Drosophila intermediate neural progenitors produce lineage-dependent related series of diverse neurons

Abstract

Drosophila type II neuroblasts (NBs), like mammalian neural stem cells, deposit neurons through intermediate neural progenitors (INPs) that can each produce a series of neurons. Both type II NBs and INPs exhibit age-dependent expression of various transcription factors, potentially specifying an array of diverse neurons by combinatorial temporal patterning. Not knowing which mature neurons are made by specific INPs, however, conceals the actual variety of neuron types and limits further molecular studies. Here we mapped neurons derived from specific type II NB lineages and found that sibling INPs produced a morphologically similar but temporally regulated series of distinct neuron types. This suggests a common fate diversification program operating within each INP that is modulated by NB age to generate slightly different sets of diverse neurons based on the INP birth order. Analogous mechanisms might underlie the expansion of neuron diversity via INPs in mammalian brain.

Keywords: Adult brain; Cell lineage analysis; Drosophila type II neuroblasts; Intermediate neural progenitors; Neuronal cell fate; Temporal identity.

Fig. 1.

Targeting Drosophila type II NBs for twin-spot clonal labeling. (A) Schematic illustration of the serial recombinase-dependent transgene activations via excision of the corresponding stop cassettes, enabling a pan-neuronal LexA::P65 driver specifically in the eight type II NB lineages (blue) due to stg14-triggered dpn-dependent Cre activity in the type II NBs (orange). (B-F) stg14-GAL4-dependent activation of dpn>stop>LexA::P65 allows selective labeling of type II NB lineages in larval brains. Note the GFP expression in various type II NB subsets across the brain lobes immunostained with anti-Dpn (red) and anti-Ase (blue). Magnified views show the Dpn-positive, Ase-negative DM1 NB (yellow dashed circle) in the left hemisphere. Note an Ase-positive INP (magenta dashed circle) lying adjacent to the DM1 NB. (G) The percentage of specific type II or all type I NB clones among the 291 NB clones recovered from 285 mosaic brains carrying the stg14^dpn lineage-restricted driver and experiencing clone induction at the first instar stage. (H-W) The twin-spot MARCM clones of type II lineages, induced shortly after larval hatching, consist of the first larval-born INP clone (green) paired with its parental NB clone (red) in nc82-counterstained adult brains (blue). The cell body numbers of the full-size INP clones are indicated. Note that the lone neuron in the DL2 INP clone selectively targets the tips of the paired mushroom body (MB) α lobes (W). W is an enlargement of the boxed area in S. Scale bars: 50 μm in B; 10 μm in C; 100 μm in H.

Fig. 2.

Stereotypy and uniqueness of INP1 sublineages. (A-F) Two examples of DM1 twin-spot MARCM clones induced at early first instar carry indistinguishable full-size INP1 clones (green) paired with the remaining DM1 lineages (red). Note that the INP1-innervated SPS domain (white arrows) receives no innervation from the post-INP1 DM1 lineage (yellow arrows). (G-L) Two examples of DM6 twin-spot MARCM clones induced at early first instar carry indistinguishable full-size INP1 clones (green) paired with the remaining DM6 lineages (red). Note that the INP1-unique EB and broad OL elaborations (white arrows) are undetectable in the post-INP1 NB clones (yellow arrows). Adult brains were counterstained with nc82 monoclonal antibody (blue). SPS, superior posterior slope; EB, ellipsoid body; OL, optic lobe. Scale bar: 100 μm.

Fig. 3.

Mapping neurons serially made by INP1 and INP2 of the DM1 lineage. (A,B) Full-size INP1 and INP2 clones of the DM1 lineage, accompanied by DM1 NB clones (not shown), exhibit common (white arrows) as well as unique (yellow arrows) domains of neurite elaborations. (C-O) Identification of INP subclones derived from DM1 INP1 or INP2 as judged from the presence of INP1- or INP2-characteristic neurite elaborations. The serially derived GMC clones (red) were paired with INP subclones of decreasing size (green), as schematized on the left. The offspring of GMC1 (red, D versus E), GMC4 (red in M) and GMC5 (O) are undistinguishable between the INP1 and INP2 sublineages, leaving the sublineage origin for the three last-born neurons (M,O) undetermined. By contrast, the GMC2 and GMC3 clones of INP1 versus INP2 (red, G versus H or J versus K) elaborate differentially due to fate differences in one of the two postmitotic neurons made by a given GMC (see Fig. 4). RUB, rubus; PLP, posterior lateral protocerebrum; PVLP, posterior ventrolateral protocerebrum; VES, vest; IPS, inferior posterior slope; GOR, gorget; SEG, subesophageal ganglion; FB, fan-shaped body. The numbers of samples are indicated. Scale bars: 100 μm.

Fig. 4.

Sibling INPs produce related invariant sequences of diverse neurons. Neurons made by the serial GMCs derived from the first two INPs of the larval DM1 lineage were individually traced and registered into a preselected adult fly brain template. Insets are magnified views of selected CX neurons. Known type II NB and INP temporal factors (Bayraktar and Doe, 2013) are shown. Scale bar: 100 μm; 20 μm in insets.