Role of Notch signaling in establishing the hemilineages of secondary neurons in Drosophila melanogaster

Abstract

The secondary neurons generated in the thoracic central nervous system of Drosophila arise from a hemisegmental set of 25 neuronal stem cells, the neuroblasts (NBs). Each NB undergoes repeated asymmetric divisions to produce a series of smaller ganglion mother cells (GMCs), which typically divide once to form two daughter neurons. We find that the two daughters of the GMC consistently have distinct fates. Using both loss-of-function and gain-of-function approaches, we examined the role of Notch signaling in establishing neuronal fates within all of the thoracic secondary lineages. In all cases, the 'A' (Notch(ON)) sibling assumes one fate and the 'B' (Notch(OFF)) sibling assumes another, and this relationship holds throughout the neurogenic period, resulting in two major neuronal classes: the A and B hemilineages. Apparent monotypic lineages typically result from the death of one sibling throughout the lineage, resulting in a single, surviving hemilineage. Projection neurons are predominantly from the B hemilineages, whereas local interneurons are typically from A hemilineages. Although sibling fate is dependent on Notch signaling, it is not necessarily dependent on numb, a gene classically involved in biasing Notch activation. When Numb was removed at the start of larval neurogenesis, both A and B hemilineages were still generated, but by the start of the third larval instar, the removal of Numb resulted in all neurons assuming the A fate. The need for Numb to direct Notch signaling correlated with a decrease in NB cell cycle time and may be a means for coping with multiple sibling pairs simultaneously undergoing fate decisions.

Fig. 1.

Relationship of sibling differences to overall lineage phenotypes. (A,B) z-projection of a lineage 6 neuroblast (A) and GMC (B) clone. The primary neurites from the two siblings form the six contralateral intermediate (6ci) and six contralateral dorsal (6cd) bundles. The schematics show how the primary neurites relate to the segmental commissures and leg neuropils (LNp). (C,D) The appearance of Notch immunostaining in the nuclei of two young neurons in a lineage 1 MARCM clone. (C) A z-projection of the cell cluster. (D) An optical section showing two neurons with nuclear Notch immunostaining; insets are magnified views of the neurons in this and the adjacent section. Yellow arrows: nuclear Notch staining. Commissures: ad, anterior dorsal; pd, posterior dorsal; ai, anterior intermediate; pi, posterior intermediate; v, ventral.

Fig. 2.

Summary of the results of removing Notch or having constitutively active Notch on the phenotypes of NB MARCM clones. (A) The frequency of particular lineages (for monotypic lineages) or hemilineage bundles under the two conditions. (B) The frequency of ‘disembodied’ lineages that included the NBs, GMCs and a few young neurons, but few or no neurites emerging from the cluster. The NB was identified by the location of the cluster and the lack of its characteristic neurotactin-positive, neurite bundle. Notch null, white bars; constitutively active Notch (Notch^CA), black bars. Numbers refer to the NBs; 20/22, combined data for lineages 20 to 22; in, local leg interneurons, mn, motoneurons. Other designations are neurite bundles: c, contralateral; ci, contralateral intermediate; cd, contralateral dorsal; d, dorsal; i, ipsilateral; id, ipsilateral dorsal; ii, ipsilateral intermediate; il, ipsilateral lateral; v, ventral.

Fig. 3.

MARCM clones showing the effects of manipulating Notch signaling on lineages that produce two major classes of interneurons. Green, anti-CD8; magenta, anti-neurotactin showing the arrangement of lineage bundles. Insets are a reduced grayscale image of each clone. (A-D) Wild-type NB clones. (E-H) NB clones that are homozygous Notch null show a single neurite bundle. (I,J) Examples of Notch null, GMC clones for lineages 12 and 8, showing that both siblings have the same neurite projection. (K-N) NB clones that express Notch^CA. M and N contain lineages 8 and 12 clones and the grayscale image is shown at full magnification. Both show one axon (8c, 12c) of the other sibling phenotype. Neurite bundle names are as in Fig. 2.

Fig. 4.

Effects of Notch manipulation on monotypic lineages. Examples of MARCM clones for lineages that are monotypic (A,B) or produce a major and minor class of neuron (C,D). Green, anti-CD8; magenta, anti-neurotactin. Insets are a reduced grayscale image of each clone, except for F, which shows the neurotactin channel. (A-D) Wild-type NB clones. (E-H) NB clones that are homozygous Notch null. (E) A lineage 0 clone with the neuroblast (N) associated with a compact cell cluster containing a neurite bundle that dwindles as it enters the neuropil. (F) A lineage 2 clone consisting of the NB and a few associated cells but no emerging neurites. The neurotactin-positive bundle of its wild-type, contralateral homolog is indicated by the yellow arrowhead; this bundle is missing on the side with the clone. (G,H) NB clones showing just the rare cell-type and a slightly separated NB with a small compact ball of associated cells. (I-L) NB clones that express Notch^CA. Neurite bundle names are as in Fig. 2.

Fig. 5.

Examples of MARCM NB clones that are null for the caspase dronc. (A-E) Lineages in which one cell type is typically represented by only one or two individuals. Blocking cell death results in the addition of more neurites from the cells of the rare phenotype (red arrow). (A) Lineage 0, (B) lineage 4, (C) lineage 14, (D) two examples of lineage 21, (E) lineage 20/22. (F-L) Monotypic lineages having one neurite bundle (yellow arrow) acquire a new neuronal class (red arrow) when cell death is blocked. (F) Lineage 5, (G) lineage 15, (H) lineage 2, (I) lineage 10, (J) lineage 17, (K) lineage 24, (L) lineage 23. Wild-type examples are included in Fig. 9.

Fig. 6.

Examples of MARCM NB clones that are homozygous for a numb null mutation (numb²). (A,B) Bundles from both sibling types are evident in numb clones induced at 24 hours AEL and examined at wandering. The lineage 1 clone (A) was among clones from other lineages, but the 1i and 1c bundles are readily distinguishable. (C,D) numb clones induced at 72 hours AEL and examined at wandering showed only the A sibling in lineage 8 (D), or only a few axons of the B type (bundle 1i) and the remainder of the A type (bundle 1c) in lineage 1 (C). Neurite bundle names are as in Fig. 2.

Fig. 7.

Effects of Notch activation on NB duplication. MARCM NB clones showing that loss of numb (A-D) or constitutive Notch signaling (E) results in clusters with multiple NBs. (A) Optical section through a clone (green) showing the expression of grainy head (magenta) in the supernumerary NBs. (B,C) Examples of lineages 3 and 9, which have supernumerary NBs (insets); each NB is at the end of a discrete cell cluster (arrows) and their fasciculated neurites join at the neuropil into a common bundle that projects to their normal targets. (D,E) Examples of lineage 13 clones that are numb null or express Notch^CA, respectively. Both treatments induced supernumerary NBs (insets). With loss of numb (D), the 13c bundle was of normal size, whereas 13i was hypertrophied. With Notch^CA expression (E), bundle 13i was hypertrophied but 13c was missing. N, neuroblast.

Fig. 8.

Manipulations that cause NB duplication. Summary of the analysis of NB MARCM clones that were (A) null for numb function or had (B) constitutive Notch signaling. White bars, clones with a single NB; black bars, clones with supernumerary NBs.

Fig. 9.

Summary of the role of Notch signaling in the 25 lineages of secondary neurons in the ventral CNS. The images show the wild-type morphology of each lineage. The diagrams shows the path of the neurite bundle from each hemilineage (see Fig. 1). Green, Notch-on fate; magenta, Notch-off fate. Open cell bodies show cell types that do not appear in the wild-type clones. Ones that are known to undergo programmed cell death are designated by a cross. When only one cell is filled, the oldest few neurons in the hemilineage survive while the remainder die.