) a0 /b0 , (iii) pioneer (p) a/b, (iv) typical (t) a/b and core (c) a/b neurons]. (b) A neuroblast (Nb) of a MB lineage sequentially deposits different types of MB neurons (g, a0 /b0 and a/b) during different developmental periods (from left to right: late embryo, first-instar larvae, second-instar larvae, third-instar larvae, pupa and eclosion of an adult fly). Abbreviations: ALH, after larval hatching; d, days. www.sciencedirect.com
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production of a large number of ta/b neurons (80 per MB Nb lineage) commences followed by the deposition of the ca/b neurons (26 per MB Nb lineage) towards the end of development (ChihFei Kao, Suewei Lin and Tzumin Lee, unpublished observation). Because these protracted lineages are so easily accessible with mosaic analysis, they should serve as a great model system for elucidating the underlying mechanisms of birth order- or birth time-dependent neuronal cell fate specification. Antennal lobe projection neurons The antennal lobe (AL) projection neurons (PNs) serve as relays for olfactory information from olfactory receptor neurons to the MBs and the lateral horns (LHs), and recent studies that utilized analogous lineage and single-cell analysis of PNs revealed orderly derivation of distinct PNs in the adPN lineage [14] (Figure 2). Like most other non-MB Nbs that experience two neurogenic periods [24], the adPN Nb does not resume self-renewing asymmetric divisions until the second instar stage and completes its neurogenesis before pupal formation [14]. In this three-day window (as opposed to nine days for post-embryonic MB neurogenesis), one adPN Nb makes 75 post-mitotic neurons that together innervate 15 of the 40+ identifiable AL glomeruli [29]. Each adPN targets dendrites only to one
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glomerulus [14], and PNs with distinct glomerular targets acquire different characteristic axon arborization patterns in the LHs [30,31] (Figure 2). The distinct PNs are latent and morphologically similar before pupal formation, and although they do not acquire their stereotypical projection patterns until early metamorphosis [32] they derive from the adPN Nb in an invariant sequence [14]. Taking these three things together, one adPN Nb sequentially makes 15 types of monoglomerular PNs, which, like MB development, is representative of another example of birth order- or birth time-dependent neuronal diversification (Figure 2). Compared with the MB neuronal lineages, the adPN lineage yields many more neuron types in a much shorter period of time. Further, the adPN Nb makes distinct PNs in a fast but steady tempo (Figure 2) with each type of PN consisting of about five post-mitotic neurons (Sen-Lin Lai and Tzumin Lee, unpublished observation). By contrast, MB Nbs alter their competence status (i.e. switch to produce different types of MB neurons) at an irregular pace and more in a stage-specific manner (Figure 1). The discovery of these lineage differences presents exciting opportunities for comparative analyses. We hope that elucidating how post-mitotic neurons acquire different cell fates based on their temporal origins in such diverse
Figure 2. Orderly production of distinct PNs in the Drosophila anterodorsal PN (adPN) lineage. (a) The positions of antennal lobe (AL, labeled in purple), MB and lateral horn (LH) are illustrated in the adult fly brain. Different types of PNs possess their own axon and dendrite projection patterns, and three types of PNs (DL1, D and VA1lm) are shown in the drawings. (b) A neuroblast (Nb) of an adPN lineage rests at the post-embryonic stage, resumes its proliferation at the second-instar larval stage, and stops depositing PNs around the puparium formation. About fifteen different types of PNs are produced within a relatively short period in the adPN lineage as compared to fewer types of MB neurons produced in the longer MB lineage (shown in Figure 1). www.sciencedirect.com
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neuronal lineages sheds light on the different modalities providing for temporal identity specification in the developing nervous system. Protein concentrations of Chinmo as novel temporal codes MARCM allows one to label single neurons and at the same time learn the developmental history (e.g. lineage and temporal origins) of the labeled cells (see Box 1). If desired, MARCM permits manipulation of gene functions (by loss of heterozygosity or induction of dominant transgenes) selectively in the labeled neurons [33,34]. In fact, MARCM analysis of lethal mutations in the MB lineages has allowed us to identify genes, including usp [35], Dscam [15], babo [36], dSmad2 [36], cul3 [37], ph [38] and chinmo [18], that control various aspects of neural development. Among them, the Chinmo BTB-zinc finger nuclear protein was recovered as a master regulator for MB neuronal temporal identity at the larval stages. Interestingly, levels of Chinmo exist in a gradient through larval MB neurogenesis (high to low through time) and the levels of Chinmo in newly derived post-mitotic neurons dictate their temporal cell fates [18] (Figure 3). Briefly, early larval-born MB neurons contain
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more Chinmo than their later-derived siblings during the initial development of individual cells; and a reduction or an increase in the expression of Chinmo can accelerate or delay neuronal temporal cell fate transitions. Taken together, these observations suggest concentration of Chinmo might act as a novel temporal code in the combinatorial specification of neuronal cell fates. As revealed by immunostaining, Chinmo broadly exists and gradually decreases through larval development of the Drosophila CNS [18]. It has the potential for being a universal determinant of neuronal temporal identity among larval-derived post-mitotic neurons in the way that Hb, Kr, Pdm and Cas confer temporal identity in diverse embryonic neuronal lineages. However, distinct neuron types arise in different temporal patterns in different post-embryonic neuronal lineages, as illustrated above. In the adPN lineage, although the first-larval-born adPN does require Chinmo for proper temporal cell fate specification [18], it remains unclear whether Chinmo also exits in a gradient in the developing adPN lineage and whether a similar gradient alone is sufficient to specify all the distinct adPN neurons. It is possible that distinct, perhaps multiple, gradients could exist in the lineages that use
Figure 3. A model for formation of the Chinmo gradient and an illustration of the precocious shift in neuronal temporal identity upon loss of chinmo. (a) There is no detectable gradient in the transcription of chinmo as reflected by abundant chinmo mRNA in precursor cells. However, a gradual reduction in the Chinmo expression level is observed among sequentially deposited post-mitotic neurons, suggesting a post-transcriptional control over the production of Chinmo. An unidentified negative (or positive) post-transcriptional regulator with a gradually increasing (or decreasing) influence might govern the production of the Chinmo gradient in post-mitotic neurons. (b) A hypothetical neuronal lineage is used to illustrate how the Chinmo gradient controls the production of sequentially derived neurons (A to D from early-born to lateborn). When chinmo is knocked out of early-born neurons in single-cell MARCM analyses (green circle cells), they adopt the fate of late-born neurons. Abbreviation: wt, wild type. www.sciencedirect.com
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different temporal strategies in their orderly derivation of multiple neuron types. Alternatively, an analogous gradient could have different consequences in different lineages. Furthermore, multiple mechanisms might act independently and/or in concert to simultaneously and/or sequentially govern neuronal temporal identity. For example, a non-Chinmo mechanism is apparently required for proper specification of distinct subtypes of MB a/b neurons and gradients of Chinmo might operate collaboratively with additional mechanisms to increase neuronal diversity in the adPN lineage. Given that levels of Chinmo dictate neuronal temporal identity, the elucidation of how the high to low gradient of Chinmo is established through development of a neuronal lineage is an important task for our understanding the origins of neuronal temporal identity. Several interesting observations have been made [18]. First, locating endogenous Chinmo proteins versus chinmo transcripts revealed that the gradient expression of chinmo is only evident at the protein level (Figure 3). This implies involvement of post-transcriptional regulation in the establishment of gradients of the Chinmo protein. Second, transgenic chinmo with the endogenous 50 UTR faithfully recapitulated the high to low Chinmo gradient in both wild-type and chinmo mutant MB Nb clones. By contrast, chinmo transcripts lacking the 50 UTR were constitutively translated, resulting in high Chinmo expression throughout MB lineage development. These observations indicate that a 50 UTR-dependent translational control underlies the initial specification of MB neuronal temporal cell fates. Although several Caenorhabditis elegans heterochronic mutants carry mutations in miRNA genes [39–41], it is uncommon for miRNAs to suppress gene expression through 50 UTR [42]. In fact, no putative miRNA target sequences could be identified in the chinmo 50 UTR and abolition of miRNA or siRNA processing did not affect MB neuronal temporal cell fate specification (ChihFei Kao and Tzumin Lee, unpublished results). Given that 50 UTR-dependent translational control often involves RNA-binding proteins [43,44], it is more likely that such neuronal temporal cell fates governed by a Chinmo gradient are originally specified by differential expression of some RNAbinding proteins (Figure 3). Identifying additional temporal codes and elucidating how they are derived promise to unravel how distinct lineages generate multiple neuron types in different temporal patterns. New possibilities and further challenges Single-cell analysis of diverse Drosophila neuronal lineages has clearly revealed the broad involvement of birth order- or birth time-dependent cell fate determinants in the derivation of multiple neuron types from a common progenitor. This lays a solid ground for elucidating various modalities used in the Drosophila nervous system for temporal cell fate specification. However, identifying temporal codes and determining their origins remain challenging especially in non-MB neuronal lineages. For instance, in lineages that quickly produce different types of cells, it is impossible to assure any temporal cell fate transformation in a given mutant single-cell clone, because in current MARCM one cannot determine exactly which www.sciencedirect.com
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neuronal precursor has undergone mitotic recombination to give rise to a particular MARCM-labeled neuron. Newer, more sophisticated MARCM techniques that incorporate finer temporal control over the induction of MARCM clones or methodology for identifying when mitotic recombination has occurred are needed for detecting subtle neuronal temporal identity phenotypes in fast-tempo neuronal lineages. In addition, most temporal codes likely exist transiently during the initial specification of neuronal cell fates. To locate them and determine how they are generated, we must better characterize neuronal precursors. Though some progress has been made along this line [45], it remains extremely challenging to identify specific neuron progenitors and their immediate derivatives throughout development of the Drosophila brain (except the MB lineages). Systematic efforts should be made to study the development of the Drosophila brain at the level of individual cells so that we can elucidate the diverse strategies used in the Drosophila nervous system for temporal cell fate specification. We hope that the lessons learned from the highly complex Drosophila brain will facilitate the study of vastly more complex brains, ultimately leading us to a better understanding of how multitudes of neuron types derive from a limited number of neural progenitors in higher organisms. Acknowledgements We would like to thank C.T. Zugates and members in the Lee laboratory for helpful discussions regarding the manuscript. The work of the authors is supported by the National Institutes of Health and March of Dimes Birth Defects Foundation.
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