Fd4 is expressed in NB7-1 after the expression of spatial factors Vnd and En

(A) Expression of Fd4, En, Vnd and Wor in a segment during embryonic stage 9, 11, 13 and 15. Anterior, up. Dashed lines, ventral midline. Scale bar: 10 μm. (B) Quantification of NB7-1 expressing various combination of En, Vnd, and Fd4. (C) Summary of expression for Fd4, and En Vnd double positive cells. STF: spatial transcription factors. (D) Proposed function of spatial transcription factors and Fd4 in neuroblast identity.

Fd4 is expressed in NBs, GMCs, and new-born neurons, but not in differentiated neurons

(A) Expression of Fd4, Worniu (Wor), Prospero (Pros), and Elav in a hemisegment of a stage 12 embryo. Posterior view with dorsal oriented upwards and ventral downwards. The left 4 panels show marker expression, and the fifth panel (3D Recon.) shows a 3D reconstruction of Fd4+ cells and adjacent Fd4-Elav+ cells from the left 4 panels using Imaris Spots function. The right-most image shows the expression profiles of different cell types. Yellow dashed lines outline the cells expressing Fd4. Scale bar: 2 μm. (B) Representative images of Fd4 expression in U motor neurons (UMN) in the stage 9, 10, 11, 12, and 13 embryos. Yellow dashed lines outline the UMNs. Green squares box the UMNs that express FD4. Scale bar: 1 μm (C) Quantification of Fd4 intensity in UMNs. Each dot is a measurement of Fd4 fluorescence intensity in a UMN normalized to the fluorescent intensity in a Fd4-negative cell. Yellow diamond represents the average intensity, and error bars are standard deviation. a.u., artificial unit. Number of hemisegment with UMNs measured: stage 9, 21; stage 10, 24; stage 11, 20; stage 12, 26; stage 13, 18. (D) Summary of Fd4 expression in Eve+ cells during early NB7-1 lineage. The intensity of green represents the expression levels of Fd4. (E) Proposed functions of Fd4 and terminal selector genes in neuron identity.

Fd4 is required for late-born neuron specification

(A) Eve and Dbx expression in the NB7-1 lineage of wild type and fd4/fd5 mutant stage 17 embryos. Posterior view with dorsal oriented upwards, and ventral downwards. The NB7-1 lineage is outlined with yellow dashed line. Rightmost panels are 3D reconstruction of the NB7-1 lineage from the left three panels with Imaris Spots function. Scale bar: 2 μm. (B-D) Quantification of total (B), Eve+ (C) and Dbx+ (D) cells in each lineage. Each dot represents an individual lineage. Yellow diamond, mean; error bars, standard deviation; n, number of lineages analyzed; p, the p-value of Student’s t-test. (E) UMNs in wild type and fd4/fd5 mutant stage 17 embryos. Identity of UMNs is determined by the expression of marker Hb or Runt, and the relative position of the cell within the hemisegment. The rightmost panels are summary cartoon from the left panels. Scale bar: 2 μm. (F) Quantification of each individual UMNs in each lineage. Each dot represents an individual lineage. Yellow diamond, mean; error bars, standard deviation; n, number of lineages analyzed; p, the p-value of Student’s t-test. (G) Dbx and Cas expression in the NB7-1 lineage of wild type and fd4/fd5 mutant stage 17 embryos. Dorsal view with anterior oriented upwards, and posterior downwards. Rightmost panels are 3D reconstruction of the NB7-1 lineage from the left three panels with Imaris Spots function. Scale bar: 2 μm. (H-I) Quantification. Each dot represents an individual lineage. Yellow diamond, mean; error bars, standard deviation; n, number of lineages analyzed; p, the p-value of Student’s t-test. Genotypes: wild type: fd4-gal4,fd51nt, UAS-myr-sfGFP; fd4/fd5 mutant: fd4-gal4,fd51nt/Df(3R)BSC493, UAS-myr-sfGFP. (J) Summary of functions of spatial TFs, Fd4 and terminal selector genes in neuron identity.

Wild spread of neuronal markers Eve and Dbx following ubiquitous Fd4 misexpression

(A-B) Eve and Dbx expression in wild type. En as a marker for segment boundary. Scale bars: 10 μm. (C-D) Eve and Dbx expression following Fd4 misexpression. En as a marker for segment boundary. Scale bars: 10 μm. (E-F) Quantification of Eve+ and Dbx+ cells in each hemisegment. Each dot represents an individual hemisegment. Yellow diamond, mean; error bars, standard deviation; n, number of lineages analyzed; p, the p-value of Student’s t-test. Genotypes: wild type: y-w-; Fd4 misexpression: sca-gal4,UAS-fd4.

Fd4 misexpression in NB5-6 induces NB7-1 lineage markers and represses NB5-6 lineage markers

(A) Expression of Ap and Eve in wild type (top row) or following Fd4 misexpression (bottom row) in NB5-6 using the NB5-6-specific lbe-gal4 driver. Scale bar: 2 μm (B-C) Quantification of the number of RedStinger+ (B), Ap+ (C) and Eve+ (C) cells. Each dot represents an individual lineage. Yellow diamond, mean; error bars, standard deviation; n, number of lineages analyzed; p, the p-value of Student’s t-test. (D) Expression of NB7-1 markers (Hb, Runt, Eve) in wild type NB5-6 lineage and following Fd4 misexpression in the NB5-6 lineage. Scale bar: 2 μm (E) Quantification of Hb+Eve+ and Runt+Eve+ cells. Each dot represents an individual lineage. Yellow diamond, mean; error bars, standard deviation; n, number of lineages analyzed; p, the p-value of Student’s t-test. (F) Lateral view of lbe-gal4+ axon projection in wild type (top panel) and Fd4 misexpressed (bottom panel) embryos. The axons (white, arrowhead) was overlaying the body wall muscles (blue), and the muscles were labeled with antibody against Tropomyosin 1 (Tm1). Scale bars: 20 μmm. (G) Quantification of percent of axons projected out of CNS to the body wall muscles in wild type and Fd4 misexpressed embryos. (H) Expression of motor neuron marker pMad in ectopic Eve+ cells in Fd4 misexpressed NB5-6 lineage in newly hatched larvae. The number in pMad panel shows the average number of pMad+ Eve+ cells per hemisegment. Scale bar: 2 μm. Genotypes: (A-E, H) wild type: lbe-gal4,UAS-RedStinger; Fd4 misexpression (+UAS-fd4): lbe-gal4,UAS-RedStinger,UAS-fd4; (F-G) wild type: 10xUAS-myr-smGdP.HA,lbe-gal4. Fd4 misexpression (+UAS-fd4): 10xUAS-myr-smGdP.HA, lbe-gal4, UAS-fd4

Fd4 misexpression in NB7-3 induces NB7-1 lineage markers and represses NB7-3 lineage markers

(A-B) Expression of serotonin (5-HT), Corazonin (Crz) and Eve in wild type (A) and following Fd4 misexpression in the NB7-3 lineage (B). Scale bar: 2 μm. (C) Quantification. Each dot represents an individual lineage. Yellow diamond, mean; error bars, standard deviation; n, number of lineages analyzed; p, the p-value of Student’s t-test. Genotypes: wild type: eg-gal4,UAS-myr-sfGFP; Fd4 misexpression (+UAS-fd4): eg-gal4,UAS-myr-sfGFP,UAS-fd4.

Fd4 misexpression in NB7-3 lineage dorsalizes motor neuron projections

(A) Expression of motor neuron markers Nkx6, Eve, and pMad in wild type (left column) and Fd4 misexpressed (right column) motor neuron in newly hatched larvae. Scale bars: 2 μm. (B) Quantification. Each dot represents an individual lineage. Yellow diamond, mean; error bars, standard deviation; n, number of lineages analyzed; p, the p-value of Student’s t-test. (C) Dorsal view of three segments of wild type (top panel) and Fd4 misexpressed (bottom panel) neuronal projections. Yellow arrows indicate the fascicles projecting out from the neuropil. White arrowheads, ventral midline. Scale bars: 20 μm. (D) Quantification of fascicles exiting nervous system. Wild type (1±0; top panel). Fd4 misexpression (1.6±0.5; bottom panel). (E, F) Lateral view of eg-gal4+ motor neuron axon projection in wild type (E; left panels) and Fd4 misexpressed (F; right panels) embryos. Top two panels are the maximum projections of confocal image stacks of eg-Gal4+ neurons. Middle and bottom panels are eg-Gal4+ neuron axons reconstructed with Imaris (white), overlaying the body wall muscles (red). The muscles are labeled with antibody against Tropomyosin 1 (Tm1). The dashed lines indicate the boundary between dorsal and longitudinal (middle panels), and longitudinal and ventral muscles (middle and bottom panels). Scale bars: 50 μm. (G) Quantification of motor neuron axon lengths. Each black dot represents the length of an axon measured from the VNC. White diamonds indicate the average length. The error bars are standard deviation. (H) Summary. Genotypes: wild type: eg-gal4,UAS-myr-sfGFP; Fd4 misexpression (+UAS-fd4): eg-gal4,UAS-myr-sfGFP,UAS-fd4.

Model We propose a three-step model for the specification and maintenance of neuroblast and neuron identity.

(A) Spatial transcription factors (e.g. Vnd, En) are expressed transiently in rows and columns of neuroectoderm where they act combinatorially to specify neuroblast identity. (B) neuroblast identity transcription factors (e.g. Fd4 or Lbe) are expressed in single neuroblasts and their new-born progeny throughout their lineage and act downstream of spatial factors to maintain neuroblast identity and upstream of terminal selector genes to specify late-born progeny. (C) Terminal selector genes (e.g. Eve, Dbx) are expressed in neurons where they permanently maintain neuron identity and functions.

Fd4 expression follows the expression of spatial factors En and Vnd

Quantification of Fd4, En, Vnd-GFP and Wor fluorescence intensity in NB7-1. Each dot is a measurement of protein fluorescence intensity in a NB7-1 normalized to the fluorescent intensity in a Fd4, En, and Vnd-GFP triple negative neuroblast (Wor+). Yellow diamond represents the average intensity, and error bars are standard deviation. a.u., artificial unit. Number of NB7-1 measured: stage 9, 20; stage 11, 22; stage 13, 30; stage 15, 34.

fd4 and fd5 coexpress in the ventral nerve cord during embryogenesis

Expression of fd4 and fd5 in a stage 13 embryo. Posterior view with dorsal (D) oriented upwards, and ventral (V) downwards. White dashed lines indicate the segment midline. Scale bar: 10 μm.

Fd4 does not regulate spatial patterning genes or temporal transcription factors

(A) Schematic of spatial factor expression in one hemisegment of ventral nerve cord. (B) Fd4 misexpression in En+ cells in a stage 9 embryo. Overlayed green color shows Vnd expression domain, and overlayed red color shows Ind expression domain. Blue dash-lines outline the region where Fd4 is misexpressed by en-gal4. White dashed lines, ventral midline. Genotype: en-gal4,UAS-fd4,vnd-GFP-FPTB. (C-D) Fd4 misexpression in Vnd+ cells in a stage 9 embryo. Blue dash-lines outline the region where Fd4 is misexpressed by vnd-T2A-gal4. White dashed lines, ventral midline. Genotype: vnd-T2A-gal4,UAS-fd4. (E) Schematic of temporal transcription factor expression from stage 9 to stage 12. (F) Fd4 misexpression in En+ cells in a stage 9 (left) and a stage 12 (right) embryo. Overlayed blue color shows the cells with Fd4 misexpressed with en-gal4. White dashed lines, ventral midline. Genotype: en-gal4,UAS-fd4. Scale bar: 5 μm for all panels.