A novel temporal identity window generates alternating cardinal motor neuron subtypes in a single progenitor lineage

doi:10.21203/rs.3.rs-26008/v1

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A novel temporal identity window generates alternating cardinal motor neuron subtypes in a single progenitor lineage

https://doi.org/10.21203/rs.3.rs-26008/v1

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published 28 Jul, 2020

Read the published version in Neural Development →

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Background: Spatial patterning specifies neural progenitor identity, with further diversity generated by temporal patterning within individual progenitor lineages. These mechanisms generate cardinal classes of motor neurons, sharing a transcription factor identity and common muscle group targets). In Drosophila , two cardinal classes are Even-skipped (Eve)+ motor neurons projecting to dorsal longitudinal muscles and Nkx6+ motor neurons projecting to ventral oblique muscles. The Drosophila neuroblast 7-1 (NB7-1) lineage generates distinct Eve+ motor neurons via the temporal transcription factor (TTF) cascade Hunchback (Hb)-Krüppel (Kr)-Pdm-Castor (Cas). Methods: Here we use sparse labelling and molecular markers to identify a novel VO motor neuron subtype in the NB7-1 lineage, and birth-date this neuron to a Kr+ Pdm+ temporal identity window. We selectively drive overexpression of Kr and Pdm in the NB7-1 lineage, and assay the production and axonal targeting of ectopic VO neurons. We then use gain- and loss-of-function strategies to show that the identity and targeting specificity of the VO neuron is dependent on the transcription factor Nkx6. Results: Here we show that a newly discovered Kr/Pdm temporal identity window gives rise to an Nkx6+ Eve- motor neuron projecting to ventral oblique muscles, resulting in alternation of cardinal motor neuron subtypes from a single progenitor (Eve>Nkx6>Eve). We show that co-overexpression of Kr/Pdm generates ectopic VO motor neurons within the NB7-1 lineage – the first evidence that this TTF combination specifies neuronal identity. Moreover, we show that the Kr/Pdm combination promotes Nkx6 expression, which itself is necessary and sufficient for ventral oblique muscle targeting, thereby linking temporal patterning to motor neuron synaptic target selection. Conclusions: We show that one neuroblast lineage generates interleaved cardinal motor neurons fates; that the Kr/Pdm TTFs form a novel temporal identity window that promotes expression of Nkx6; and that the Kr/Pdm>Nkx6 pathway is necessary and sufficient to specify VO motor neuron identity and morphology.

Developmental Neuroscience

temporal transcription factor

motor neuron

temporal identity

Nkx6

Eve

Figure S1. NB7-1 sequentially expresses Kr, Kr/Pdm, and Pdm.

(A-A’’) NB7-1 is labelled by GFP (green) (NB7-1-gal4 UAS-GFP). In the early stage 11 embryo NB7-1 expresses Kr (grey) and Asense (blue), but is Pdm (red) negative. This indicates that NB7-1 is in the Kr temporal identity window. Scale bar: 5 µm.

(B-B’’) NB7-1 is labelled by GFP (green) in the mid-stage 11 embryo. NB7-1 expresses Kr (grey), Pdm (red) and Asense (blue) simultaneously. This demonstrates the existence of a distinct Kr+ Pdm+ temporal identity window of the NB7-1 lineage.

(C-C’’) By late stage 11, NB7-1 (green) has entered the Pdm temporal identity window. NB7-1 expresses Pdm (red) and Asense (blue), but no Kr (grey) is detected.

Figure S2. Methodology for quantifying ISNd and SNc motor neuron localization.

(A,A') The volume of the ISNd was normalized to that of SNc to account for slight differences in embryo staging. ISNd and SNc were identified by the pan-motor axon marker FasII (magenta) in embryos expressing GFP (green) in the NB7-1 lineage (NB7-1-gal4 UAS-GFP).

(B,B') FasII (magenta) was used to identify SNc in a maximum intensity projection, and the volume quantified using the Imaris Surface function.

(C,C') FasII (magenta) was used to identify ISNd in a maximum intensity projection, and the volume quantified using the Imaris Surface function.

Figure S3. Nkx6 induces ectopic VO motor neurons targeting ventral oblique muscles.

(A) Control NB7-1 progeny (NB7-1-gal4 UAS-GFP) show precise innervation of the ISNd and ventral oblique muscles (arrowhead). Scale bar: 15 µm.

(B) NB 7-1 progeny misexpressing Kr and Pdm (NB7-1-gal4 UAS-GFP UAS-Kr UAS-Pdm) demonstrate overexpansion of the ISNd domain and unusual increase in ISNd size in most hemisegments (arrowhead).

(C) Overexpression of Nkx6 in the NB7-1 lineage (NB7-1-gal4 UAS-Nkx6 UAS-GFP) leads to excessive, broad, and disorganized innervation of ventral oblique muscles (arrowhead). (D) Knockdown of Nkx6 in the NB7-1 lineage (NB7-1-gal4 UAS-Nkx6-RNAi) results in loss of ventral motor projections to ISNd (yellow arrow).

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Journal Publication

published 28 Jul, 2020

Read the published version in Neural Development →

Review #2 received at journal
07 Jun, 2020
Editorial decision: Minor revision
07 Jun, 2020
Review #1 received at journal
26 May, 2020
Reviewer #2 agreed at journal
25 May, 2020
Reviewers invited by journal
16 May, 2020
Reviewer #1 agreed at journal
16 May, 2020
Editor assigned by journal
30 Apr, 2020
First submitted to journal
29 Apr, 2020
Submission checks completed at journal
29 Apr, 2020
Editor invited by journal
29 Apr, 2020

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A novel temporal identity window generates alternating cardinal motor neuron subtypes in a single progenitor lineage

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