Hierarchical deployment of factors regulating temporal fate in a diverse neuronal lineage of the Drosophila central brain

Abstract

The anterodorsal projection neuron lineage of Drosophila melanogaster produces 40 neuronal types in a stereotypic order. Here we take advantage of this complete lineage sequence to examine the role of known temporal fating factors, including Chinmo and the Hb/Kr/Pdm/Cas transcriptional cascade, within this diverse central brain lineage. Kr mutation affects the temporal fate of the neuroblast (NB) itself, causing a single fate to be skipped, whereas Chinmo null only elicits fate transformation of NB progeny without altering cell counts. Notably, Chinmo operates in two separate windows to prevent fate transformation (into the subsequent Chinmo-indenpendent fate) within each window. By contrast, Hb/Pdm/Cas play no detectable role, indicating that Kr either acts outside of the cascade identified in the ventral nerve cord or that redundancy exists at the level of fating factors. Therefore, hierarchical fating mechanisms operate within the lineage to generate neuronal diversity in an unprecedented fashion.

Figure 1. Loss of Chinmo results in the disappearance of AL innervations by eight adPN types

(A) The genetic design and clonal patterns of twin-spot MARCM. In this study, chinmo mutant clones are consistently labeled with mCD8::GFP, while their wild-type sister clones express rCD2::RFP. Shown on the right are two possible patterns of chinmo mutant clones generated by twin-spot MARCM. Note that one of adPN daughters from GMC clones die prematurely (black crosses). (B) and (C) The largest wild-type and chinmo¹ mutant adPN NB clones labeled with GAL4-GH146. Focal sections are shown from anterior to posterior of the AL (from 1 to 4). AL glomerular innervations of individual adPN types are outlined by dot lines. Arrowheads mark the missing AL glomerular innervations. The scale bar in all figures equals 10 µm. (D) Twin-spot MARCM clones generated at mid-larval stage and labeled with Acj6-GAL4. The clone was generated during birth of the last wild-type VA1lm adPN (GMC clone; axon projections shown in inset). In the associated chinmo¹ mutant NB clone, both the clone size (n=32.4±2.2; N=5) and AL glomerular innervations are comparable to wild-type controls (not shown). (E) Summary of the missing temporal fates in adPN lineage.

Figure 2. adPN temporal fate transformations in chinmo mutant GMC clones

(A–E) Selected wild-type adPNs are revealed in twin-spot MARCM GMC clones. [1]: the merged image of GMC clone (rCD2:RFP; magenta) and nc82 staining (cyan) at AL region. [2]: the axonal projections of indicated adPN type at LH. Distinct adPN types innervate different AL glomeruli and acquire distinguishable axon arborization patterns. Note that the paired chinmo mutant NB clones are not shown. Further, the development of wild-type GMC clone is not affected by the associated mutant NB clone. (F–J) chinmo mutant adPNs (green) with the prospective cell fates judged from their associated wild-type NB clones (magenta). Illustrations show temporal fate transformations as evidenced by the actual AL glomerular targets (green) versus the prospective glomerular targets (magenta). [1]: the merged image of chinmo mutant GMC clone (mCD8:GFP; green) and nc82 staining (cyan) at AL region. The innervated glomeruli are outlined by dot lines and indicated by arrowhead(s). Note partial fate transformation in the cases where one ‘uniglomerular’ PN innervated two glomeruli (F1 and G1). [2]: single focal planes of the paired wild-type NB clone that reveals the time of clone induction. Focal planes from anterior to posterior are shown. The glomerular innervations by adPNs born right after the mutant GMC clone (orange arrowheads) and the void of innervations from the mutant adPN and its preceding type (white arrowheads) are indicated. [3]: the axonal projections of chinmo^−/− adPN at LH. Note that unlike embryonic-born adPNs that are individually unique (except the two VM3-targeting adPNs), all the larval-derived adPN types consist of multiple indistinguishable neurons born contiguously. (K) Summary of chinmo-elicited temporal cell fate transformations of adPNs.

Figure 3. Selective requirement of Kr for the VA7l adPN temporal fate

(A) The mutant alleles used and phenotypes observed in largest mutant adPN NB clones are summarized in the table. (B) The largest Kr¹ mutant adPN NB clone labeled with GAL4-GH146. One focal section from anterior part of the AL shows lack of VA7l glomerular innervation. (C) Temporal fate transformation of the Kr mutant prospective VA7l adPN. The twin-spot MARCM clone was generated at the birth of prospective VA7l GMC (C1), as timed by the earliest adPN type present in the associated wild-type NB clone, VA2 adPN (C2). The axon projections at LH are shown in (C3): Kr-null prospective VA7l and (C4): wild-type VA2 adPN. (D) Summary of the VA7l-to-VA2 temporal fate transformation in the Kr mutant GMC that normally yields a VA7l adPN.

Figure 4. Kr and Chinmo govern temporal fate specification via distinct mechanisms

(A) Full-size wild-type (left) and Kr¹-null adPN NB clones (right) marked by GR40B05-GAL4 (three consecutively born embryonic adPNs are labeled: VA2, DM4, and DL5). Clones were generated in early embryos. The clone cell numbers are presented as mean±S.D (n) derived from the indicated number of samples (N). (B) Full-size wild-type (left) and chinmo¹-null adPN NB clones (right) marked by GR46E07-GAL4 (six larval-born adPN types are labeled: DL1, DA3, DC2, D, VA3, and VC3). Clones were generated shortly after larval hatching. Note no change in the total cell count despite loss of three glomerular targets in the chinmo mutant clone. (C) Temporal requirements and hypothetical model of Kr and Chinmo function in adPN neurogenesis. While only one temporal fate (VA7l fate) is affected by loss of Kr, Chinmo is required in two separate windows to support eight temporal fates. Our mosaic studies suggest Kr acts via a hypothetical transcriptional cascade (e.g. TF[n−1]/Kr/TF[n+1]) to confer the VA7l adPN fate and regulate temporal fate transition by delaying the expression of next temporal factor (TF[n+1]) in the NB as well as the Kr-positive GMC. By contrast, Chinmo operates in newborn neurons within a defined window to suppress the following temporal fate. This is possibly achieved by Chinmo working together with TF[x], defining the window of Chinmo’s action, to repress TF[x+1] activity. It may subsequently permit further diversification through a sequential activation of multiple feed-forward transcriptional networks.