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. 2026 Feb 1;40(3-4):255–266. doi: 10.1101/gad.353223.125

Hunchback functions in the postmitotic larval MDN to restrict axon outgrowth, synapse formation, and backward locomotion

Kristen Lee 1,✉, Natalie Rico Carvajal 1, Josmarie Graciani 1, Chris Q Doe 1,✉
PMCID: PMC12863255  PMID: 41266089

In this study, Lee et al. report that, in addition to a known role in neural progenitor temporal patterning, the transcription factor Hunchback (Hb) also serves a distinct role in postmitotic mature neurons in Drosophila. Here, Hb restricts the growth and synaptic function of the moonwalker descending neuron (MDN), which tunes larval backward locomotion, elucidating how temporal transcription factors can coordinately regulate neuronal diversity and connectivity to dictate animal behavior.

Keywords: Drosophila, MDN, moonwalker descending neuron, A18b, backward locomotion, Hunchback, synapse localization

Abstract

During neurodevelopment, a single progenitor cell can generate many different neuron types. As these neurons mature, they form unique morphologies, integrate into neural circuits, and contribute to behavior. However, the integration of these developmental events is understudied. Here, we show that the same transcription factor is important for both the generation of neuronal diversity and maintaining mature neuronal identity, providing novel insights into how the generation of neuronal identity and morphology are coordinated. We utilized a previously characterized larval locomotor circuit in Drosophila, where activation of the moonwalker descending neuron (MDN) triggers backward locomotion via its presynaptic connection with the premotor neuron A18b. The MDN expresses the temporal transcription factor Hunchback (Hb), which has a well-characterized role in neural progenitors. Loss of Hb in the postmitotic MDN increases axon/dendrite branching, leading to additional functional synapses on A18b and increasing backward locomotion. We conclude that the endogenous function of Hb is to restrain axon/dendrite outgrowth, including limiting MDN–A18b synapses, thereby dampening backward locomotion. Our work provides insights into how a transcription factor can have different functions throughout life; that is, Hb generates neuronal diversity in the progenitor and regulates neuronal connectivity in the mature neuron to generate an appropriately tuned behavior.

A major goal of neuroscience is to understand the relationship between mechanisms that generate molecular neuronal diversity and those determining circuit assembly and behavior. Work over the past decade has shown that spatial patterning is used to specify distinct progenitor pools in mammals or even single progenitors (neuroblasts) in flies (Briscoe et al. 2000; Skeath and Thor 2003). A second step in generating diversity within individual progenitor lineages is temporal patterning, in which successively born neurons acquire a distinct identity based on a transcription factor cascade (Doe 2017; El-Danaf et al. 2023; Pollington et al. 2023). However, the coordination between the mechanisms generating neuronal diversity and the maintenance of neuronal circuitry in a mature animal remains largely unexplored.

The best-characterized temporal transcription factor (TTF) cascade is in the Drosophila embryonic ventral nerve cord (VNC; analogous to the mammalian spinal cord), where neuroblasts sequentially express Hunchback (Hb), Kruppel, Pdm1/2, and Castor (Cas) (Doe 2017. Similarly, the Hb mammalian ortholog Ikaros (Ikz1) and Caenorhabditis elegans ortholog hbl-1 are involved in neural progenitor temporal patterning (Lin et al. 2003; Blackshaw and Cayouette 2025). Each TTF is transiently expressed in progenitors/neuroblasts and then inherited by neuronal progeny born during its expression window; in this way, TTF expression can be maintained postmitotically. While the role of Hb in fly and mouse progenitors has been well characterized (Isshiki et al. 2001; Novotny et al. 2002; Pearson and Doe 2003; Kanai et al. 2005; Tran et al. 2010; Hirono et al. 2012; Alsiö et al. 2013; Blackshaw and Cayouette 2025), the role of Hb in larval or adult postmitotic neurons is less well understood (Goto et al. 2011; Lee et al. 2022).

Although Hb expression in postmitotic embryonic Drosophila VNC neurons does not alter motor neuronal identity (Hirono et al. 2012), Hb appears to function differentially in postmitotic central brain descending interneurons. Recently, we have shown that Hb is expressed in the postmitotic GABAergic Pair1 descending neuron, part of a larval backward locomotion circuit, and that Hb has a role in maintaining Pair1 synapse number, connectivity, and behavior (Lee et al. 2022). Interestingly, the Hb ortholog in C. elegans, hbl-1, is also required in the GABAergic DD motor neuron to regulate synapse number (Thompson-Peer et al. 2012). Additionally, in worms, flies, and mice, Hb (or its respective ortholog) is expressed in postmitotic neurons with long projections (Thompson-Peer et al. 2012; Alsiö et al. 2013; Lee et al. 2022; Javed et al. 2023). While these mammalian neurons are both excitatory and inhibitory, the postmitotic function of Ikaros, the mouse Hb ortholog, remains unknown (Alsiö et al. 2013; Javed et al. 2023). Taken together, these findings suggest that postmitotic Hb regulation of synapses in long-projecting neurons may be evolutionarily conserved. Here, we expand on this hypothesis by investigating the role of Hb in the postmitotic moonwalker descending neuron (MDN), a cholinergic excitatory long-projecting neuron in Drosophila, with the goal that our findings can be translated to mammals.

There are four MDNs in the brain (two in each brain lobe), which trigger backward locomotion when activated (Carreira-Rosario et al. 2018). Larval MDNs provide excitatory input to the Pair1 neurons, which induces a pause in forward locomotion when activated (Carreira-Rosario et al. 2018; Tastekin et al. 2018; Lee et al. 2022). In addition, the MDNs provide direct excitatory input to the A18b premotor neurons, which are segmentally repeated premotor neurons in the VNC that are active during backward, but not forward, locomotion (Carreira-Rosario et al. 2018). While this is a simplified rendition of the larval backward locomotor circuit, extensive work has been done to characterize the neuronal circuits contributing to backward crawling, including intersegmental feedback circuits in the VNC (Kohsaka et al. 2019) and incorporation of circuits responding to averse stimuli (Omamiuda-Ishikawa et al. 2020). Although these studies are extremely valuable, they lack an interrogation of the molecular mechanisms mediating the development and maintenance of these circuits.

Here we focus on the role of Hb within the larval MDNs. We show that Hb is required to limit MDN neurite outgrowth and prevent the MDN from forming functional ectopic synapses with its circuit partners, thereby revealing that Hb functions to prevent abnormally robust backward locomotor behavior. Understanding whether Hb is functioning similarly among postmitotic central brain neuronal cell types will provide important information about how early developmental mechanisms can have long-term influences on neuronal identity.

Results

Hunchback is required in the larval MDN to restrain backward locomotion

Here we focus on a specific neuron in the backward locomotor circuit: MDN, which (like its partner neuron, Pair1) is born during the embryonic Hb temporal window (Fig. 1A) and maintains Hb expression throughout larval life (Fig. 1B). There are two pairs of MDNs in the larval brain, and all four MDNs express Hb. To determine whether Hb may also be functioning in postmitotic MDNs, we knocked down Hb specifically in larval postmitotic MDNs and assayed locomotor behavior. Fortunately, the larval MDN split-Gal4 driver turns on ∼4 h after larval hatching, ensuring that all our manipulations are in the postmitotic MDN (Carreira-Rosario et al. 2018). We previously showed that our UAS-HbRNAi transgene was highly efficient at knocking down Hb to low levels both panneuronally and in the Pair1 neuron (Lee et al. 2022) and here found the same strong Hb knockdown (>60% reduced expression) when expressing UAS-HbRNAi in the MDN neuron (Fig. 1C).

Figure 1.

Figure 1.

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Hunchback is required in the larval MDN to restrain backward locomotion. (A) Percentage of MDNs expressing Hunchback (Hb), Krüppel (Kr), Castor (Cas), and Grainy head (Grh) in 0–2 h old larvae. n = 8–12 animals. (B) Hunchback (magenta in the composite images; white in the grayscale images) expression in the MDN (green in the composite images; yellow outline in the grayscale images) at 24 and 72 h after larval hatching. The genotype used was UAS-myr::smGdp-HA; GMR22H02_AD; GMR23E07_DBD. Scale bar, 5 µm. (C) Compared with controls (white bar), Hb RNAi (blue bar) in the MDN (green in the composite image) significantly reduces but does not eliminate Hb expression (magenta in the composite image). The control genotype used was UAS-myr::smGdp-HA; GMR22H02_AD; GMR23E07_DBD, UAS-Luc RNAi TRiP.JF01355. The Hb RNAi genotype used was UAS-myr::smGdp-HA; GMR22H02_AD; GMR23E07_DBD, UAS-Hb RNAi TRiP.HMS01183. Scale bar, 5 µm. Statistics were as follows: t-test, P < 0.0001, n = 6–7 animals. (D, left panel) Directional speed over time before, during, and after optogenetic activation via a red light stimulus (red square) of control (gray) and Hb RNAi (blue) animals. (Middle panel) Total distance traveled (positive = forward distance and negative = backward distance) during optogenetic activation of control (white) and Hb RNAi (blue) animals. Statistics were as follows: t-test, P = 0.0336, n = 12–14 animals. (Right panel) Average speed during optogenetic activation of control (white) and Hb RNAi (blue) animals. Statistics were as follows: t-test, P = 0.0003, n = 12–14 animals. The control genotype used was UAS-CsChrimson::mVenus; GMR22H02_AD; GMR23E07_DBD, UAS-Luc RNAi TRiP.JF01355. The Hb RNAi genotype used was UAS-CsChrimson::mVenus; GMR22H02_AD; GMR23E07_DBD, UAS-TRiP.HMS01183 Hb RNAi. (E) Choline acetyltransferase (ChAT) expression (magenta in the composite images; white in the grayscale images) in the MDN (green in the composite images; yellow outline in the grayscale images) at 72 h after larval hatching does not change between control (white bar) and Hb RNAi (blue bar) animals. The control genotype used was UAS-myr::smGdp-HA; GMR22H02_AD; GMR23E07_DBD, UAS-Luc RNAi TRiP.JF01355. The Hb RNAi genotype used was UAS-myr::smGdp-HA; GMR22H02_AD; GMR23E07_DBD, UAS-Hb RNAi TRiP.HMS01183. Statistics were as follows: t-test, P = 0.2677, n = 8 animals. Scale bar, 5 µm.

In control animals, when the larval MDN is optogenetically activated, the animals move backward (Fig. 1D). Interestingly, optogenetic activation of the larval MDN following Hb knockdown promotes larvae to crawl backward a significantly longer distance than controls (Fig. 1D, middle panel) and at a significantly faster average velocity (Fig. 1D, right panel). The difference in backward distance traveled is likely due to both the change in speed and a second bout of backward locomotion during the optogenetic activation window (Fig. 1D, left panel). We conclude that reducing Hb levels in the larval MDNs results in significantly more backward locomotion, suggesting that the normal function of Hb is to restrain the backward locomotor circuit.

We hypothesized that Hb is required to maintain aspects of larval MDN identity, similar to its function in the Pair1 neuron. First, we assayed neurotransmitter expression. The MDN is an excitatory cholinergic neuron in the larvae and adults (Bidaye et al. 2014; Carreira-Rosario et al. 2018), and we used hybridization chain reaction (HCR) to assay RNA levels of choline acetyltransferase (ChAT) in control and Hb knockdown animals (Fig. 1E). We opted to use in situ hybridization for these experiments because the ChAT antibody was unreliable. When Hb was knocked down, the larval MDN still expressed ChAT at levels similar to controls (Fig. 1E), showing that Hb is not regulating MDN neurotransmitter identity. We conclude that Hb restrains MDN-induced backward locomotion but is not required for MDN neurotransmitter choice.

Hunchback restrains larval MDN axon/dendrite branching

Hb is not required for axon or dendrite morphology in the larval Pair1 neuron (Lee et al. 2022). To see whether Hb regulates axon or dendrite morphology in larval MDNs, we knocked down Hb and labeled single MDNs using multicolor flip-out (MCFO) (Fig. 2; Nern et al. 2015). We used the image analysis software Imaris to trace each neuron (Fig. 2A, “filaments”), allowing us to quantify the branching and length of dendrites and axons. The MDN has an ipsilateral dendrite and crosses the midline to form a contralateral dendrite before sending a descending axon to segment A4 in the VNC (Fig. 2A; Carreira-Rosario et al. 2018). When Hb was knocked down, both the ipsilateral and contralateral dendrites had significantly more branches (Fig. 2A–D) and an increased summed neurite length (Fig. 2F,G). Similarly, there were significantly more axon branches and greater total length (Fig. 2A,B [yellow arrowhead] E,H). We conclude that Hb is required in MDNs to limit branch number and total axon/dendrite length. These results differ from our previous findings on Hb function in Pair1 but are consistent with a role for Hb in restraining backward locomotion as described in the previous section.

Figure 2.

Figure 2.

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Hunchback restrains larval MDN axon/dendrite branching. (A,B) Representative images of single labeled MDNs in control (A) and Hb RNAi (B) animals. In vivo (left) and Imaris image analysis software tracing (“filaments”; right) images are shown. Scale bar, 50 µm. The yellow dashed box indicates the zoomed-in dendritic region. Scale bar, 10 µm. Images are rotated in the Z-plane for clarity. The yellow arrowhead highlights the extended axon projection. The ipsilateral (ipsi.) dendrite is on the same side of the cell body, the contralateral (contra.) dendrite is on the opposite side from the cell body, and the axon descends into the ventral nerve cord on the contralateral side. (C–E) Branch numbers for the ipsilateral dendrite (C), contralateral dendrite (D), and axon (E) in control (white bars) and Hb RNAi (colored bars) animals. Statistics for C–E were as follows: (C) t-test, P = 0.0019, n = 6–10 neurons; (D) t-test, P = 0.0045, n = 5–10 neurons; (E) t-test, P = 0.0017, n = 5–8 neurons. (F–H) Summed lengths of all traced neurites of the ipsilateral dendrite (F), contralateral dendrite (G), and axon (H) in control (white bars) and Hb RNAi (colored bars) animals. Statistics for F–H wer as follows: (F) t-test, P = 0.0027, n = 6–10 neurons; (G) t-test, P < 0.0001, n = 5–10 neurons; (H) t-test, P = 0.0045, n = 6–8 neurons. The control genotype used was hs-Flp-G5::Pest; 10X UAS(frt.Stop)myr::smGDP-V5-THS-10XUAS(frt.Stop)myr::GDP-FLAG/GMR22H02_AD; GMR23E07_DBD, UAS-Luc RNAi TRiP.JF01355. The Hb RNAi genotype used was hs-Flp-G5::Pest; 10X UAS(frt.Stop)myr::smGDP-V5-THS-10XUAS(frt.Stop)myr::GDP-FLAG/GMR22H02_AD; GMR23E07_DBD, UAS-Hb RNAi TRiP.HMS01183.

Hunchback prevents axon and presynapse targeting to the lower abdominal segments

To characterize the effect of Hb knockdown on MDN morphology in more detail, we focused on its descending axon. The MDN-Gal4 driver labels two descending neurons: the MDN and a more medial Hb-negative neuron, which we have termed the Hb-negative descending neuron (DN) (Fig. 3A). Here we used the Hb-negative DN as an internal control, as it should not be affected by Hb knockdown and has a morphology unique from MDN, and both the DN and MDN terminate in a similar location in the abdominal ganglion (Fig. 3A,B). To confidently identify abdominal segments A1–A7, we used the segmentally repeated cluster of lateral Even-skipped (Eve)-positive neurons (Fig. 3C,D). In controls, the MDN descending axon invariably terminated in segment A4, though it occasionally extended slightly into segment A5 but never into segments A6 and A7 (Fig. 3C [white arrowhead], quantified in E); the same is true for the Hb-negative DN internal control neuron (Fig. 3C [yellow arrowhead], quantified in E). In contrast, Hb knockdown resulted in the MDN axon fully extending into segments A5–A7 (Fig. 3D [white arrowhead], quantified in E). Importantly, the Hb-negative DN control neuron still primarily targeted the A4 segment (Fig. 3D [yellow arrowhead], quantified in E), thereby highlighting both the specificity of the Hb knockdown and its role in MDN axon mistargeting.

Figure 3.

Figure 3.

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Hunchback is required for larval MDN axon targeting and synapse localization. (A) Schematic of the SS01613-Gal4 (referred to here as “MDN-Gal4”) expression pattern in both the larval CNS and a hemisegment of a third instar larval VNC. The MDN axon (white) is anatomically distinct from the Hunchback-negative off-target axon (yellow). (B) The Hb-negative descending neuron (Hb-neg DN; green) does not express Hb (magenta) in the larvae. Scale bar, 5 µm. (C–E) MDN-Gal4 (green) showing the MDN axon (white arrowhead) and off-target axon (yellow arrowhead) in control (C) and Hb RNAi (D) animals. Even-skipped (eve; magenta) labeling of abdominal segments A1–A7 is shown. Scale bar, 25 µm. (E) Quantification of abdominal segment termination of the MDN (left) and off-target (right) axons in control (white) and Hb RNAi (blue) animals. Statistics were as follows: two-way ANOVA: genotype, F(1,90) = 13, P = 0.0003; neuron, F(1,90) = 20, P < 0.0001; interaction, F(1,90) = 13, P = 0.0102; Bonferroni's multiple comparisons between genotypes for each neuron: MDN, P < 0.0001; off-target, P = 0.9241; n = 18–38 VNC hemisegments. The control genotype used was UAS-myr::smGdp-HA; GMR22H02_AD; GMR23E07_DBD, UAS-Luc RNAi TRiP.JF01355. The Hb RNAi genotype used was UAS-myr::smGdp-HA; GMR22H02_AD; GMR23E07_DBD, UAS-Hb RNAi TRiP.HMS01183. (F–H) MDN-Gal4 (green) showing the MDN axon (white arrowhead) and off-target axon (yellow arrowhead) in control (F) and Hb RNAi (G) animals. Labeling of presynapses in the MDN-Gal4 pattern using Bruchpilot (Brp-sh; magenta in the composite images). The yellow dashed line marks the boundary between the A4 and A5 segments designated by Eve staining. Scale bar, 10 µm. (H) Quantification of the number of MDN presynapses anterior to A5 (A1–A4; left) and posterior to A5 (A5–A7; right) in control (white) and Hb RNAi (purple) animals. Statistics were as follows: two-way ANOVA: genotype, F(1,56) = 8.1, P = 0.0060; abdominal segment, F(1,56) = 757, P < 0.0001; interaction, F(1,56) = 3.1, P = 0.0794; Bonferroni's multiple comparisons between genotypes for each abdominal segment location: A1–A5, P = 0.9032; A5–A7, P = 0.0035; n = 12–18 VNC hemisegments. The control genotype used was UAS-myr::smGdp-HA; GMR22H02_AD, UAS-brp-D3-mStrawberry; GMR23E07_DBD, UAS-Luc RNAi TRiP.JF01355. Hb RNAi genotype: UAS-myr::smGdp-HA; GMR22H02_AD, UAS-brp-D3-mStrawberry; GMR23E07_DBD, UAS-Hb RNAi TRiP.HMS01183.

To determine whether the MDN had presynapses in the ectopic A5–A7 axon extension, we used MDN-Gal4 to express a nonfunctional presynaptic marker, Bruchpilot-short (Brp-short), and assayed the number of synapses on the MDN axon posterior or anterior to A5. This boundary was determined by Eve staining (Fig. 3F,G, dashed line) and by the Hb-negative DN internal control axon, which showed unchanged termination in A4 (Fig. 3F,G, yellow arrowhead). In controls, the MDN axon primarily terminated at segment A4, as expected (Fig. 3F), with Brp+ presynapses distributed along the axon (Fig. 3F, Brp-sh). Hb knockdown showed no change in the number of presynapses in segments A1–A4; however, we detected Brp+ synapses in the ectopic posterior axonal domain (Fig. 3G, quantified in H). We conclude that Hb acts in MDNs to prevent ectopic synapse formation either directly or indirectly by restraining axon extension. These findings are consistent with a role for Hb in restraining backward locomotion as described above.

Hunchback prevents MDN innervation of A18b dendritic regions in posterior abdominal segments

The larval MDN circuit is well characterized (Fig. 4A; Carreira-Rosario et al. 2018; Lee and Doe 2021; Lee et al. 2022). One neuron in this circuit is A18b, a cholinergic premotor neuron downstream from the MDN located in the VNC, which is active only during backward locomotion (Carreira-Rosario et al. 2018). Given that larval Hb knockdown leads to an increase in backward locomotion and MDN axon mistargets to abdominal segments A5–A7, we hypothesized that Hb knockdown may result in ectopic MDN–A18b synapses in A5–A7.

Figure 4.

Figure 4.

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Hb prevents MDN innervation of A18b+ dendrite volume in A5–A7. (A) Schematic of the core MDN larval circuit. The MDN forms synapses with Pair1 in the central brain and with A18b in the VNC. (B) A18b neurons (green; asterisks mark the cell body) are present in abdominal segments A4–A7 (Eve; magenta) at 72 h after larval hatching. Scale bar, 30 µm. (C) Quantification of the number of A18b neurons that the MDN axon contacts within the VNC at 72 h after larval hatching in control (white bar) and Hb RNAi (blue bar) animals. Statistics were as follows: t-test, P = 0.0013, n = 10–13 VNC hemisegments. (D,D′) The region of MDN and A18b dendritic innervation in control animals. (D) Anterior to posterior view of MDN-Gal4 (white in the grayscale image; green in the composite image) innervating A18b dendrites (magenta in the composite image) in segments A4–A7 in control animals at 72 h after larval hatching. (White arrowhead) MDN axon, (yellow arrowhead) Hb-negative DN off-target axon. Scale bar, 10 µm. (D′) Ventral to dorsal view of MDN-Gal4 (green in the composite image; white in the grayscale image) innervating the A18b dendritic region (magenta in the composite image; white in the grayscale image; outlined in magenta for clarity) in the A4 segment of the VNC. (White arrowhead) MDN axon, (yellow arrowhead) Hb-negative DN axon. Scale bar, 10 µm. The genotype used was UAS-myr::smGdp-HA, LexAop-myr::smGdp-V5; GMR22H02_AD, 94E10-LexA; GMR23E07_DBD, UAS-Luc RNAi TRiP.JF01355. (E,E′) The region of MDN and A18b dendritic innervation in Hb RNAi animals. (E) Anterior to posterior view of MDN-Gal4 (white in the grayscale image; green in the composite image) innervating A18b dendrites (magenta in the composite image) in segments A4–A7 in Hb animals at 72 h after larval hatching. (White arrowhead) MDN axon, (yellow arrowhead) Hb-negative DN axon. Scale bar, 10 µm. (E′) Ventral to dorsal view of MDN-Gal4 (green in the composite image; white in the grayscale image) innervating the A18b dendritic region (magenta in the composite image; white in the grayscale image; outlined in magenta for clarity) in the A4–A6 segments of the VNC. (White arrowhead) MDN axon, (yellow arrowhead) Hb-negative DN axon. Scale bar, 10 µm. The genotype used was UAS-myr::smGdp-HA, LexAop-myr::smGdp-V5; GMR22H02_AD, 94E10-LexA; GMR23E07_DBD, UAS-Hb RNAi TRiP.HMS01183.

To test this hypothesis, we first confirmed that A18b neurons are located in A4–A7 in the third instar larval VNC (Fig. 4B, asterisk). Next, we asked whether Hb knockdown increased MDN innervation of the A18b dendritic volume in segments A4–A7 by quantifying the number of A18b neurons that the MDN axon overlaps with (Fig. 4C). In controls, MDN innervates the A18b dendritic volume primarily in segment A4 but occasionally also in segment A5 (Fig. 4D [white arrowhead], quantified in C). In addition to segment A4, Hb RNAi resulted in a robust innervation of the A18b dendritic volume in the more posterior segments, A5–A7 (Fig. 4E [white arrowhead], quantified in C). Importantly, the Hb-negative DN does not contact A18b (Fig. 4D,E, yellow arrowhead). We conclude that Hb RNAi results in the MDN extending its axon into the more posterior segments, A5–A7, where the MDN axon innervates the A18b dendritic domains. This raised the possibility that Hb RNAi may lead to ectopic MDN–A18b synapse formation.

Hunchback prevents ectopic MDN–A18b putative synapses in posterior abdominal segments

To test the hypothesis that the MDN forms ectopic synapses with more posterior A18b neurons when Hb is knocked down, we first needed a reliable way to label MDN synapses. Due to genetic limitations, we could not label both MDN synapses and A18b membranes in control and Hb knockdown animals. Instead, we utilized a previously published method showing that axonal varicosities strongly correlate with synapse location (Sales et al. 2019). We validated this method by demonstrating that the nonfunctional presynaptic marker Brp-short colocalizes exclusively with MDN axonal varicosities (Fig. 5A). To determine whether we could unbiasedly label these varicosities, we increased the contrast of the axonal image and then used the “spots” function in Imaris image analysis software to label the varicosities. We found that this technique revealed a strong correlation between MDN axonal varicosities and the Brp-short signal, thus allowing us to confidently label MDN synapses (Fig. 5A′).

Figure 5.

Figure 5.

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Hunchback prevents ectopic MDN–A18b putative synapses in posterior abdominal segments. (A,A′) MDN axon varicosities can be used to label MDN presynapses. (A) MDN axon (green in the composite image; white in the grayscale image) colocalized with Bruchpilot (Brp-sh; magenta in the composite images; white in the grayscale image; white arrowheads show location). Scale bar, 5 µm. (A′) MDN axon varicosities (white in the grayscale image; green in the composite image) were used as a reference for 3D spots (blue in the composite image). These spots colocalized with Bruchpilot (Brp-sh; magenta in the composite image; white in the grayscale image). Scale bar, 5 µm. (B,C) Single slice images of the MDN (white in the grayscale image; outlined in green for reference; green in the composite image) and A18b (white in the grayscale image; magenta in the composite image) in control animals (B) and Hb RNAi animals (C). Scale bar, 7 µm. (D) Putative MDN synapses with A18b neurons in control animals at 72 h after larval hatching. (Left panel) MDN putative synapses (green) and A18b neurons within abdominal segments A4–A7 (magenta). Scale bar, 10 µm. The yellow box indicates the zoomed-in region in the four right panels. (Right four panels) MDN putative synapses (green in the composite image) and A18b neurons within abdominal segments A4 and A5 (magenta in the composite image); the dashed line separates the A18b neuron in A4 from A5. Spots (green dots in the composite image) indicate the locations of MDN putative synapses (white in the composite image). MDN spots (green dot in the composite image and standalone image) touching the A18b surface (magenta in the composite image) were assayed. Scale bar, 5 µm. (E) Putative MDN synapses with A18b neurons in Hb RNAi animals at 72 h after larval hatching. (Left panel) MDN putative synapses (green) and A18b neurons within abdominal segments A4–A7 (magenta). Scale bar, 10 µm. The yellow box indicates the zoomed-in region in the four right panels. (Right four panels) MDN putative synapses (green in the composite image) and A18b neurons within abdominal segments A4–A6 (magenta in the composite image); the dashed line separates A18b neurons within unique segments. Spots (green dots in the composite image) indicate the location of MDN putative synapse (white in the composite image). MDN spots (green dots in the composite image and standalone image) touching the A18b surface (magenta in the composite image) were assayed. Scale bar, 5 µm. (F) Quantification of the number of MDN putative synapse spots touching the A18b neuronal surface in control (white bar) and Hb RNAi (blue bar) animals. Statistics were as follows: two-way ANOVA: genotype, F(1,88) = 27, P < 0.0001; abdominal segment, F(3,88) = 23, P < 0.0001; interaction, F(3,88) = 2.3, P = 0.0777; Bonferroni's multiple comparisons between genotypes within each abdominal segment: A4, P = 0.9830; A5, P = 0.0022; A6, P = 0.0002; A7, P = 0.5511; n = 11–13 VNC hemisegments. The control genotype used was UAS-myr::smGdp-HA, LexAop-myr::smGdp-V5; GMR22H02_AD, 94E10-LexA; GMR23E07_DBD, UAS-Luc RNAi TRiP.JF01355. The Hb RNAi genotype used was UAS-myr::smGdp-HA, LexAop-myr::smGdp-V5; GMR22H02_AD, 94E10-LexA; GMR23E07_DBD, UAS-Hb RNAi TRiP.HMS01183.

Next, we used this method to assay where the MDN is forming synapses with A18b in control and Hb knockdown animals. First, we showed that MDN varicosities and A18b membranes overlap in a single optical slice (Fig. 5B,C). Next, we used three-dimensional data to determine how many synapses the MDN was forming with each A18b neuron in abdominal segments A4–A7. We assayed MDN synapse number using axonal varicosities, as described above. To unbiasedly determine how many synapses the MDN was forming with A18b, we used Imaris image analysis software to put a “surface” over the A18b dendritic domains. This allowed us to assay how many “spots” were touching a “surface.” In control animals, we found that the MDN formed —three to six total synapses with A18b neurons exclusively in the A4–A5 segments, with most residing in the A4 segment (Fig. 5D, quantified in F). In contrast, Hb knockdown increased MDN synapses with A18b to an average of seven to 12 total synapses between the A4–A7 abdominal segments (Fig. 5E, quantified in F). There was a significant increase in MDN–A18b synapse number in segments A5 and A6 when Hb was knocked down (Fig. 5F), which correlated with an increased likelihood that the MDN axon targeted these regions when Hb was knocked down (Figs. 3, 4), demonstrating that the MDN is forming ectopic synapses with A18b neurons. Therefore, Hb normally functions to limit the number of MDN–A18b synapses.

Hunchback prevents ectopic MDN–A18b functional synapses in posterior abdominal segments

Next, we wanted to determine whether Hb knockdown resulted in functional synapses with A18b in A5–A7. To test this, we expressed a CaMPARI2 (calcium-modulated photoactivatable ratiometric integrator) transgene in A18b neurons (Moeyaert et al. 2018). CaMPARI functions as a photoconvertible protein, converting from green to red when exposed to 405 nm illumination and concurrent elevation of calcium levels; that is, neuronal activity (Fig. 6A). The CaMPARI2 transgene specifically allows antibody detection of the original green (via anti-FLAG) and photoconverted red (via anti-CaMPARI2) signals independently, allowing increased sensitivity when imaging. As described previously (Carreira-Rosario et al. 2018), we activated the MDN via a noxious head poke, concurrently exposed the larvae to 405 nm illumination, and assayed the number and segmental location of A18b neurons photoconverted to red in both control and Hb knockdown conditions (Fig. 6B). As anticipated, all A18b neurons expressed the green CaMPARI2 signal (Fig. 6C,D, asterisk). In control animals, A18b neurons in segment A4, and occasionally in segment A5, were photoconverted to red, showing that MDN activation is causing increased activity in A18b neurons in this region (Fig. 6C [white arrowheads], quantified in E). When Hb was knocked down in MDNs, A18b neurons in the more posterior segments, A5–A7, were also photoconverted to red (Fig. 6D [white arrowheads], quantified in E), showing that MDN activation is causing increased activity in A18b neurons in this region. A posterior neuron that is not A18b was also consistently activated in the Hb knockdown animals (Fig. 6D, yellow arrowhead). Importantly, in both control and Hb knockdown animals, A18b was only photoconverted when a noxious head poke was administered but not when the stimulus was absent (Fig. 6E), showing that the experimental setup alone does not photoconvert A18b neurons to red. Taken together, these results show that the MDN forms ectopic functional connections with A18b following Hb knockdown in the MDN. Thus, when Hb is reduced, the MDN axon extends into more posterior segments but still prioritizes synaptic connections with neurons within its established locomotor circuit—its partner, A18b (Fig. 6F). We conclude that Hb is required in MDNs to restrict the number and function of MDN–A18b synaptic contacts and thus dampen backward locomotor behavior.

Figure 6.

Figure 6.

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Hunchback prevents ectopic MDN–A18b functional synapses in posterior abdominal segments. (A) Schematic of how the CaMPARI transgene determines whether a neuron experienced increased activity during exposure to photoconvertible light. If a neuron did not experience increased activity during the light exposure, it remained green. If a neuron was active during the light exposure, it photoconverted (PC) to red (depicted as magenta). (B) Schematic of the experimental protocol. A noxious head poke was used to manually induce backward (BWD) locomotion, causing the MDN to fire. Photoconversion using the CaMPARI transgene was used to determine whether the A18b neurons were activated in response to MDN activity. (C,C′) A18b photoconversion during MDN activation (i.e., poke) in control animals at 72 h after larval hatching. (C) Three-dimensional projection of CaMPARI2 signal showing all A18b cell bodies labeled green (white and marked with asterisks in the grayscale image; green in the composite image) and the cells that photoconverted to red (white in the grayscale image; magenta in composite image). Abdominal segments A4–A7 are labeled. The white arrowhead shows positive CaMPARI2 PC signal. Scale bar, 15 µm. (C′) Single slices of A18 cell bodies in abdominal segments A4–A7 from the 3D projection image. The genotype used was LexAop-CaMPARI2; GMR22H02_AD, 94E10-LexA; GMR23E07_DBD, UAS-Luc RNAi TRiP.JF01355. (D,D′) A18b photoconversion during MDN activation (i.e., poke) in Hb RNAi animals at 72 h after larval hatching. (D) Three-dimensional projection of CaMPARI2 signal showing all A18b cell bodies labeled green (marked with asterisks; white in the grayscale image; green in the composite image) and the cells that photoconverted to red (white in the grayscale image; magenta in the composite image). Abdominal segments A4–A7 are labeled. The white arrowhead shows positive CaMPARI2 PC signal in A18b. The yellow arrowhead shows positive CaMPARI2 PC signal in off-target neurons. Scale bar, 30 µm. (D′) Single slices of A18 cell bodies in abdominal segments A4–A7 from the 3D projection image. The genotype used was LexAop-CaMPARI2; GMR22H02_AD, 94E10-LexA; GMR23E07_DBD, UAS-Hb RNAi TRiP.HMS01183. (E) Quantification of the normalized red to green ratio of the lateral region of the VNC containing the A18b cell bodies at 72 h after larval hatching in control RNAi (Luc RNAi; white bar) and Hb RNAi (blue bar) animals. Statistics were as follows: two-way ANOVA: genotype, F(1,35) = 49, P < 0.0001; treatment, F(1,35) = 320, P < 0.0001; interaction, F(1,35) = 58, P < 0.0001; Bonferroni's multiple comparisons: no poke control versus no poke Hb RNAi, P > 0.9999; poke control versus poke Hb RNAi, P < 0.0001; n = 9–10 VNC hemisegments. (F) Schematic of all results in wild-type (right) and Hb knockdown (left) animals. (Green) MDN, (magenta) A18b. The lightning bolt represents MDN activation.

Discussion

Here we showed that the MDN, a Drosophila central brain descending neuron required for initiating backward locomotion, is born in the Hb TTF window during neurogenesis. The MDN expresses Hb throughout larval life, allowing us to assay its role in larval postmitotic MDN neuronal identity maintenance. Larval MDN Hb knockdown results in increased backward distance traveled when the MDN is ontogenetically activated. We showed that in control animals, the MDN forms synapses with the premotor neuron A18b in abdominal segment A4. However, when Hb is knocked down, the MDN axon mistargets to the more posterior abdominal segments, A5–A7, and forms functional ectopic synapses with the A18b neurons in these segments. Thus, Hb restricts MDN–A18b synaptic contacts and dampens backward locomotor behavior.

These data add to a growing body of evidence suggesting that TTFs may be functioning differentially in the Drosophila stem cells versus postmitotic neurons (Hirono et al. 2017; Lee et al. 2022). Specifically, overexpression of Hb in an embryonic VNC neuronal progenitor resulted in large-scale morphological, circuitry, and behavioral changes in its neuronal progeny (Pollington and Doe 2025). Similarly, temporal transcription factors in the Drosophila visual system neuronal progenitors determine axonal targeting, synapse localization, and behavior (Holguera et al. 2025). Although temporal patterning function is better understood in VNC and visual system neuroblast lineages (Doe 2017 Pollington et al. 2023; Holguera et al. 2025), this work highlights the importance of studying the role of maintained temporal patterning genes to better understand how these early developmental mechanisms can shape neuronal identity throughout life. To our knowledge, in all central brain neurons with maintained Hb expression, Hb has been required to maintain the identity of these cells, suggesting that these findings are broadly applicable to other central brain neurons with maintained TTF expression (Goto et al. 2011; Lee et al. 2022). Follow-up studies investigating additional TTFs postmitotically in the VNC and visual system should be done to further understand this phenomenon.

We were surprised to find that Hb is functioning differently in postmitotic MDNs and Pair1 neurons in the larval central brain. In Pair1 neurons, Hb does not alter morphology but is required for normal synapse number and behavior (Lee et al. 2022). While Hb is still required in MDNs for synapse number, our major finding was that Hb is functioning to regulate axon outgrowth/guidance. While we do not know for certain, this difference between Hb function in MDNs and Pair1 may be due to a few key differences between these cells. First, MDNs and Pair1 express different neurotransmitters. Strikingly, both Pair1 in flies and the DD neurons in C. elegans are GABAergic and do not have morphology changes when Hb/hbl-1 expression is reduced, instead showing synapse number changes (Thompson-Peer et al. 2012; Lee et al. 2022). Perhaps the MDN's excitatory profile, in conjunction with Hb expression, is contributing to its morphology changes. Indeed, the mammalian Hb ortholog Ikaros is expressed in both excitatory and inhibitory neurons in mice (Alsiö et al. 2013; Javed et al. 2023), suggesting that Ikaros may be differentially regulating morphology and synapse number postmitotically in these cells as well. Taken together, this may suggest that Hb is working in conjunction with a terminal selector gene to specify neurotransmitter identity and perhaps also maintain neuronal morphology. Work in the Drosophila medulla has demonstrated that temporal transcription factors regulate terminal selectors that are required for neurotransmitter identity in progenitors/immature neurons (Simon et al. 2025). Additional research is needed to determine whether these genes also maintain neuronal morphology postmitotically.

A second potential difference between the larval MDN and Pair1 is Hb expression levels, which are significantly different between these cells throughout life (data not shown). Previous work has shown that Hb can be present in “high” or “low” quantities in a neuronal stem cell, leading to the generation of unique neuronal types (Pearson and Doe 2003; Gabilondo et al. 2011). Work investigating the Hox gene Antennapedia (Antp) in postmitotic Drosophila leg motor neurons has also demonstrated that Antp levels specify axon targeting, suggesting that TF expression levels are functionally relevant in postmitotic neurons (Baek et al. 2013), driving the hypothesis that Hb levels may also be relevant postmitotically. Last, building on this work investigating Hox genes, the larval MDN and Pair1 are located in different regions of the brain, enabling unique Hox genes to function in combination with temporal factors to maintain MDN morphology. Perhaps Hox genes are the true determinators of axon targeting, and Hb is just functioning to maintain their expression postmitotically. Indeed, in larval Pair1, Hb is needed to maintain the expression of the Hox gene Bicoid (Lee et al. 2022).

In both the larval MDNs and Pair1 neurons, Hb is required for normal behavior. In larval Pair1, reduced Hb expression led to an increase in pausing and therefore an increase in head casting; both these behaviors are associated with two different neuronal circuits downstream from Pair1 (Tastekin et al. 2018; Lee et al. 2022). Interestingly, reduced Hb expression caused a second bout of backward locomotion during larval MDN optogenetic activation. Here, we showed that Hb knockdown in the larval MDN alters the number of premotor A18b neurons that the MDN is able to form functional synapses with. Given that two A18b neurons are present in each abdominal segment in the larval VNC, it is interesting to speculate that A18b neurons in different abdominal segments may be integrated into different backward locomotor circuits with different kinetics, thus causing the two bouts of backward locomotion observed when Hb is knocked down in the MDN.

During development, conserved axon guidance cues (i.e., attractive or repulsive signals) allow the axon to travel to the correct location within the nervous system (Evans and Bashaw 2010). Mutation of the axon guidance cue Round-about (Robo) leads to abnormal behavior in larvae (Berni 2015), demonstrating that axon pathfinding is important for whole-animal function. The axon typically travels in multiple bouts of smaller distances between “guidepost” cells before settling in a final location. Guidepost cells in insects, or intermediate targets in vertebrates, are specialized to provide local cues to either promote or inhibit the growth of a specific neuronal axon (Araújo and Tear 2003). Although many genes have been implicated in axon pathfinding (Kraut et al. 2001), our work is the first to show that the TTF Hb may also be involved in some aspect of this process—whether it is axon pathfinding specifically or axonal adhesion/stabilization after the initial pathfinding occurs. We hypothesize that Hb may be required for the expression of an axon guidance cue, allowing the MDN axon to traverse the VNC and terminate on A18b in abdominal segment A4. Perhaps A18b neurons are acting as a guidepost cell for MDN axon pathfinding.

Materials and methods

Fly husbandry

All flies were reared with a 12 h light/dark cycle in a 25°C room at 50% relative humidity unless noted otherwise. All comparisons between groups were based on studies with flies grown, handled, and tested together.

Fly stocks

Fly stocks used were as follows:

  • GMR22H02_AD (attp40); GMR23E07_DBD (attp2) (SS01613-Gal4) (gift from J. Truman, University of Washington),

  • LexAop-myr::smGdp-V5, UAS-myr::smGdp-HA;; (Bloomington Drosophila Stock Center 64092),

  • UAS-Hb RNAi TRiP.HMS01183 (attp2) (Bloomington Drosophila Stock Center 34704),

  • UAS-Luc RNAi TRiP.JF01355 (attp2) (Bloomington Drosophila Stock Center 31603),

  • UAS-Chrimson::mVenus (attp8);; (Bloomington Drosophila Stock Center 55134),

  • 10X UAS(frt.Stop)myr::smGDP-V5-THS-10XUAS(frt.Stop)myr::GDP-FLAG; (Bloomington Drosophila Stock Center 62124),

  • hs-FlpG5::Pest;; (Bloomington Drosophila Stock Center 77140),

  • UAS-brp::sh-mStrawberry (Bloomington Drosophila Stock Center 80571),

  • 94E10-LexA (attp40) (made in the Doe laboratory), and

  • LexAop-CaMPARI2;; (Janelia 3028062).

Immunohistochemistry

Standard confocal microscopy and immunocytochemistry methods were performed. In short, larval CNSs were dissected in ice-cold hemolymph-like buffer and fixed with 4% paraformaldehyde for 12–23 min, depending on age. The tissue was exposed to normalized donkey serum block for either 40 min at room temperature or overnight at 4°C. Next, the tissue was exposed to a primary antibody solution overnight at 4°C. After being washed with 0.3% PBST, samples were exposed to a secondary antibody solution overnight at 4°C. After a series of dehydrations, tissue was mounted with DPX; the only exception was the CaMPARI experiments, which were mounted with ProLong glass antifade mountant (Thermo Fisher). Primary antibodies used were as follows: rabbit anti-Hunchback (1:400; Doe laboratory), rat anti-HA (1:100; Sigma 11867423001), chicken anti-V5 (1:800; Bethyl Laboratories A190-218A), mouse anti-FLAG (1:1000; Sigma F1804), rabbit anti-Eve (1:500; Doe laboratory), rabbit anti-DsRed (1:200; Abcam ab62341), and mouse anti-CaMAPRI2 (1:1000; Absolute Antibody). Secondary antibodies were from Jackson ImmunoResearch (donkey at 1:400).

Image acquisition and processing

Confocal image stacks were acquired on a Zeiss 900 AiryScan microscope using Nyquist sampling. All images were processed in either FIJI or Imaris. Methodology for specific analyses using these software packages is described below. Figures were made using Adobe Illustrator.

Quantification of pixel intensity

All pixel intensity quantification was done manually using the “measure” feature in FIJI. The “freehand” tool was used to outline the cell body. Measurements were set to “area” and “raw integrated density.” A middle slice of the total cell body was measured. For each cell body, the “raw integrated density” was divided by the “area.” The sum of these values is reported.

Behavior

Embryos were transferred to a food bottle and aged until 48 h after larval hatching, and then the larvae were transferred to apple caps with a food mixture of 0.5 µM all-trans retinol (ATR) or 0.5 µM ethanol (sans ATR control), 6 mL of deionized water, and 5 mL of yeast grain for 24 h. Behavior experiments were all done with third instar larvae aged 72–76 h after larval hatching. Larvae were transferred to 1.3% agar sheets on the FIM table. The FIM table used frustrated total internal reflection (FTIR) to capture high-resolution and high-control videos for experiments. After a 2 min accumulation, larva locomotion was recorded at four frames per second for a 150 frame video (37.5 sec) with a Basler camera. At 50 frames, the red light was turned on for 50 frames (turned off at 100 frames). Next, the behavior video was uploaded to FIJI to crop the video and increase the contrast, creating a darker background. Then, it was uploaded to FIMTracker to track larval behavior. Directional information was acquired using the “mov_direction” calculations. Distance traveled and acceleration were reported.

Multicolor FlpOut and quantification of morphology

Stage 16/17 embryos were heat-shocked for 8–10 min at 37°C in a water bath. After heat shock, embryos recovered for an equal amount of time at 18°C. The CNS was dissected from 70–74 h old animals. Images of the brains were imported into the Imaris image analysis software. The “filament” tool was used to trace the morphology of MCFO-labeled MDN. The largest diameter was set to 3.5 µm, and the smallest diameter was 0.9 µm. We used low threshold settings to remain unbiased and keep the analysis sensitive. It was not clear whether the small processes are stable or dynamic. Defining the primary neurite as the neurite extending from the cell body, the ipsilateral dendrite was the material ipsilateral to, but not including, the primary neurite. The contralateral dendrite was the material contralateral to, but not including, the primary neurite. The primary neurite was the neurite extending from the cell body. The first projection off the descending neurite was used as a landmark for the beginning of the axon region and the end of the contralateral dendrite region. This landmark has been characterized previously (Carreira-Rosario et al. 2018).

Abdominal segment identification

We used Even-skipped (Eve), which is expressed in a lateral cluster of 10 neurons per abdominal segment, to identify segments A1–A7. Boundaries were defined by the most posterior region of each Eve-positive cluster.

Presynapse quantification with Brp-short

Using Imaris image analysis software, the “filament” tool was used to trace the MDN axon, as described above in “Multicolor FlpOut and Quantification of Morphology.” The “spots” tool was then used to unbiasedly label the Brp-short staining/puncta with a three-dimensional spot. The spot's diameter was dependent on each image, but most were 1.3 µm on average. The “spots close to the filament” tool was then used to detect the number of spots located on the filament, specifically on the axon portion, with a threshold of 1. The number of spots on the axon filament was then recorded.

Presynapse quantification with MDN axon varicosities

Using Imaris image analysis software, we increased the contrast within the MDN axon so that the varicosities were isolated. The “spots” tool was then used to unbiasedly label the varicosities with a three-dimensional spot. The average diameter was set to 1.3 µm. The surface tool was used to label the A18b neurons. The “spots close to surface” tool was then used to detect the number of spots made close to the A18b surface, with a threshold of 1. We used the Even-skipped (Eve) staining to determine the abdominal segments, as defined above.

CaMPARI experiments

Animals were reared in the dark, with minimal light exposure during handling. At 72 h after larval hatching, five animals were placed on a firm agar cap and exposed to the photoconverting light (405 nm) for 2 min while being monitored under a stereoscope. If the animals received a noxious head poke, they were poked in the head (mouth hooks used to determine the head) with a paint brush approximately —four to five times, ensuring each head poke resulted in backward crawling. If the animals did not receive the noxious stimulus, they were allowed to crawl on the plate uninterrupted and observed to ensure that backward locomotion did not occur during light exposure. The central nervous system was immediately dissected after photoconvertible light exposure and underwent the immunohistochemistry protocol outlined above. We stained the tissue for FLAG and CaMPARI2 as outlined previously (Moeyaert et al. 2018) and found that the green signal and anti-FLAG signal overlapped. We opted to only show the endogenous green (488 nm) signal in our figures. The CaMPARI2 signal was visualized using an Alexa fluor 555 secondary antibody. All samples were handled together and imaged on the same day to avoid batch effects.

Average intensity projections of the red:green signal ratio were calculated for each VNC hemisegment. The experimental region of interest (ROI) started at the A4 A18b cell body and ended at the tip of the VNC. To calculate the background, the same ROI was shifted medially. For each signal, the background ROI fluorescence was subtracted from the experimental ROI fluorescence, before being divided by the area of the ROI. To calculate the red:green ratio, the background-subtracted red value was divided by the background-subtracted green value.

Statistics

When applicable, values were normalized to the average of the control. All statistical analyses were performed with Prism 10 (GraphPad Software). Numerical data in graphs show individual measurements (dots) and means (bars). The number of replicates (n) and definition of measurement reporter (i.e., animal or axon) for each data set are in the corresponding figure legend.

Acknowledgments

We thank Jim Truman for reagents, Heather Pollington and Ben Brissette for comments on the manuscript, and Heather Pollington for help making schematics in Adobe Illustrator. Antibodies obtained from the Developmental Studies Hybridoma Bank, created by the National Institute of Child Health and Human Development (NICHD) of the National Institutes of Health (NIH), and maintained at the University of Iowa, Department of Biology, were used in this study. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. Funding was provided by Howard Hughes Medical Institute (HHMI; to C.Q.D.) and NICHD (F32 HD105344 to K.L.). This article is subject to HHMI's Open Access to Publications policy. HHMI laboratory heads have previously granted a nonexclusive CC BY 4.0 license to the public and a sublicensable license to HHMI in their research articles. Pursuant to those licenses, the author-accepted manuscript of this article can be made freely available under a CC BY 4.0 license immediately upon publication.

Author contributions: K.L. conceptualized the study, curated and analyzed the data, validated the results, and wrote and edited the manuscript. N.R.C. and J.G. curated and analyzed the data and edited the manuscript. C.Q.D. conceptualized and supervised the study and wrote and edited the manuscript.

Footnotes

Article published online ahead of print. Article and publication date are online at http://www.genesdev.org/cgi/doi/10.1101/gad.353223.125.

Competing interest statement

The authors declare no competing interests.

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