Cerebellar nuclei (CN) neurons serve as the primary output of the cerebellum and originate from the cerebellar primordium at the early stages of cerebellar development. Employing various methodologies, we have characterized a specific subset of CN neurons that do not originate from the rhombic lip and the ventricular zone of the cerebellar primordium. Embryos were collected from timed pregnant mice at early stages of development and processed for immunohistochemistry (IHC), Western blotting, in situ hybridization (ISH), embryonic culture, DiI labeling, or flow cytometry analysis (FCM). Our findings indicate that a subset of CN neurons expressing α-synuclein (SNCA), OTX2, MEIS2, and p75NTR (NGFR) are located in the rostro-ventral (rv) region of the nuclear transitory zone (NTZ), while CN neurons derived from the rhombic lip are positioned in the caudo-dorsal (cd) area of the NTZ in the cerebellar primordium. Utilizing Otx2-GFP and Atoh1-/- mice, we have determined that these cells do not originate from the germinal zone of the cerebellar primordium. These results suggest the existence of a novel extrinsic germinal zone for the cerebellar primordium, possibly the mesencephalon, from which early CN neurons originate.The cerebellum contains a variety of distinct neuronal populations, each playing a significant role in its function within the brain. This research demonstrates that a particular subset of cerebellar nuclei neurons originates from a previously unrecognized germinal zone specific to the cerebellar primordium, independently of Atoh1's influence.

The male-specific dosage compensation complex (DCC), which consists of five proteins and two non-coding roX RNAs, is necessary for the transcriptional enhancement of X-linked genes to compensate for the sex chromosome monosomy in Drosophila XY males compared with XX females. The MSL1 and MSL2 proteins form the heterotetrameric core of DCC and are critical for the specific recruitment of the DCC to the high-affinity "entry" sites (HAS) on the X chromosome. In this study, we demonstrated that the N-terminal region of MSL1 is critical for stability and functions of MSL1. Amino acid deletions and substitutions in the N-terminal region of MSL1 strongly affect both the interaction with roX2 RNA and DCC binding to HAS on the X chromosome. In particular, substitution of the conserved N-terminal amino-acids 3-7 in MSL1 (MSL1GS) affects dosage compensation similar to the inactivation of genes encoding roX RNAs. In addition, MSL1GS binds to promoters such as MSL1WT but does not co-bind with MSL2 and MSL3 to X chromosomal HAS. However, overexpression of MSL2 partially restores the functional activity of MSL1GS in dosage compensation. Thus, the interaction of MSL1 with roX RNA is critical for the efficient assembly of DCCs on HAS of the male X chromosome.

Ataxin-2 (ATXN2) is a gene implicated in spinocerebellar ataxia type II (SCA2), amyotrophic lateral sclerosis (ALS) and Parkinsonism. The encoded protein is a therapeutic target for ALS and related conditions. ATXN2 (or Atx2 in insects) can function in translational activation, translational repression, mRNA stability and in the assembly of mRNP-granules, a process mediated by intrinsically disordered regions (IDRs). Previous work has shown that the LSm (Like-Sm) domain of Atx2, which can help stimulate mRNA translation, antagonizes mRNP-granule assembly. Here we advance these findings through a series of experiments on Drosophila and human Ataxin-2 proteins. Results of Targets of RNA Binding Proteins Identified by Editing (TRIBE), co-localization and immunoprecipitation experiments indicate that a polyA-binding protein (PABP) interacting, PAM2 motif of Ataxin-2 may be a major determinant of the mRNA and protein content of Ataxin-2 mRNP granules. Experiments with transgenic Drosophila indicate that while the Atx2-LSm domain may protect against neurodegeneration, structured PAM2- and unstructured IDR- interactions both support Atx2-induced cytotoxicity. Taken together, the data lead to a proposal for how Ataxin-2 interactions are remodelled during translational control and how structured and non-structured interactions contribute differently to the specificity and efficiency of RNP granule condensation as well as to neurodegeneration.

Recent findings indicate that the translation elongation rate influences mRNA stability. One of the factors that has been implicated in this link between mRNA decay and translation speed is the yeast DEAD-box helicase Dhh1p. Here, we demonstrate that the human ortholog of Dhh1p, DDX6, triggers deadenylation-dependent decay of inefficiently translated mRNAs in human cells. DDX6 interacts with the ribosome through the Phe-Asp-Phe (FDF) motif in its RecA2 domain. Furthermore, RecA2-mediated interactions and ATPase activity are both required for DDX6 to destabilize inefficiently translated mRNAs. Using ribosome profiling and RNA sequencing, we identified two classes of endogenous mRNAs that are regulated in a DDX6-dependent manner. The identified targets are either translationally regulated or regulated at the steady-state-level and either exhibit signatures of poor overall translation or of locally reduced ribosome translocation rates. Transferring the identified sequence stretches into a reporter mRNA caused translation-and DDX6-dependent degradation of the reporter mRNA. In summary, these results identify DDX6 as a crucial regulator of mRNA translation and decay triggered by slow ribosome movement and provide insights into the mechanism by which DDX6 destabilizes inefficiently translated mRNAs.

Communication between distant cells can be mediated by extracellular vesicles (EVs) that deliver proteins and RNAs to recipient cells. Little is known about how EVs are targeted to specific cell types. Here, we identify the Drosophila cell-surface protein Stranded at second (Sas) as a targeting ligand for EVs. Full-length Sas is present in EV preparations from transfected Drosophila Schneider 2 (S2) cells. Sas is a binding partner for the Ptp10D receptor tyrosine phosphatase, and Sas-bearing EVs preferentially target to cells expressing Ptp10D. We used co-immunoprecipitation and peptide binding to show that the cytoplasmic domain (ICD) of Sas binds to dArc1 and mammalian Arc. dArc1 and Arc are related to retrotransposon Gag proteins. They form virus-like capsids which encapsulate Arc and other mRNAs and are transported between cells via EVs. The Sas ICD contains a motif required for dArc1 binding that is shared by the mammalian and Drosophila amyloid precursor protein (APP) orthologs, and the APP ICD also binds to mammalian Arc. Sas facilitates delivery of dArc1 capsids bearing dArc1 mRNA into distant Ptp10D-expressing recipient cells in vivo.

Full text Figures and data Side by side Abstract eLife assessment Introduction Results Discussion Materials and methods Data availability References Peer review Author response Article and author information Abstract Touch sensation is primarily encoded by mechanoreceptors, called low-threshold mechanoreceptors (LTMRs), with their cell bodies in the dorsal root ganglia. Because of their great diversity in terms of molecular signature, terminal endings morphology, and electrophysiological properties, mirroring the complexity of tactile experience, LTMRs are a model of choice to study the molecular cues differentially controlling neuronal diversification. While the transcriptional codes that define different LTMR subtypes have been extensively studied, the molecular players that participate in their late maturation and in particular in the striking diversity of their end-organ morphological specialization are largely unknown. Here we identified the TALE homeodomain transcription factor Meis2 as a key regulator of LTMRs target-field innervation in mice. Meis2 is specifically expressed in cutaneous LTMRs, and its expression depends on target-derived signals. While LTMRs lacking Meis2 survived and are normally specified, their end-organ innervations, electrophysiological properties, and transcriptome are differentially and markedly affected, resulting in impaired sensory-evoked behavioral responses. These data establish Meis2 as a major transcriptional regulator controlling the orderly formation of sensory neurons innervating peripheral end organs required for light touch. eLife assessment This fundamental study identifies the homeodomain transcription factor Meis2 as a transcriptional regulator of maturation and end-organ innervation of low-threshold mechanoreceptors (LTMRs) in the dorsal root ganglia (DRG) of mice. The authors use histology, behavioral tests, RNA-sequencing, and electrophysiological recordings to provide evidence that conditional deletion of Meis2 in postmitotic DRG neurons causes gene expression changes together with targeting errors and altered sensory neuron responses, ultimately resulting in reduced sensitivity to light touch in mutant animals. The data presented are convincing, the discussion comprehensive, and the conclusions drawn justified. https://doi.org/10.7554/eLife.89287.3.sa0 About eLife assessments Introduction Tactile stimuli like brush, light pressure, or roughness engage highly specialized and diverse arrays of mechanoreceptors in both the hairy and glabrous skin (Abraira and Ginty, 2013; Delmas et al., 2011; Handler and Ginty, 2021; Lechner and Lewin, 2013; Wu et al., 2021; Zimmerman et al., 2014; Li et al., 2011). Somatosensory perception via these mechanoreceptors involves primary sensory neurons whose cell bodies reside within dorsal root ganglia (DRG) and cranial sensory ganglia. These sensory neurons within the DRG can be broadly classified as nociceptors, mechanoreceptors, or proprioceptors, and each group is characterized by the expression of specific combination of genes and have distinctive physiological properties and projections within the spinal cord and periphery (Lallemend and Ernfors, 2012; Marmigère and Ernfors, 2007; Vermeiren et al., 2020). Cutaneous mechanoreceptors or low threshold mechanoreceptors (LMTRs) exhibit a variety of specialized terminal endings in the hairy and glabrous skin with strikingly unique morphologies (Abraira and Ginty, 2013; Delmas et al., 2011; Handler and Ginty, 2021; Lechner and Lewin, 2013; Zimmerman et al., 2014; Li et al., 2011; Sharma et al., 2020; Schwaller et al., 2021). LTMRs projecting to the glabrous skin innervate Merkel cell complexes or Meissner corpuscles at the dermal–epidermal border. Those innervating Merkel cells in the glabrous or hairy skin have large thickly myelinated axons (Aβ-fibers) and are characterized as slowly adapting mechanoreceptors responding to skin movement and static displacement (also referred to as Aβ-SAIs). On the other hand, Meissner corpuscles are mechanoreceptors which are only sensitive to skin movement or vibration (rapidly adapting mechanoreceptors) and are referred to as Aβ-RAs. LTMRs innervating hair follicles in the hairy skin can form lanceolate endings or circumferential endings. Virtually all mechanoreceptors innervating hairs show rapidly adapting properties and respond only to hair movement, but not to static displacement (Lechner and Lewin, 2013). LTMRs with large myelinated axons innervating hairy skin are characterized as Aβ-RAs, and a specialized population of slowly conducting myelinated fibers called D-hair mechanoreceptors (or Aδ-RAs) also form lanceolate endings on small hairs. D-hair mechanoreceptors are most sensitive to low-velocity stroking, have large receptive fields, and are directionally tuned (Li et al., 2011; Walcher et al., 2018). A small number of LTMRs in the hairy skin are not activated by hair movement but show properties of rapidly adapting mechanoreceptors (Lewin and McMahon, 1991). These were originally characterized as so-called field receptors (Lewin and McMahon, 1991; Burgess and Horch, 1973) and were recently shown to form circumferential endings around hair follicles (Bai et al., 2015). LTMRs tuned to high-frequency vibration are called Aβ-RAII and innervate Pacinian corpuscles deep in the skin or on the bone (Schwaller et al., 2021). Ruffini endings that are thought to be innervated by stretch-sensitive mechanoreceptors (Aβ-SAII) remain poorly characterized in mice (Handler and Ginty, 2021). Recent advances in combining single-cell transcriptomic and deep RNA sequencing with genetic tracing have tremendously extended the classical subtypes repertoire and clustered at least 20 different subtypes of LTM neurons (Wu et al., 2021; Sharma et al., 2020; Bai et al., 2015; Usoskin et al., 2015; Zheng et al., 2019). Cracking the transcriptional codes supporting sensory neuron identity and diversification has been the object of tremendous efforts in the last decades (Wu et al., 2021; Lallemend and Ernfors, 2012; Marmigère and Ernfors, 2007; Sharma et al., 2020; Usoskin et al., 2015; Zheng et al., 2019). For instance, the functions of specification factors or terminal selectors, like Maf, Shox2, Runx3, Pea3, and ER81, have been functionally implicated in LTMR segregation (Lallemend and Ernfors, 2012; Marmigère and Ernfors, 2007; Scott et al., 2011; Abdo et al., 2011; Arber et al., 2000; Hu et al., 2012). Whereas the specific function of adhesion molecules in shaping the assembly of touch circuitry is being unraveled (Meltzer et al., 2023), the transcriptional control of target cell innervation within the skin and of the establishment of specialized peripheral end-organ complexes is less understood. Meis2 is another TF expressed in LTMRs (Sharma et al., 2020; Usoskin et al., 2015; Zheng et al., 2019). Its mutation in humans causes severe neurodevelopmental defects (Gangfuß et al., 2021; Giliberti et al., 2020; Shimojima et al., 2017), and somatic mutations of its DNA consensus binding site are associated with neurodevelopmental defects (Bae et al., 2022). It belongs to a highly conserved homeodomain family containing three members in mammals, Meis1, Meis2, and Meis3 (Geerts et al., 2003; Longobardi et al., 2014), and Meis1 is necessary for target-field innervation of sympathetic peripheral neurons (Bouilloux et al., 2016). We thus wondered if Meis2 could also be a pertinent regulator of late primary sensory neuron differentiation. Here, we show that Meis2 regulates the innervation of specialized cutaneous end organs important for LTMR function. We confirmed that Meis2 expression is restricted to LTMR subclasses at late developmental stages compatible with functions in specification and/or target-field innervation. We generated mice carrying an inactive Meis2 gene in postmitotic sensory neurons. While these animals are healthy and viable and do not exhibit any neuronal loss, they display tactile sensory defects in electrophysiological and behavioral assays. Consistent with these findings, we identified major morphological alterations in LTMR end-organ structures in Meis2 null sensory neurons. Finally, transcriptomic analysis at late embryonic stages showed dysregulation of synapse and neuronal projection-related genes that underpin these functional and behavioral phenotypes. Results Meis2 is expressed by cutaneous LTMRs We analyzed Meis2 expression using in situ hybridization (ISH) at various developmental stages in both mouse and chick lumbar DRG, combined with well-established molecular markers of sensory neuron subclasses (Figure 1A, Figure 1—figure supplements 1 and 2). In mouse, Meis2 mRNA was first detected at embryonic day (E) 11.5 in a restricted group of large DRG neurons. This restricted expression pattern was maintained at E14.5, E18.5, and adult stages (Figure 1A). In chick, Meis2 was expressed in most DRG neurons at Hamburger–Hamilton stage (HH) 24, but later becomes restricted to a well-defined subpopulation in the ventrolateral part of the DRG where LTMRs and proprioceptors are located (Rifkin et al., 2000; Figure 1—figure supplement 2A). In both species, Meis2-positive cells also expressed the pan-neuronal marker Islet1, indicating that they are postmitotic neurons. In chick, we estimated that Meis2-positive cells represented about 15% of Islet1-positive DRG neurons at HH29 and HH36, respectively, suggesting a stable expression in given neuronal populations during embryonic development. Double ISH for Meis2 and Ntrk2, Ntrk3 or Ret mRNAs in E14.5 and E18.5 mouse embryonic DRG (Figure 1—figure supplement 1A and B) showed a large co-expression in Ntrk2- and Ntrk3-positive neurons confirming that Meis2-positive neurons belong to the LTMR and proprioceptive subpopulations. Finally, double ISH for Meis2 and Ret in E14.5 mouse DRG showed that virtually all large Ret-positive neurons representing part of the LTMR pool co-expressed Meis2 at this stage before the emergence of the small nociceptive Ret-positive population. Similar results were found in chick at HH29 (Figure 1—figure supplement 2B). Figure 1 with 3 supplements see all Download asset Open asset Meis2 is expressed in subclasses of dorsal root ganglia (DRG) cutaneous mechanoreceptive neurons in mouse embryos. (A) In situ hybridization (ISH) for Meis2 mRNA showed expression in a subpopulation of DRG sensory neurons at embryonic stages E11.5, E14.5, and E18.5, at P0, and at adult stages. Dashed lines delineate the DRG. Scale bar = 50 µm. (B) IF for Meis2 (red) and c-Maf, Ntrk2, or Ntrk3 (blue) at P7 following injection of cholera toxin B subunit (CTB in green) in the skin of newborn pups. Note that Meis2+/CTB+ retro-traced sensory neurons co-expressed c-Maf, Ntrk2, or Ntrk3 (arrows). Scale bar = 50 µm. We estimated that 30.5 ± 3.5% (mean ± SEM; n = 3) of Meis2-positive neurons co-expressed Ntrk2, and that 39.5 ± 5.4% co-expressed Ntrk3. Conversely, Meis2 was co-expressed in 53.6 ± 9.4% of Ntrk2-positive neurons, and in 78.5 ± 5.0% of Ntrk3-positive neurons. Meis2 expression depends on target-derived signals (C, D). (C) Representative images of Meis2 mRNA expression (blue or pseudo-colored in red) and islet1 (green) in DRGs of Hamburger–Hamilton stage (HH) 36 chick embryos on the ablated and contralateral sides. Box plots showing the number of Islet1+/Meis2+ DRG neurons per section at stage HH36 following limb bud ablation. For Islet1-positive neurons, the contralateral side was considered as 100% of neurons per section. For Meis2-positive neurons, values represent the percentage of Meis2+ over Islet1+ neurons. (D) Representative images of Meis2 mRNA expression (blue or pseudo-colored in red) and islet1 (green) in DRGs of HH29 chick embryos on the ablated and contralateral sides. Box plots showing the quantification of Islet1+/Meis2+ neurons number per section at stage HH29 on the contralateral and ablated sides. Arrowheads point at remaining Meis2-positive VL neurons. Dashed lines encircle the DRGs. **p≤0.005; ***p≤0.0005; ns = not significant following Student's t-test. n = 3 chick embryos. Scale bar = 100 µm. Altered touch perception in Meis2 mutant mice (E–H). (E) Box plots showing the responses following application of Von Frey filaments of different forces. Isl1+/Cre::Meis2LoxP/LoxP mice exhibited a significantly reduced sensitivity to the 0.16, 0.4, and 0.6 g Von Frey filaments but not to higher forces filaments compared to WT and Isl1+/Cre littermates. * p≤0.05; ** p≤0.005; *** p≤0.001 following Kruskal–Wallis statistical analysis. (F) Box plots showing the dynamic touch responses when the hind paw palms of individual mice were stroked with a tapered cotton swab. Analysis showed that Isl1+/Cre::Meis2LoxP/LoxP mice were less responsive to the stimulus than WT and Isl1+/Cre littermates. *** p≤0.0001 following a one-way ANOVA statistical analysis. (G) Box plots indicating that the latency to the first signs of aversive behavior in the hot plate test is similar in all groups of mice. WT, n = 19; Isl1+/Cre, n = 16; Isl1+/Cre::Meis2LoxP/LoxP, n = 9. (H) Box plots showing the number of bouts when a sticky paper tape was applied on the back skin of mice. Analysis indicated a significant decrease in the number of bouts in Isl1+/Cre::Meis2LoxP/LoxP mice compared to WT and Isl1+/Cre littermates. * p≤0.05 following a one-way ANOVA statistical analysis. Figure 1—source data 1 Isl1+/Cre::Meis2LoxP/LoxP adult mice exhibit normal locomotion. Table recapitulating different Catwalk two-paw analysis parameters in 3-month-old female mice. Several recordings were performed for each mouse. Only sequences when mice showed a constant and straight locomotion with an average speed between 25 and 55 cm s-1 were selected for analysis. Student's t-test analysis showed no significant differences for any of the parameters. https://cdn.elifesciences.org/articles/89287/elife-89287-fig1-data1-v1.docx Download elife-89287-fig1-data1-v1.docx In mouse, comparison of Meis2 mRNA expression to Ntrk1, a well-established marker for early nociceptive and thermo-sensitive neurons, showed that only a few Meis2-positive neurons co-expressed Ntrk1 at E11.5 and E18.5 (Figure 1—figure supplement 1C). In chick HH29 embryos, Meis2 expression was fully excluded from the Ntrk1 subpopulation (Figure 1—figure supplement 2C). In adult mouse DRG, comparison of Meis2 mRNA to Ntrk1, Calca, and TrpV1 immunostaining confirmed that Meis2-expressing neurons are largely excluded from the nociceptive and thermosensitive populations of DRG neurons. Instead, a large proportion of Meis2-positive neurons co-expressed Nefh, a marker for large myelinated neurons including LTMR and proprioceptors, and Pvalb, a specific marker for proprioceptors (Figure 1—figure supplement 1D). Finally, Meis2 expression in LTMRs projecting to the skin was confirmed by retrograde-tracing experiments using cholera toxin B subunit (CTB) coupled with a fluorochrome injected into hind paw pads of P5 newborn mice. Analyses of CTB expression in lumbar DRG 3 d later at P8 showed that many retrogradely labeled sensory neurons were also immunopositive for Meis2, Maf, Ntrk2, and Ntrk3 (Figure 1B). Altogether, our results on Meis2 co-localization with Nefh, Ntrk2, Ntrk3, Ret, Pvalb, and Maf at different embryonic and postnatal stages are consistent with previous reports on restricted Meis2 expression to the Aβ-field, Aβ-SA1 and Aβ-RA subclasses of LTMR neurons and proprioceptive neurons (Sharma et al., 2020; Usoskin et al., 2015; Zheng et al., 2019; Shin et al., 2020). The relatively lower coincidence of Meis2 and Ntrk2 expressions compared to Ntrk3 is consistent with Meis2 being excluded from the Aδ-LTMRs (D-hair mechanoreceptors). The lack of co-expression with Ntrk1 and TrpV1 also confirmed Meis2 exclusion from peptidergic and non-peptidergic subpopulations. Target-derived signals are necessary to maintain Meis2 expression The requirement for extrinsic signals provided by limb mesenchyme and muscles for proprioceptor and LTMR development has been documented (Sharma et al., 2020; Arber et al., 2000; Shin et al., 2020; Patel et al., 2003; de Nooij et al., 2013; Poliak et al., 2016; Wang et al., 2019). To test the influence of target-derived signals on Meis2 expression in sensory neurons, limb buds were unilaterally ablated in HH18 chick embryos. Embryos were harvested at HH29 and HH36, before and after ventrolateral neurons are lost, respectively (Oakley et al., 1995; Oakley et al., 1997; Calderó et al., 1998; Figure 1C and D, Figure 1—figure supplement 2D). In HH36 embryos, about 65% of Meis2-positive neurons were lost on the ablated side compared to the contralateral side (Figure 1C). This is consistent with a 30% loss of all sensory DRG neurons represented by the pan-neuronal marker Islet1, and the 50 and 65% loss of Ntrk2 and Ntrk3-positive VL-neurons, respectively (Figure 1—figure supplement 2D). The number of Ntrk2-positive DL neurons was not significantly affected. In HH27 embryos, while no significant loss of Islet1-positive neurons was detected following limb ablation, about 40% of Meis2-positive neurons were lost, and remaining Meis2-positive neurons expressed very low levels of Meis2 mRNAs (Figure 1D). These results indicate that target-derived signals are necessary for the maintenance but not the induction of Meis2 expression in sensory neurons. Meis2 gene inactivation in postmitotic sensory neurons induces severe behavioral defects We next asked whether Meis2 inactivation would induce changes in LTMR structure and function. We generated a conditional mouse mutant strain for Meis2 (Meis2LoxP/LoxP) in which the first coding exon for the homeodomain was flanked by LoxP sites (Figure 1—figure supplement 3A). To validate the use of our strain, we first crossed the Meis2LoxP/LoxP mice with the Wnt1Cre strain. This crossing efficiently inactivated Meis2 in the neural crest, and Wnt1Cre::Meis2LoxP/LoxP newborn pups exhibited a cleft palate as previously reported in another conditional Meis2 mouse strain (Machon et al., 2015; Figure 1—figure supplement 3B). They were, however, not viable, precluding functional and anatomical analyses at adult stages. To bypass this neural crest phenotype and more specifically address Meis2 function in postmitotic neurons, we crossed the Meis2LoxP/LoxP mice with the Isl1Cre/+ strain and focused our analysis on the Isl1Cre/+::Meis2LoxP/LoxP strain. Mutant pups were viable, appeared healthy, and displayed a normal palate, allowing sensory behavior investigations. We monitored tactile-evoked behaviors in adult WT, Isl1Cre/+ and Isl1Cre/+::Meis2LoxP/LoxP mice using stimuli applied to both glabrous and hairy skin. We used Von Frey filaments to apply a series of low forces ranging from 0.008 to 1.4 g to the hind paw and found the frequency of withdrawal responses to be significantly decreased in Isl1+/Cre::Meis2LoxP/LoxP mice compared to control WT and Isl1+/Cre mice between 0.16 and 0.6 g (Figure 1E), indicating that mutant mice are less responsive to light touch. No differences were observed between WT and Isl1+/Cre mice for any of the stimuli applied. Behaviors evoked from stimulation of the glabrous skin were next assessed using the 'cotton swab' dynamic touch assay (Bourane et al., 2015). Here, responses were significantly decreased in Isl1+/Cre::Meis2LoxP/LoxP mice compared to control WT and Isl1+/Cre littermates (Figure 1F). We also used the hot plate assay to assess noxious heat-evoked behaviors and found no difference in response latencies between WT, Isl1Cre/+ and Isl1Cre/+::Meis2LoxP/LoxP mice (Figure 1G). Finally, we compared the sensitivity of mice to stimuli applied to the hairy skin using the sticky tape test. Placing sticky tape on the back skin evoked attempts to remove the stimulus in a defined time window, and we found that such bouts of behavior were significantly reduced in Isl1Cre/+::Meis2LoxP/LoxP mice compared to WT and Isl1Cre/+ control mice (Figure 1H). Finally, although Meis2 and Isl1 are both expressed by spinal motor neurons and proprioceptors (Catela et al., 2016; Dasen et al., 2005; Ericson et al., 1992), we did not observe obvious motor deficits in Isl1Cre/+::Meis2LoxP/LoxP mice. Thus, in a catwalk analysis we found no differences in any of the gait parameters measured between WT and mutant mice (Figure 1—source data 1). Overall, these behavioral analyses indicate that Meis2 gene inactivation specifically affects light touch sensation both in the glabrous and the hairy skin. The impaired behavioral response to light touch in Meis2 mutant suggests that Meis2 gene activity is necessary for the anatomical and functional maturation of LTMRs. Meis2 is dispensable for LTMR specification and survival To investigate whether Meis2 gene inactivation interfered with LTMR survival during embryonic development, we performed histological analysis of the Isl1Cre/+::Meis2LoxP/LoxP and Wnt1Cre::Meis2LoxP/LoxP strains (Figure 2). There was no difference in the size of the DRGs between E16.5 WT and Isl1Cre/+::Meis2LoxP/LoxP embryos as well as in the number of Ntrk2 and Ntrk3-positive neurons (Figure 2A), suggesting no cell loss. In E18.5 embryonic DRGs, the number of LTMR and proprioceptors identified as positive for Ntrk2, Ntrk3, Ret, and Maf was unchanged following Meis2 inactivation (Figure 2C). Consistent with the lack of Meis2 expression in nociceptors, the number of Ntrk1-positive neurons was also unaffected (Figure 2B). Finally, quantification of DRG neuron populations at P0 in Wnt1Cre::Meis2LoxP/LoxP mice showed similar results with no differences in the number of Ntrk2 and Ntrk3-positive neurons (Figure 2C). At this stage, phospho-Creb (pCreb) expression in Ntrk2 and Ntrk3-positive neurons was similar in WT and mutants (Figure 2—figure supplement 1), suggesting that Ntrk signaling is not affected. Altogether, these results show that Meis2 is dispensable for LTMR and proprioceptor survival and specification during embryogenesis. Figure 2 with 1 supplement see all Download asset Open asset Meis2 is dispensable for low-threshold mechanoreceptor (LTMR) neuron survival and specification. (A) Box plots showing that the dorsal root ganglia (DRG) volumes along the rostrocaudal axis are similar in embryonic day (E) 16.5 WT and Isl1Cre/+::Meis2LoxP/LoxP embryos. IF for Ntrk2 or Ntrk3 (red) and Islet1 (blue) and box plots analysis indicating that the percentage of Ntrk2+ and Ntrk3+ neurons is not affected in E16.5 Isl1Cre/+::Meis2LoxP/LoxP. Dashed lines encircle the DRGs. n = 4; n.s. = not significant. Scale bar = 20 µm. (B) Representative images showing IF for Ntrk1, Ret, Ntrk2, Ntrk3, and Maf in E18.5 WT and Isl1Cre/+::Meis2LoxP/LoxP DRGs. Box plots showing that the number of Ret+, Ntrk2+, Ntrk3+, and Maf+ LTMR neurons and of Ntrk1+ nociceptive neurons are similar in E18.5 WT and Isl1Cre/+::Meis2LoxP/LoxP DRGs. n = 3; n.s. = not significant. Scale bar = 100 µm. (C) Representative images showing IF for Ntrk2 or Ntrk3 (green) with Pvalb or Maf (red) in P0 WT and Wnt1Cre::Meis2LoxP/LoxP DRGs. Box plots showing that the number of Ntrk2+ and Ntrk3+ neurons is unchanged in P0 WT and Wnt1Cre::Meis2LoxP/LoxP DRGs. n = 3, n.s. = not significant. Scale bar = 20 µm. Meis2 is necessary for normal end-organ innervation To better understand the molecular changes underlying tactile defects in Meis2 mutant mice, we performed RNAseq analysis on DRGs dissected from WT, Isl1Cre/+, and Isl1Cre/+::Meis2LoxP/LoxP E18.5 embryos. For all analyses, consistent with the changes measured for Meis2 and Isl1 genes (Figure 3—figure supplement 1A and B), only DEGs with a minimal fold change of 20% and a p-value <0.05 were considered. Analyses of the dataset (n = 3; p<0.05; Figure 3, Figure 3—figure supplement 1, Figure 3—source data 1) identified 43 differentially expressed genes (DEGs) in the WT vs Isl1+/Cre::Meis2LoxP/LoxP comparison, 107 DEGs in the Isl1+/Cre vs Isl1+/Cre::Meis2LoxP/LoxP comparison, and 109 DEGs in the WT vs Isl1+/Cre comparison. Among them, only 10 DEGs were found in both WT vs Isl1+/Cre::Meis2LoxP/LoxP and Isl1+/Cre vs Isl1+/Cre::Meis2LoxP/LoxP comparisons (Figure 3A). Half of them were down- or upregulated (Figure 3—figure supplement 1C), and eight were found to be expressed in sensory neurons expressing Meis2 (Figure 3—figure supplement 1D). These include three ncRNA (A230077H06Rik, Gm20163, Gm42418), the Adhesion G Protein-Coupled Receptor G3 (Adgrg3, also known as GPR97), the Cellular Repressor of E1A Stimulated Genes 2 (Creg2), predicted to be located in Golgi apparatus and endoplasmic reticulum, the Tubulin Alpha 8 (Tuba8) mutated in Polymicrogyria, a developmental malformation of the cortex (Abdollahi et al., 2009), the Hes Family BHLH Transcription Factor 5 (Hes5) activated downstream of the Notch pathway and largely involved in neuronal differentiation, the mitochondrial ribosomal protein s28 (Mrps28) whose mutation severely impairs the development of the nervous system (Pulman et al., 2019), the Phospholipase C Delta 1 (Plcd1) important for neuronal development and function of mature neurons, and the Pyridoxamine 5'-Phosphate Oxidase (Pnpo) involved in the synthesis of vitamin B6 and whose mutation causes a form of neonatal epileptic encephalopathy and motor neuron disease. Gene Ontology (GO) analysis for the 43 DEGs in the WT vs Isl1+/Cre::Meis2LoxP/LoxP comparison and for the 107 DEGs in the Isl1+/Cre vs Isl1+/Cre::Meis2LoxP/LoxP comparison revealed significant relevant hits with many terms associated with neuronal projections and functions (Figure 3B and C, Figure 3—source data 2, Figure 3—figure supplements 2 and 3). These include subsets for the GO term associated with synapse, dendrite, and axons and more specifically with GABAergic synapses, dendritic shaft, or postsynaptic membrane. None of these GO terms were significantly enriched in the WT vs Isl1+/Cre comparison (Figure 3B, Figure 3—figure supplements 2 and 3) which overall showed lower enrichment scores and p-values than in the two other datasets. It is important to note that many of the genes associated with neuron projection or synapse that were present in either WT vs Isl1+/Cre::Meis2LoxP/LoxP dataset or Isl1+/Cre vs Isl1+/Cre::Meis2LoxP/LoxP dataset, such as Oprd1, Calb2, Whrn, Lrp2, Lypd6, Grid1, and Rps21, failed to enter the list of the 10 best DEGs either because their fold changes were below the cutoff or their p-values were close to but higher than 0.05. Interestingly, a significant association with the GO term Cadherin in the Isl1+/Cre vs Isl1+/Cre::Meis2LoxP/LoxP comparison points at the protocadherin family in which several members were downregulated (Figure 3C). Finally, comparing these genes to single-cell RNAseq (scRNAseq) analysis in adult DRG neurons (Usoskin et al., 2015) showed that most of them are expressed by Meis2-expressing DRG sensory neuron subtypes (Figure 3—figure supplement 4). These molecular analyses strongly support the role of Meis2 in regulating embryonic target-field innervation. We thus investigated this hypothesis, and in P0 Wnt1Cre::Meis2LoxP/LoxP, Nefh staining in the hind paws showed strong innervation deficits as reflected by a paucity of neurofilament-positive myelinated branches in both the glabrous and hairy skin (Figure 3E). In WT newborn mice, numerous Nefh+ sensory fibers surround all dermal papillae of the hairy skin and footpad of the glabrous skin, whereas in Wnt1Cre::Meis2LoxP/LoxP littermates, very few Nefh+ sensory fibers are present and they poorly innervate the dermal papillae and footpads. Figure 3 with 4 supplements see all Download asset Open asset Meis2 inactivation dysregulates genes linked to neuronal projections and synaptogenesis. (A) Venn diagram comparing the number of differentially expressed genes (DEGs) between each genotype (n = 3; p<0.05). This comparison identified 10 DEGs that were differentially expressed compared to both control genotypes (WT or Isl1+/Cre embryos), and a total of 140 genes that were differentially expressed in Meis2 mutant compared to either WT or Isl1+/Cre embryos. (B) Gene Ontology (GO) analysis for the three paired-analysis (WT vs Isl1+/Cre::Meis2LoxP/LoxP; Isl1+/Cre vs Isl1+/Cre::Meis2LoxP/LoxP, and WT vs Isl1+/Cre) datasets using DAVID and the full RNAseq gene list as background. Graphs show the comparison of the fold enrichment and the -log10(p value) of selected significant (p<0.05) GO or KEGG_PATHWAY terms associated to synapse and neuron projections whatever the number of genes. Blue dotted line indicates a p-value of 0.05. Note that following DAVID analysis GO terms associated to synapse and neuron projections were overrepresented in the WT vs Isl1+/Cre::Meis2LoxP/LoxP and the Isl1+/Cre vs Isl1+/Cre::Meis2LoxP/LoxP datasets compared to the WT vs Isl1+/Cre dataset. (C) Heat maps showing the DEGs related to the GO terms synapse, neuron projection including dendrite, and protocadherin. (D) Representative images showing a strong overall deficit of Nefh+ (red) sensory projections innervating the dermal papillae in the hairy and the foot pads in the glabrous skin of P0 Wnt1Cre::Meis2LoxP/LoxP neonates forepaw compared to WT littermates. Dashed lines delineate the hair follicle and the epidermis. Scale bar = 50 µm. Figure 3—source data 1 Table showing the results of the bulk RNAseq analysis. https://cdn.elifesciences.org/articles/89287/elife-89287-fig3-data1-v1.xlsx Download elife-89287-fig3-data1-v1.xlsx Figure 3—source data 2 Table showing the results of the GO terms analysis performed with AVID. https://cdn.elifesciences.org/articles/89287/elife-89287-fig3-data2-v1.xlsx Download elife-89287-fig3-data2-v1.xlsx Meis2 gene is necessary for SA-LTMR morphology and function only in the glabrous skin LT

Habituation, the process by which animals learn to ignore insignificant stimuli, facilitates engagement with salient features of the environment. However, neural mechanisms underlying habituation also allow responses to familiar stimuli to be reinstated when such stimuli become potentially significant. Thus, the habituated state must allow a mechanism for habituation override. The remarkably precise knowledge of cell identity, connectivity, and information coding in Drosophila sensory circuits, as well as the availability of tools to genetically target these cells, makes Drosophila a valuable and important organism for analysis of habituation and habituation-override mechanisms. Studies of olfactory and gustatory habituation in Drosophila suggest that potentiation of GABAergic neurons underlies certain timescales of habituation and have specified some elements of a gustatory habituation-override pathway. More detailed understanding of gustatory habituation and habituation-override mechanisms will benefit from access to robust behavioral assays for (a) the proboscis extension reflex (PER) elicited by a sweet stimulus, (b) exposure paradigms that result in PER habituation, and, most critically, (c) manipulations that result in PER-habituation override. Here, we describe simple protocols for persistent sucrose exposure of tarsal hairs that lead to habituation of proboscis extension and for presentation of a novel appetitive stimuli that reinstate robust PER to habituated flies. This detailed protocol of gustatory habituation provides (a) a simple method to induce habituation by continuous exposure of the flies to sucrose for 10 min without leading to ingestion and (b) a novel method to override habituation by presenting yeast to the proboscis.

SUMMARYHow compartment-specific local proteomes are generated and maintained is inadequately understood, particularly in neurons, which display extreme asymmetries. Here we show that local enrichment of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in axons of Drosophila mushroom body neurons is necessary for cellular plasticity and associative memory formation. Enrichment is achieved via enhanced axoplasmic translation of CaMKII mRNA, through a mechanism requiring the RNA-binding protein Mub and a 23-base Mub-recognition element in the CaMKII 3’UTR. Perturbation of either dramatically reduces axonal, but not somatic, CaMKII protein without altering the distribution or amount of mRNA in vivo and both are necessary and sufficient to enhance axonal translation of reporter mRNA. Together, these data identify elevated levels of translation of an evenly distributed mRNA as a novel strategy for generating subcellular biochemical asymmetries. They further demonstrate the importance of distributional asymmetry in the computational and biological functions of neurons.

Habituated animals retain a latent capacity for robust engagement with familiar stimuli. In most instances, the ability to override habituation is best explained by postulating that habituation arises from the potentiation of inhibitory inputs onto stimulus-encoding assemblies and that habituation override occurs through disinhibition. Previous work has shown that inhibitory plasticity contributes to specific forms of olfactory and gustatory habituation in Drosophila Here, we analyze how exposure to a novel stimulus causes override of gustatory (proboscis extension reflex; PER) habituation. While brief sucrose contact with tarsal hairs causes naive Drosophila to extend their proboscis, persistent exposure reduces PER to subsequent sucrose stimuli. We show that in so habituated animals, either brief exposure of the proboscis to yeast or direct thermogenetic activation of sensory neurons restores PER response to tarsal sucrose stimulation. Similar override of PER habituation can also be induced by brief thermogenetic activation of a population of tyrosine hydroxylase (TH)-positive neurons, a subset of which send projections to the subesophageal zone (SEZ). Significantly, sensory-neuron induced habituation override requires transmitter release from these TH-positive cells. Treatments that cause override specifically influence the habituated state, with no effect on the naive sucrose response across a range of concentrations. Taken together with other findings, these observations in female flies are consistent with a model in which novel taste stimuli trigger activity in dopaminergic neurons which, directly or indirectly, inhibit GABAergic cells that drive PER habituation. The implications of these findings for general mechanisms of attentional and sensory override of habituation are discussed.SIGNIFICANCE STATEMENT Habituation can be overcome when a new context requires an enhanced response to a familiar stimulus. However, the underlying mechanisms remain incompletely understood. Previous studies have provided evidence that habituation of the sucrose-induced proboscis extension reflex (PER) in Drosophila occurs through potentiation of inhibition onto the PER pathway. This work defines controlled protocols for override of PER habituation and uses them to outline the underlying circuit mechanisms. The results presented support a model in which novel taste stimuli cause dishabituation by activating a subset of tyrosine hydroxylase (TH)-expressing neurons that inhibit GABAergic neurons whose potentiation underlies PER habituation. At a general level, these findings further highlight a central role for inhibition and disinhibition in the control of behavioral flexibility.

Blood cells arise from diverse pools of stem and progenitor cells. Understanding progenitor heterogeneity is a major challenge. The Drosophila larval lymph gland is a well-studied model to understand blood progenitor maintenance and recapitulates several aspects of vertebrate hematopoiesis. However in-depth analysis has focused on the anterior lobe progenitors (AP), ignoring the posterior progenitors (PP) from the posterior lobes. Using in situ expression mapping and developmental and transcriptome analysis, we reveal PP heterogeneity and identify molecular-genetic tools to study this abundant progenitor population. Functional analysis shows that PP resist differentiation upon immune challenge, in a JAK-STAT-dependent manner. Upon wasp parasitism, AP downregulate JAK-STAT signaling and form lamellocytes. In contrast, we show that PP activate STAT92E and remain undifferentiated, promoting survival. Stat92E knockdown or genetically reducing JAK-STAT signaling permits PP lamellocyte differentiation. We discuss how heterogeneity and compartmentalization allow functional segregation in response to systemic cues and could be widely applicable.