The Hunchback temporal transcription factor establishes, but is not required to maintain, early-born neuronal identity
© The Author(s). 2017
Received: 11 January 2017
Accepted: 26 January 2017
Published: 31 January 2017
Drosophila and mammalian neural progenitors typically generate a diverse family of neurons in a stereotyped order. Neuronal diversity can be generated by the sequential expression of temporal transcription factors. In Drosophila, neural progenitors (neuroblasts) sequentially express the temporal transcription factors Hunchback (Hb), Kruppel, Pdm, and Castor. Hb is necessary and sufficient to specify early-born neuronal identity in multiple lineages, and is maintained in the post-mitotic neurons produced during each neuroblast expression window. Surprisingly, nothing is currently known about whether Hb acts in neuroblasts or post-mitotic neurons (or both) to specify first-born neuronal identity.
Here we selectively remove Hb from post-mitotic neurons, and assay the well-characterized NB7-1 and NB1-1 lineages for defects in neuronal identity and function.
We find that loss of Hb from embryonic and larval post-mitotic neurons does not affect neuronal identity. Furthermore, removing Hb from post-mitotic neurons throughout the entire CNS has no effect on larval locomotor velocity, a sensitive assay for motor neuron and pre-motor neuron function.
We conclude that Hb functions in progenitors (neuroblasts/GMCs) to establish heritable neuronal identity that is maintained by a Hb-independent mechanism.
We suggest that Hb acts in neuroblasts to establish an epigenetic state that is permanently maintained in early-born neurons.
Development of the central nervous system (CNS) is multi-step process. In both mammals and Drosophila, the earliest steps are spatial patterning to define a neuroectodermal territory, followed by more precise spatial patterning to generate distinct progenitor domains (reviewed in [23, 43]). Subsequently, both mammals and Drosophila progenitors can sequentially express temporal transcription factors that specify neural identity based on birth-order (mouse: [2, 14, 32]) (fly: [3–7, 9, 18, 22, 24, 25, 34, 35, 37, 46]). The integration of spatial and temporal cues results in the production of a specific neuronal identity (reviewed in [1, 15, 38, 40]). Lastly, terminal selector genes regulate gene expression conferring distinct neural subtypes (e.g. dopaminergic neurons or cholinergic neurons); the defining feature of terminal selector genes is that their expression is maintained for the life of the neuron where it maintains the functional properties of the neuron (reviewed in [1, 21]).
Here we determine the role of the Hb temporal transcription factor in post-mitotic neurons of the well-characterized NB7-1 lineage [12, 17, 18, 22, 25, 28, 29, 33, 35, 37], with additional analysis of the role of Hb in post-mitotic neurons of the NB1-1 lineage . The NB7-1 lineage is shown in Fig. 1a: it produces five GMCs that each express the homeodomain protein Even-skipped (Eve) just before they divide to produce the Eve+ U1-U5 motor neurons and their Eve- sibling interneurons [12, 37]. U1 maintains high Hb protein levels and represses Zfh2 expression; U2 maintains lower Hb protein levels and has low Zfh2 levels; U3-U5 have no Hb protein and have high Zfh2 levels . Similar Eve, Hb, and Zfh2 expression patterns are observed for the NB1-1 and NB4-2 lineages . Thus, Zfh2 expression can be used to distinguish first-born from later-born neurons in multiple neuroblast lineages. In addition, the first-born neurons in all three lineages have axon projections to dorsal body wall muscles, whereas later-born neurons project to more ventral muscles or are interneurons [22, 30].
The role of Hb in the NB7-1, NB1-1, and NB4-2 lineages has been well characterized. Loss of Hb throughout these lineages leads to failure to produce Eve+ early-born neurons (NB7-1) or failure to repress the late-born marker Zfh2 (NB1-1 and NB4-2). Conversely, misexpression of Hb throughout these lineages leads to ectopic Zfh2- neurons with axons targeting dorsal muscles similar to endogenous first-born neurons [22, 37]. Thus, Hb represses Zfh2 and specifies early-born neuronal identity in multiple neuroblast lineages. Hb is expressed in neuroblasts, GMCs, and neurons – which of these cell types requires Hb to establish or maintain early-born neuronal identity? Hb could be required in all cells (Fig. 1b, i); Hb could be required only in post-mitotic neurons (its transient expression in neuroblasts could be simply a mechanism to restrict its expression to early-born neurons; Fig. 1b, ii) or Hb could be required only in neuroblasts/GMCs (Fig. 1b, iii). Here we use cell type specific hb RNAi to selectively remove Hb from post-mitotic neurons, and show that Hb in neuroblasts/GMCs is sufficient for stable and long-lasting early-born neuronal identity (even when the neurons lack Hb protein). This is the first analysis of any Drosophila temporal transcription factor function in post-mitotic neurons, and it shows that the Hb temporal transcription factor functions differently from terminal selector genes, which are required to maintain post-mitotic neuronal properties (reviewed in [1, 21]).
en-gal4 – Bloomington stock 1973. Expressed in row 6/7 neuroectoderm and neuroblasts (A. Brand and K. Yoffe, unpublished) .
CQ2-gal4 (II) – Bloomington stock 7468. Expressed just prior to U1 birth.
elav-gal4 – Bloomington stock 8760. Expressed all neuroblasts and neurons.
UAS-hb RNAi – Bloomington stock 34704. hunchback RNAi transgene.
yellow white (yw) – Bloomington stock 6598. Control for cell fate assays.
UAS-mCherry RNAi – Bloomington stock 35785. Control for behavior.
hb P1 , hb FB /TM3 ftzlacZ – used to specifically remove hb CNS expression; hb P1 is a transgene that recapitulates blastoderm hb expression; hb FB is a hb null allele .
Immunostaining, Imaging, and Figure preparation
Antibody staining was performed according to standard methods . Primary antibodies, dilutions and sources were: chicken anti-GFP 1:500 (Aves Labs, Inc., Tigard, OR USA); rabbit anti-Hb 1:200 for embryos, 1:400 for larvae ; mouse anti-Even-skipped (3C10-c for embryos with 1:50, 2B8 for larvae with 1:75) and mouse anti-Engrailed (4D9) 1:5 (Developmental Studies Hybridoma Bank, University of Iowa, IA, USA); rat anti-Zfh2 1:500 . Donkey anti-chicken Alexa Fluor 488-, donkey anti-rat Alexa Fluor 488-, donkey anti-rabbit Alexa Fluor 488-, donkey anti-mouse Alexa Fluor 647-, and donkey anti-rabbit Alexa Fluor 647-conjugated secondary antibodies were from Jackson ImmunoResearch (West Grove, PA USA). Goat anti-rabbit Alexa Fluor 555-, goat anti-mouse Alexa Fluor 555- and goat anti-rat Alexa Fluor 555-conjugated secondary antibodies were from Invitrogen (Eugene, OR USA). Confocal image stacks were collected using Zeiss LSM 700 confocal microscope, processed using ImageJ (NIH) and Photoshop (Adobe Systems Inc., Mountain View, CA USA); in some cases images were brightened using linear gain adjustment using the Levels command in Photoshop; when used, the entire panel was processed identically. Figures were assembled in Illustrator (Adobe Systems Inc., Mountain View, CA USA).
Larval locomotor assays
Bright-field whole larval behavioral recordings were done using newly hatched first instar larvae. Behavior arenas were made of 6% agar in grape juice, 2 mm thick and 5.5 cm in diameter. The arenas were placed under a Leica S8APO dissecting microscope and red light (700 nm, Metaphase Technologies) illuminated a single larva. The microscope was equipped with a Scion1394 monochrome CCD Camera, using Scion VisiCapture software in ImageJ. Larva were tracked using FIM Tracker software . For each larva, speed was calculated by dividing total distance traveled by the larval centroid by the time elapsed during the recording period.
Loss of Hunchback from both neuroblast and neurons eliminates early-born neuronal identity
We next determined whether en-gal4 UAS-hb RNAi could mimic the hb mutant phenotype in the NB7-1 lineage. In wild type stage 16 embryos, the Eve+ U1 motor neuron is Hb+ Zfh2- due to Hb repression of zfh2 (Fig. 2c, quantified in 2e). In en-gal4 UAS-hb RNAi stage 16 embryos, the Eve+ U1 motor neuron lacks all detectable Hb protein and has de-repressed Zfh2 (Fig. 2d, quantified in 2e). This is a slightly weaker phenotype than genetic ablation of Hb expression in the NB7-1 lineage , perhaps due to the low levels of Hb in the neuroblast (see Discussion). We examined earlier stage embryos to determine when Hb was lost from the U1 neuron, and found that Hb is undetectable from the time of U1 birth at stage 11, although it takes several hours for Zfh2 to become de-repressed (Fig. 2e). We conclude that hb RNAi is capable of removing some Hb protein from NB7-1 and all detectable Hb protein from the U1 neuron, resulting in abnormal U1 neuronal identity.
Loss of Hunchback from neurons does not alter early-born neuronal identity
To determine how long CQ2-gal4 UAS-hb RNAi can keep Hb levels off in the U1 motor neuron, and how long Zfh2 remains repressed in the absence of Hb, we examined CQ2-gal4 UAS-hb RNAi animals at the second larval instar stage. In wild type larvae, the Eve+ U1 neuron is Hb+ Zfh2- (Fig. 3c, quantified in 3e). In CQ2-gal4 UAS-hb RNAi larvae, the Eve+ U1 neuron is Hb- yet Zfh2 is still repressed (Fig. 3d, quantified in 3e). Thus, even prolonged loss of Hb does not lead to zfh2 de-repression.
Loss of Hunchback from neurons only does not alter early-born neuronal morphology
To determine if loss of Hb from the post-mitotic U1 neuron leads to alteration in neuronal morphology, we used CQ2-gal4 to drive expression of UAS-hb RNAi (to remove Hb protein) and UAS-myristoylated (myr):GFP (to reveal U1-U5 neuronal morphology). Although myr:GFP was expressed in most or all U1-U5 motor neurons, only the U1 motor neuron projects to the most dorsal muscles of the body wall [22, 30], which allows us to detect any defects in U1 morphology.
Loss of Hunchback from U1/U2 neurons or all neurons does not alter larval locomotor velocity
Here we show that the temporal transcription factor Hb, despite being continuously expressed in the U1 motor neuron, is not required to maintain U1 neuronal identity. We conclude that Hb acts transiently in the neuroblast, GMC, or new-born neuron to establish the U1 neuronal identity, and that this identity is subsequently maintained by a Hb-independent mechanism. Our conclusion is also supported by the observation that Hb, like many temporal transcription factors, are re-used in other cell types or tissues to specify different cell fates, showing that cellular context shapes the response to Hb. It is likely that progenitors and post-mitotic neurons provide different contexts for Hb action; the role of Hb in early-born post-mitotic neurons has yet to be defined. For example, in the neuroblast/GMC progenitors, Hb confers temporal identity , in the early embryo Hb specifies anterior-posterior identity , and in adult male neurons Hb confers male-specific morphology . Similar findings are observed for other embryonic temporal transcription factors such as Kr, Pdm, and Castor [10, 22, 42, 44, 46].
Interestingly, our attempts to remove Hb from the entire NB7-1 lineage using en-gal4 UAS-hbRNAi resulted in residual Hb protein in NB7-1 and a weaker phenotype than complete genetic removal of Hb from the NB7-1 lineage . For example, both hb RNAi and hb null mutants resulted in U1 motor neurons that de-repressed zfh2 (; Fig. 2) but only genetic hb null mutants result in absence of early-born Eve+ neurons (; Fig. 2). This suggests that the Hb protein present in the NB7-1 following hb RNAi is sufficient to produce long-lasting expression of Eve in the U1 motor neuron.
We conclude that Hb has no detectable function in post-mitotic U1 neurons. Might this lack of phenotype be due to low levels of residual Hb protein in neurons? Although we can't formally rule this out, there are several reasons to discount this possibility. First, we stain for Hb protein and find most U1 neurons have no detectable Hb protein compared to background. Second, RNAi knockdown of Hb in neuroblasts produces a strong phenotype which would not be expected if very low levels of Hb are functional. Finally, we don't expect Hb protein to persist following loss of hb RNA, as we have previously shown that Hb protein in the CNS has a very short half life. These experiments co-stained neuroblasts and their progeny for Hb protein and active hb transcription (nuclear intron signal), and found that few or no cells had Hb protein but not hb transcription .
Our conclusion that Hb has no function in post-mitotic neurons is buttressed by our previous findings that late-born Hb-negative neurons are unaffected by forced Hb misexpression [12, 37]. We hypothesize that temporal transcription factors alter the epigenetic state of neuroblasts which is inherited by their progeny neurons. Thus, early-born neurons do not need Hb to maintain early-born identity, and are also unresponsive to forced expression of other temporal transcription factors; similarly, late-born neurons are unresponsive to forced expression of early temporal transcription factors [12, 37]. This model is supported by findings that Hb acts transiently at the cellular blastoderm stage together with the chromatin remodeler Mi-2 to permanently silence the Ubx gene . It is also supported by the observation that some temporal transcription factors are only transiently expressed in progenitors and new-born neurons, such as Pdm in embryonic lineages [18, 22] or Eyeless, Sloppy paired, Dichaete, and Tailless in larval optic lobe lineages . In these cases, the temporal transcription factor must act transiently in the neuroblast or GMC to confer long-lasting neuronal identity. Our findings raise the possibility that all temporal transcription factors are required transiently in progenitors to specify permanent temporal identity, despite many of these factors being maintained in post-mitotic neurons. If our findings can be extended to other temporal transcription factors, it would highlight the differences between spatial or temporal patterning genes (required transiently in progenitors) and terminal selector genes (required permanently in post-mitotic neurons). It would also highlight the importance of properly linking spatial/temporal patterning to terminal selector gene expression, an important area for future investigation.
We can’t rule out the possibility that Hb is required in post-mitotic neurons for aspects of neuronal function that we did not assay. In fact, post-embryonic expression of Hb is required for proper Fruitless + male neurons morphogenesis; following hb RNAi these neurons are transformed to a female-like morphology . Although we did not detect striking axon or dendrite changes in the U neurons following hb RNAi, we did observe a slight decrease in neuronal projections in connectives (Fig. 5). Although Hb is not required to maintain dorsal axon projections in embryonic or larval U1 motor neurons, but it may be required for proper ion channel or neurotransmitter production. Furthermore, mammalian post-mitotic neurons can be reprogrammed to another neuronal identity for a short time after their birth . Temporal transcription factors like Hb may stabilize neuronal identity to prevent such transformations; in this case, loss of neuronal Hb would only show a strong phenotype upon misexpression of a “reprogramming factor,” such as a later temporal transcription factor or a terminal selector gene for a different neural subtype.
Perhaps the strongest evidence we have against a Hb function in post-mitotic neurons is our finding that elimination of Hb protein from all post-mitotic neurons (elav-gal4 UAS-hb RNAi , Fig. 6) has no larval locomotor phenotype. Similar experiments driving pan-neuronal expression of neuronal silencers or activators leads to larval paralysis [11, 13, 45]. Thus, it is highly unlikely that loss of Hb alters early-born interneuron or motor neuron neurotransmitter phenotypes or membrane properties. In the future, it would be interesting to use transcriptional profiling to compare Hb+ and Hb- early-born neurons– our results suggest that there would be little transcriptional effect from removing Hb from post-mitotic neurons.
We conclude that the Hb functions in progenitors (neuroblasts/GMCs) to establish heritable neuronal identity that is maintained by a Hb-independent mechanism.
We suggest that Hb acts in neuroblasts to establish an epigenetic state that is permanently maintained in early-born neurons.
Central nervous system
We thank Sen-Lin Lai for comments on the manuscript, and the Bloomington stock center for fly stocks.
This work was funded by NIH grant HD27056 to C.Q.D., who is an Investigator of the Howard Hughes Medical Institute.
Availability of data and materials
All reagents are available upon request.
All experiments except those in Figs. 4 and 6 were done by KH, those in Fig. 4 were done by MK, those in Fig. 6 were done by MQC and ESH, and the manuscript was written by CQD All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
All authors consent to publication and have approved the manuscript.
Ethics approval and consent to participate
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- Allan DW, Thor S. Transcriptional selectors, masters, and combinatorial codes: regulatory principles of neural subtype specification. Wiley Interdiscip Rev Dev Biol. 2015;4:505–28.View ArticlePubMedPubMed CentralGoogle Scholar
- Alsio JM, Tarchini B, Cayouette M, Livesey FJ. Ikaros promotes early-born neuronal fates in the cerebral cortex. Proc Natl Acad Sci U S A. 2013;110:E716–25.View ArticlePubMedPubMed CentralGoogle Scholar
- Baumgardt M, Karlsson D, Salmani BY, Bivik C, MacDonald RB, Gunnar E, Thor S. Global programmed switch in neural daughter cell proliferation mode triggered by a temporal gene cascade. Dev Cell. 2014;30:192–208.View ArticlePubMedGoogle Scholar
- Baumgardt M, Karlsson D, Terriente J, Diaz-Benjumea FJ, Thor S. Neuronal subtype specification within a lineage by opposing temporal feed-forward loops. Cell. 2009;139:969–82.View ArticlePubMedGoogle Scholar
- Baumgardt M, Miguel-Aliaga I, Karlsson D, Ekman H, Thor S. Specification of neuronal identities by feedforward combinatorial coding. PLoS Biol. 2007;5, e37.View ArticlePubMedPubMed CentralGoogle Scholar
- Benito-Sipos J, Estacio-Gomez A, Moris-Sanz M, Baumgardt M, Thor S, Diaz-Benjumea FJ. A genetic cascade involving klumpfuss, nab and castor specifies the abdominal leucokinergic neurons in the Drosophila CNS. Development. 2010;137:3327–36.View ArticlePubMedGoogle Scholar
- Benito-Sipos J, Ulvklo C, Gabilondo H, Baumgardt M, Angel A, Torroja L, Thor S. Seven up acts as a temporal factor during two different stages of neuroblast 5–6 development. Development. 2011;138:5311–20.View ArticlePubMedGoogle Scholar
- Berger C, Renner S, Luer K, Technau GM. The commonly used marker ELAV is transiently expressed in neuroblasts and glial cells in the Drosophila embryonic CNS. Dev Dyn. 2007;236:3562–8.View ArticlePubMedGoogle Scholar
- Brody T, Odenwald WF. Programmed transformations in neuroblast gene expression during Drosophila CNS lineage development. Dev Biol. 2000;226:34–44.View ArticlePubMedGoogle Scholar
- Chang YC, Jang AC, Lin CH, Montell DJ. Castor is required for Hedgehog-dependent cell-fate specification and follicle stem cell maintenance in Drosophila oogenesis. Proc Natl Acad Sci U S A. 2013;110:E1734–42.View ArticlePubMedPubMed CentralGoogle Scholar
- Clark MQ, McCumsey SJ, Lopez-Darwin S, Heckscher ES, Doe CQ. Functional Genetic Screen to Identify Interneurons Governing Behaviorally Distinct Aspects of Drosophila Larval Motor Programs. G3 (Bethesda). 2016;6:2023–31.View ArticleGoogle Scholar
- Cleary MD, Doe CQ. Regulation of neuroblast competence: multiple temporal identity factors specify distinct neuronal fates within a single early competence window. Genes Dev. 2006;20:429–34.View ArticlePubMedPubMed CentralGoogle Scholar
- Crisp SJ, Evers JF, Bate M. Endogenous patterns of activity are required for the maturation of a motor network. J Neurosci. 2011;31:10445–50.View ArticlePubMedPubMed CentralGoogle Scholar
- Elliott J, Jolicoeur C, Ramamurthy V, Cayouette M. Ikaros confers early temporal competence to mouse retinal progenitor cells. Neuron. 2008;60:26–39.View ArticlePubMedGoogle Scholar
- Erclik T, Li X, Courgeon M, Bertet C, Chen Z, Baumert R, Ng J, Koo C, Arain U, Behnia R, Rodriguez AD, Senderowicz L, Negre N, White KP, Desplan C. Integration of temporal and spatial patterning generates neural diversity. Nature. 2017;541:365–70.View ArticlePubMedGoogle Scholar
- Goto J, Mikawa Y, Koganezawa M, Ito H, Yamamoto D. Sexually dimorphic shaping of interneuron dendrites involves the hunchback transcription factor. J Neurosci. 2011;31:5454–9.View ArticlePubMedGoogle Scholar
- Grosskortenhaus R, Pearson BJ, Marusich A, Doe CQ. Regulation of temporal identity transitions in Drosophila neuroblasts. Dev Cell. 2005;8:193–202.View ArticlePubMedGoogle Scholar
- Grosskortenhaus R, Robinson KJ, Doe CQ. Pdm and Castor specify late-born motor neuron identity in the NB7-1 lineage. Genes Dev. 2006;20:2618–27.View ArticlePubMedPubMed CentralGoogle Scholar
- Heckscher ES, Lockery SR, Doe CQ. Characterization of Drosophila larval crawling at the level of organism, segment, and somatic body wall musculature. J Neurosci. 2012;32:12460–71.View ArticlePubMedPubMed CentralGoogle Scholar
- Heckscher ES, Zarin AA, Faumont S, Clark MQ, Manning L, Fushiki A, Schneider-Mizell CM, Fetter RD, Truman JW, Zwart MF, Landgraf M, Cardona A, Lockery SR, Doe CQ. Even-skipped(+) interneurons are core components of a sensorimotor circuit that maintains left-right symmetric muscle contraction amplitude. Neuron. 2015;88:314–29.View ArticlePubMedPubMed CentralGoogle Scholar
- Hobert O. Terminal selectors of neuronal identity. Curr Top Dev Biol. 2016;116:455–75.View ArticlePubMedGoogle Scholar
- Isshiki T, Pearson B, Holbrook S, Doe CQ. Drosophila neuroblasts sequentially express transcription factors which specify the temporal identity of their neuronal progeny. Cell. 2001;106:511–21.View ArticlePubMedGoogle Scholar
- Jessell TM. Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nature reviews. Genetics. 2000;1:20–9.PubMedGoogle Scholar
- Kambadur R, Koizumi K, Stivers C, Nagle J, Poole SJ, Odenwald WF. Regulation of POU genes by castor and hunchback establishes layered compartments in the Drosophila CNS. Genes Dev. 1998;12:246–60.View ArticlePubMedPubMed CentralGoogle Scholar
- Kanai MI, Okabe M, Hiromi Y. Seven-up controls switching of transcription factors that specify temporal identities of Drosophila neuroblasts. Dev Cell. 2005;8:203–13.View ArticlePubMedGoogle Scholar
- Karlsson D, Baumgardt M, Thor S. Segment-specific neuronal subtype specification by the integration of anteroposterior and temporal cues. PLoS Biol. 2010;8, e1000368.View ArticlePubMedPubMed CentralGoogle Scholar
- Kehle J, Beuchle D, Treuheit S, Christen B, Kennison JA, Bienz M, Muller J. dMi-2, a hunchback-interacting protein that functions in polycomb repression. Science. 1998;282:1897–900.View ArticlePubMedGoogle Scholar
- Kohwi M, Hiebert LS, Doe CQ. The pipsqueak-domain proteins Distal antenna and Distal antenna-related restrict Hunchback neuroblast expression and early-born neuronal identity. Development. 2011;138:1727–35.View ArticlePubMedPubMed CentralGoogle Scholar
- Kohwi M, Lupton JR, Lai SL, Miller MR, Doe CQ. Developmentally regulated subnuclear genome reorganization restricts neural progenitor competence in Drosophila. Cell. 2013;152:97–108.View ArticlePubMedPubMed CentralGoogle Scholar
- Landgraf M, Bossing T, Technau GM, Bate M. The origin, location, and projections of the embryonic abdominal motorneurons of Drosophila. J Neurosci. 1997;17:9642–55.PubMedGoogle Scholar
- Li X, Erclik T, Bertet C, Chen Z, Voutev R, Venkatesh S, Morante J, Celik A, Desplan C. Temporal patterning of Drosophila medulla neuroblasts controls neural fates. 2013. Nature.Google Scholar
- Mattar P, Ericson J, Blackshaw S, Cayouette M. A conserved regulatory logic controls temporal identity in mouse neural progenitors. Neuron. 2015;85:497–504.View ArticlePubMedGoogle Scholar
- Mettler U, Vogler G, Urban J. Timing of identity: spatiotemporal regulation of hunchback in neuroblast lineages of Drosophila by Seven-up and Prospero. Development. 2006;133:429–37.View ArticlePubMedGoogle Scholar
- Moris-Sanz M, Estacio-Gomez A, Alvarez-Rivero J, Diaz-Benjumea FJ. Specification of neuronal subtypes by different levels of Hunchback. Development. 2014;141:4366–74.View ArticlePubMedGoogle Scholar
- Novotny T, Eiselt R, Urban J. Hunchback is required for the specification of the early sublineage of neuroblast 7–3 in the Drosophila central nervous system. Development. 2002;129:1027–36.PubMedGoogle Scholar
- Nusslein-Volhard C, Wieschaus E. Mutations affecting segment number and polarity in Drosophila. Nature. 1980;287:795–801.View ArticlePubMedGoogle Scholar
- Pearson BJ, Doe CQ. Regulation of neuroblast competence in Drosophila. Nature. 2003;425:624–8.View ArticlePubMedGoogle Scholar
- Pearson BJ, Doe CQ. Specification of temporal identity in the developing nervous system. Annu Rev Cell Dev Biol. 2004;20:619–47.View ArticlePubMedGoogle Scholar
- Risse B, Thomas S, Otto N, Lopmeier T, Valkov D, Jiang X, Klambt C. FIM, a novel FTIR-based imaging method for high throughput locomotion analysis. PLoS One. 2013;8, e53963.View ArticlePubMedPubMed CentralGoogle Scholar
- Rossi AM, Fernandes VM, Desplan C. Timing temporal transitions during brain development. Curr Opin Neurobiol. 2016;42:84–92.View ArticlePubMedGoogle Scholar
- Rouaux C, Arlotta P. Direct lineage reprogramming of post-mitotic callosal neurons into corticofugal neurons in vivo. Nat Cell Biol. 2013;15:214–21.View ArticlePubMedPubMed CentralGoogle Scholar
- Ruiz-Gomez M, Romani S, Hartmann C, Jackle H, Bate M. Specific muscle identities are regulated by Kruppel during Drosophila embryogenesis. Development. 1997;124:3407–14.PubMedGoogle Scholar
- Skeath JB, Thor S. Genetic control of Drosophila nerve cord development. Curr Opin Neurobiol. 2003;13:8–15.View ArticlePubMedGoogle Scholar
- Stratmann J, Gabilondo H, Benito-Sipos J, Thor S. Neuronal cell fate diversification controlled by sub-temporal action of Kruppel. eLife. 2016;5.
- Suster ML, Bate M. Embryonic assembly of a central pattern generator without sensory input. Nature. 2002;416:174–8.View ArticlePubMedGoogle Scholar
- Tran KD, Doe CQ. Pdm and Castor close successive temporal identity windows in the NB3-1 lineage. Development. 2008;135:3491–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Tran KD, Miller MR, Doe CQ. Recombineering Hunchback identifies two conserved domains required to maintain neuroblast competence and specify early-born neuronal identity. Development. 2010;137:1421–30.View ArticlePubMedPubMed CentralGoogle Scholar