The Hunchback temporal transcription factor establishes, but is not required to maintain, early-born neuronal identity

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Fig. 1

The NB7-1 lineage, temporal transcription factors, and models for Hunchback function in specifying early-born neuronal identity. a The neuroblast 7–1 (NB7-1) sequentially expresses the temporal transcription factors Hunchback (Hb), Kruppel (Kr), Pou domain proteins Nubbin/Pdm2 (Pdm), and Castor (Cas). Each factor is maintained into the post-mitotic neurons, although Pdm neuronal expression is transient. The U1-U5 motor neurons are Eve+; all but the first-born U1 express the late-born neuronal marker Zfh2. b Models for Hb specification of early-born neuronal identity. (i) Hb may be required in the neuroblast, GMC, and post-mitotic neurons, matching its expression pattern. (ii) Hb expression in post-mitotic neurons may be sufficient to maintain their identity; in this case the transient neuroblast expression may be a mechanism to limit Hb to early-born neurons. (iii) Hb expression in neuroblasts may be sufficient to heritably specify identity of their neuronal progeny, perhaps by initiating a transcriptional cascade or creating epigenetic modifications

Fig. 2

Loss of Hunchback from neuroblasts and neurons eliminates early-born neuronal identity. a, b Using engrailed-gal4 (en-gal4) to drive UAS-hunchback RNAi (UAS-hb ^RNAi) results in reduced Hb protein levels in row 6 and row 7 neuroblasts. Scale bars, 10 μm. c Wild type (y w) stage 16 embryo, three segments shown. U1 motor neurons (circled) are Eve+ Hb+ and Zfh2-. d en-gal4 UAS-hb ^RNAi stage 16 embryo, three segments shown. U1 motor neurons (circled) are Eve+ Hb- and Zfh2+. Anterior up, ventral midline at center of each panel, scale bar, 10 μm. e Quantification

Fig. 3

Loss of Hunchback from post-mitotic neurons does not alter early-born neuronal identity. a Wild type (y w) stage 16 embryo, three segments shown. The U1 neuron is Eve+ Hb+ Zfh2-; one example is circled and enlarged in the inset. b CQ2-gal4 UAS-hb ^RNAi stage 16 embryo, four segments shown. The U1 neuron is Eve+ Hb- Zfh2-; one example is circled and enlarged in the inset. Note that loss of neuronal Hb does not result in Zfh2 de-repression. c Wild type (y w) second instar larval CNS, four segments shown. The U1 neuron is Eve+ Hb+ Zfh2-; one example is circled and enlarged in the inset. d CQ2-gal4 UAS-hb ^RNAi second instar larval CNS, four segments shown. The U1 neuron is Eve+ Hb- Zfh2-; one example is circled and enlarged in the inset. Note that loss of neuronal Hb does not result in Zfh2 de-repression. Anterior up, ventral midline at center of each panel, scale bar, 10 μm. e Quantification

Fig. 4

Transient Hunchback in neuroblasts is sufficient to specify early-born neuronal identity. a Wild type (y w) stage aCC/pCC sibling neurons (circle) are Eve+ Hb+ and Zfh2-. b hunchback CNS null mutant aCC/pCC sibling neurons (circle) are Eve+ Hb- and Zfh2+. Note that loss of Hb throughout the neuroblast lineage results in zfh2 de-repression. c hunchback CNS null mutant with sca-gal4 UAS-hb driving expression of Hb in neuroblasts. aCC/pCC sibling neurons (circle) are Eve+ Hb- and Zfh2-. The Hb protein made in the parental neuroblast is not maintained in the aCC/pCC neuronal progeny. Note that Hb expression in the neuroblast maintains zfh2 repression despite lack of Hb in the neurons. In all panels, stage 15 embryo, three segments shown, anterior to top, ventral midline at center of panel. Scale bars, 5 μm. d Quantification

Fig. 5

Loss of Hunchback from post-mitotic neurons only does not alter embryonic neuronal morphology. U1-U5 motor neuron morphology detected by CQ2-gal4 driving expression of UAS-myristoylated:GFP (green). Only the Hb+ U1 and U2 motor neurons project to the most dorsal muscles [22, 30]. a, b Wild type CQ2-gal4;UAS-myr:GFP stage 16 embryo. a The most dorsal projecting U1/U2 motor neurons extend past the tracheal dorsal trunk (dashed lines). b Dendritic projections form a thick posterior commissural fascicle (arrow) and a thin anterior commissural fascicle (arrowhead) in each segment; note faint processes in most intersegmental connectives. c, d CQ2-gal4/+; UAS-hb ^RNAi/UAS-myr:GFP stage 16 embryo. c The most dorsal projecting U1/U2 motor neurons extend past the tracheal dorsal trunk (dashed lines). No difference is seen in axon or dendrite morphology between wild type and CQ2-gal4 UAS-hb ^RNAi embryos. d Dendritic projections form a thick posterior commissural fascicle (arrowhead) and a thin anterior commissural fascicle (arrow) in each segment; note slightly reduced processes in most intersegmental connectives. Anterior up, dorsal (a,c) or ventral (b,d) midline at center of each panel, scale bar, 10 μm. e Quantification

Fig. 6

Loss of Hunchback from U1/U2 motor neurons or all neurons does not alter larval locomotor velocity. a Wild type yw stage 16 embryo stained for Hb protein. b elav-gal4 UAS-hb ^RNAi stage 16 embryo stained for Hb protein. c Loss of Hb from the U1/U2 motor neurons or all neurons does not alter larval locomotor velocity. Genotypes: U1/U2 control = CQ2-gal4 UAS-mCherry ^RNAi. U1/U2 hunchback RNAi = CQ2-gal4 UAS-hb ^RNAi. Pan-neuronal control = elav-gal4 UAS-mCherry ^RNAi. Pan-neuronal hunchback RNAi = elav-gal4 UAS-hb ^RNAi. Average speed for each genotype (vertical line) and standard deviation (horizontal line) are shown overlaid on distance/time (n is 1–2 crawls for 5 larva of each genotype) (circles)