UNC-30/PITX coordinates neurotransmitter identity with postsynaptic GABA receptor clustering.

Terminal selectors are transcription factors that control neuronal identity by regulating expression of key effector molecules, such as neurotransmitter biosynthesis proteins and ion channels. Whether and how terminal selectors control neuronal connectivity is poorly understood. Here, we report that UNC-30 (PITX2/3), the terminal selector of GABA nerve cord motor neurons in Caenorhabditis elegans, is required for neurotransmitter receptor clustering, a hallmark of postsynaptic differentiation. Animals lacking unc-30 or madd-4B, the short isoform of the motor neuron-secreted synapse organizer madd-4 (punctin/ADAMTSL), display severe GABA receptor type A (GABAAR) clustering defects in postsynaptic muscle cells. Mechanistically, UNC-30 acts directly to induce and maintain transcription of madd-4B and GABA biosynthesis genes (e.g. unc-25/GAD, unc-47/VGAT). Hence, UNC-30 controls GABAA receptor clustering in postsynaptic muscle cells and GABA biosynthesis in presynaptic cells, transcriptionally coordinating two crucial processes for GABA neurotransmission. Further, we uncover multiple target genes and a dual role for UNC-30 as both an activator and a repressor of gene transcription. Our findings on UNC-30 function may contribute to our molecular understanding of human conditions, such as Axenfeld-Rieger syndrome, caused by PITX2 and PITX3 gene variants.

Maintenance of neuronal identity in C. elegans and beyond: Lessons from transcription and chromatin factors.

Neurons are remarkably long-lived, non-dividing cells that must maintain their functional features (e.g., electrical properties, chemical signaling) for extended periods of time - decades in humans. How neurons accomplish this incredible feat is poorly understood. Here, we review recent advances, primarily in the nematode C. elegans, that have enhanced our understanding of the molecular mechanisms that enable post-mitotic neurons to maintain their functionality across different life stages. We begin with "terminal selectors" - transcription factors necessary for the establishment and maintenance of neuronal identity. We highlight new findings on five terminal selectors (CHE-1 [Glass], UNC-3 [Collier/Ebf1-4], LIN-39 [Scr/Dfd/Hox4-5], UNC-86 [Acj6/Brn3a-c], AST-1 [Etv1/ER81]) from different transcription factor families (ZNF, COE, HOX, POU, ETS). We compare the functions of these factors in specific neuron types of C. elegans with the actions of their orthologs in other invertebrate (D. melanogaster) and vertebrate (M. musculus) systems, highlighting remarkable functional conservation. Finally, we reflect on recent findings implicating chromatin-modifying proteins, such as histone methyltransferases and Polycomb proteins, in the control of neuronal terminal identity. Altogether, these new studies on transcription factors and chromatin modifiers not only shed light on the fundamental problem of neuronal identity maintenance, but also outline mechanistic principles of gene regulation that may operate in other long-lived, post-mitotic cell types.

Molecular and Cellular Mechanisms of Motor Circuit Development.

Motor circuits represent the main output of the central nervous system and produce dynamic behaviors ranging from relatively simple rhythmic activities like swimming in fish and breathing in mammals to highly sophisticated dexterous movements in humans. Despite decades of research, the development and function of motor circuits remain poorly understood. Breakthroughs in the field recently provided new tools and tractable model systems that set the stage to discover the molecular mechanisms and circuit logic underlying motor control. Here, we describe recent advances from both vertebrate (mouse, frog) and invertebrate (nematode, fruit fly) systems on cellular and molecular mechanisms that enable motor circuits to develop and function and highlight conserved and divergent mechanisms necessary for motor circuit development.

The SWI/SNF chromatin remodeling assemblies BAF and PBAF differentially regulate cell cycle exit and cellular invasion in vivo.

Chromatin remodelers such as the SWI/SNF complex coordinate metazoan development through broad regulation of chromatin accessibility and transcription, ensuring normal cell cycle control and cellular differentiation in a lineage-specific and temporally restricted manner. Mutations in genes encoding the structural subunits of chromatin, such as histone subunits, and chromatin regulating factors are associated with a variety of disease mechanisms including cancer metastasis, in which cancer co-opts cellular invasion programs functioning in healthy cells during development. Here we utilize Caenorhabditis elegans anchor cell (AC) invasion as an in vivo model to identify the suite of chromatin agents and chromatin regulating factors that promote cellular invasiveness. We demonstrate that the SWI/SNF ATP-dependent chromatin remodeling complex is a critical regulator of AC invasion, with pleiotropic effects on both G0 cell cycle arrest and activation of invasive machinery. Using targeted protein degradation and enhanced RNA interference (RNAi) vectors, we show that SWI/SNF contributes to AC invasion in a dose-dependent fashion, with lower levels of activity in the AC corresponding to aberrant cell cycle entry and increased loss of invasion. Our data specifically implicate the SWI/SNF BAF assembly in the regulation of the G0 cell cycle arrest in the AC, whereas the SWI/SNF PBAF assembly promotes AC invasion via cell cycle-independent mechanisms, including attachment to the basement membrane (BM) and activation of the pro-invasive fos-1/FOS gene. Together these findings demonstrate that the SWI/SNF complex is necessary for two essential components of AC invasion: arresting cell cycle progression and remodeling the BM. The work here provides valuable single-cell mechanistic insight into how the SWI/SNF assemblies differentially contribute to cellular invasion and how SWI/SNF subunit-specific disruptions may contribute to tumorigeneses and cancer metastasis.

Widespread employment of conserved C. elegans homeobox genes in neuronal identity specification.

Homeobox genes are prominent regulators of neuronal identity, but the extent to which their function has been probed in animal nervous systems remains limited. In the nematode Caenorhabditis elegans, each individual neuron class is defined by the expression of unique combinations of homeobox genes, prompting the question of whether each neuron class indeed requires a homeobox gene for its proper identity specification. We present here progress in addressing this question by extending previous mutant analysis of homeobox gene family members and describing multiple examples of homeobox gene function in different parts of the C. elegans nervous system. To probe homeobox function, we make use of a number of reporter gene tools, including a novel multicolor reporter transgene, NeuroPAL, which permits simultaneous monitoring of the execution of multiple differentiation programs throughout the entire nervous system. Using these tools, we add to the previous characterization of homeobox gene function by identifying neuronal differentiation defects for 14 homeobox genes in 24 distinct neuron classes that are mostly unrelated by location, function and lineage history. 12 of these 24 neuron classes had no homeobox gene function ascribed to them before, while in the other 12 neuron classes, we extend the combinatorial code of transcription factors required for specifying terminal differentiation programs. Furthermore, we demonstrate that in a particular lineage, homeotic identity transformations occur upon loss of a homeobox gene and we show that these transformations are the result of changes in homeobox codes. Combining the present with past analyses, 113 of the 118 neuron classes of C. elegans are now known to require a homeobox gene for proper execution of terminal differentiation programs. Such broad deployment indicates that homeobox function in neuronal identity specification may be an ancestral feature of animal nervous systems.

Transcriptional mechanisms of motor neuron development in vertebrates and invertebrates.

Across phylogeny, motor neurons (MNs) represent a single but often remarkably diverse neuronal class composed of a multitude of subtypes required for vital behaviors, such as eating and locomotion. Over the past decades, seminal studies in multiple model organisms have advanced our molecular understanding of the early steps of MN development, such as progenitor specification and acquisition of MN subtype identity, by revealing key roles for several evolutionarily conserved transcription factors. However, very little is known about the molecular strategies that allow distinct MN subtypes to maintain their identity- and function-defining features during the late steps of development and postnatal life. Here, we provide an overview of invertebrate and vertebrate studies on transcription factor-based strategies that control early and late steps of MN development, aiming to highlight evolutionarily conserved gene regulatory principles necessary for establishment and maintenance of neuronal identity.

Efficacy of auxin-inducible protein degradation in C. elegans tissues using different auxins and TIR1-expressing strains.

The auxin-inducible degradation system has emerged as a powerful tool to deplete proteins of interest in cells and tissues of various model organisms, including C. elegans 2-5 . Here, we present a detailed protocol to perform AID-driven spatiotemporal depletion of specific proteins in C. elegans tissues. First, we introduced the AID degron and a fluorescent reporter at two conserved proteins: (a) the transcription factor CFI-1 (human ARID3), which is expressed in the nucleus of multiple C. elegans neurons and head muscle cells 6,7 , and (b) the broadly expressed translation initiation factor Y47D3A.21 (human DENR) that localizes in the cytoplasm. Second, we provide a step-by-step guide on how to generate C. elegans strains suitable for AID-mediated protein (CFI-1 and DENR) depletion. Third, we find that the degree of CFI-1 and DENR depletion in C. elegans tissues is comparable upon treatment with either natural auxin (indole-3-acetic acid (IAA) or a water-soluble synthetic auxin analog (K-NAA). Last, we compare the degree of AID-mediated CFI-1 depletion in C. elegans neurons when the transport inhibitor response 1 (TIR1), component of the SCF ubiquitin ligase complex, is provided in neurons or all somatic cells. Altogether, this protocol provides side-by-side comparisons of different auxins and TIR1-expressing lines. Such comparisons may benefit future studies of AID-mediated protein depletion in C. elegans . Graphical abstract: Image provided as pdf (together with Figures). Highlights: Efficient protein depletion in C. elegans tissues upon treatment with either natural or synthetic auxins. Pansomatic TIR1 expression leads to efficient depletion of CFI-1 and DENR.Panneuronal TIR1 expression leads to neuron-specific, yet variable CFI-1 depletion.The AID system is compatible with fluorescence microscopy, Western blotting and behavioral assays.

Protocol for auxin-inducible protein degradation in C. elegans using different auxins and TIR1-expressing strains.

The auxin-inducible degron (AID) system is a powerful tool to deplete proteins in vivo. Here, we present a protocol for AID-mediated depletion of two proteins (CFI-1/AT-rich interaction domain 3 [ARID3] and Y47D3A.21/density-regulated re-initiation and release factor [DENR]) in C. elegans tissues using different auxins and transport inhibitor response 1 (TIR1)-expressing strains. We describe steps for genetic crossing, sample preparation, fluorescent microscopy, and treatment with either natural (indole-3-acetic acid [IAA]) or synthetic (1-naphthaleneacetic acid, potassium salt [K-NAA]) auxins. We then detail procedures for comparing the degree of CFI-1 depletion in C. elegans neurons upon panneuronal or pansomatic TIR1 expression. For complete details on the use and execution of this protocol, please refer to Li et al.1,2.

The Iroquois (Iro/Irx) homeobox genes are conserved Hox targets involved in motor neuron development.

The Iroquois (Iro/Irx) homeobox genes encode transcription factors with fundamental roles in animal development. Despite their link to various congenital conditions in humans, our understanding of Iro/Irx gene expression, function, and regulation remains incomplete. Here, we conducted a systematic expression analysis of all six mouse Irx genes in the embryonic spinal cord. We found five Irx genes (Irx1, Irx2, Irx3, Irx5, and Irx6) to be confined mostly to ventral spinal domains, offering new molecular markers for specific groups of post-mitotic motor neurons (MNs). Further, we engineered Irx2, Irx5, and Irx6 mouse mutants and uncovered essential but distinct roles for Irx2 and Irx6 in MN development. Last, we found that the highly conserved regulators of MN development across species, the HOX proteins, directly control Irx gene expression both in mouse and C. elegans MNs, critically expanding the repertoire of HOX target genes in the developing nervous system. Altogether, our study provides important insights into Iro/Irx expression and function in the developing spinal cord, and uncovers an ancient gene regulatory relationship between HOX and Iro/Irx genes.

UNC-30/PITX coordinates neurotransmitter identity with postsynaptic GABA receptor clustering.

Terminal selectors are transcription factors that control neuronal identity by regulating the expression of key effector molecules, such as neurotransmitter (NT) biosynthesis proteins, ion channels and neuropeptides. Whether and how terminal selectors control neuronal connectivity is poorly understood. Here, we report that UNC-30 (PITX2/3), the terminal selector of GABA motor neuron identity in C. elegans , is required for NT receptor clustering, a hallmark of postsynaptic differentiation. Animals lacking unc-30 or madd-4B, the short isoform of the MN-secreted synapse organizer madd-4 ( Punctin/ADAMTSL ), display severe GABA receptor type A (GABA A R) clustering defects in postsynaptic muscle cells. Mechanistically, UNC-30 acts directly to induce and maintain transcription of madd-4B and GABA biosynthesis genes (e.g., unc-25/GAD , unc-47/VGAT ). Hence, UNC-30 controls GABA A R clustering on postsynaptic muscle cells and GABA biosynthesis in presynaptic cells, transcriptionally coordinating two critical processes for GABA neurotransmission. Further, we uncover multiple target genes and a dual role for UNC-30 both as an activator and repressor of gene transcription. Our findings on UNC-30 function may contribute to our molecular understanding of human conditions, such as Axenfeld-Rieger syndrome, caused by PITX2 and PITX3 gene mutations.

A molecular atlas of adult C. elegans motor neurons reveals ancient diversity delineated by conserved transcription factor codes.

Motor neurons (MNs) constitute an ancient cell type targeted by multiple adult-onset diseases. It is therefore important to define the molecular makeup of adult MNs in animal models and extract organizing principles. Here, we generate a comprehensive molecular atlas of adult Caenorhabditis elegans MNs and a searchable database. Single-cell RNA sequencing of 13,200 cells reveals that ventral nerve cord MNs cluster into 29 molecularly distinct subclasses. Extending C. elegans Neuronal Gene Expression Map and Network (CeNGEN) findings, all MN subclasses are delineated by distinct expression codes of either neuropeptide or transcription factor gene families. Strikingly, combinatorial codes of homeodomain transcription factor genes succinctly delineate adult MN diversity in both C. elegans and mice. Further, molecularly defined MN subclasses in C. elegans display distinct patterns of connectivity. Hence, our study couples the connectivity map of the C. elegans motor circuit with a molecular atlas of its constituent MNs and uncovers organizing principles and conserved molecular codes of adult MN diversity.

Cell context-dependent CFI-1/ARID3 functions control neuronal terminal differentiation.

AT-rich interaction domain 3 (ARID3) transcription factors are expressed in the nervous system, but their mechanisms of action are largely unknown. Here, we provide, in vivo, a genome-wide binding map for CFI-1, the sole C. elegans ARID3 ortholog. We identify 6,396 protein-coding genes as putative direct targets of CFI-1, most of which encode neuronal terminal differentiation markers. In head sensory neurons, CFI-1 directly activates multiple terminal differentiation genes, thereby acting as a terminal selector. In motor neurons, however, CFI-1 acts as a direct repressor, continuously antagonizing three transcriptional activators. By focusing on the glr-4/GRIK4 glutamate receptor locus, we identify proximal CFI-1 binding sites and histone methyltransferase activity as necessary for glr-4 repression. Rescue assays reveal functional redundancy between core and extended DNA-binding ARID domains and a strict requirement for REKLES, the ARID3 oligomerization domain. Altogether, this study uncovers cell-context-dependent mechanisms through which a single ARID3 protein controls the terminal differentiation of distinct neuron types.

Translation of dipeptide repeat proteins in C9ORF72 ALS/FTD through unique and redundant AUG initiation codons.

A hexanucleotide repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A hallmark of ALS/FTD pathology is the presence of dipeptide repeat (DPR) proteins, produced from both sense GGGGCC (poly-GA, poly-GP, poly-GR) and antisense CCCCGG (poly-PR, poly-PG, poly-PA) transcripts. Translation of sense DPRs, such as poly-GA and poly-GR, depends on non-canonical (non-AUG) initiation codons. Here, we provide evidence for canonical AUG-dependent translation of two antisense DPRs, poly-PR and poly-PG. A single AUG is required for synthesis of poly-PR, one of the most toxic DPRs. Unexpectedly, we found redundancy between three AUG codons necessary for poly-PG translation. Further, the eukaryotic translation initiation factor 2D (EIF2D), which was previously implicated in sense DPR synthesis, is not required for AUG-dependent poly-PR or poly-PG translation, suggesting that distinct translation initiation factors control DPR synthesis from sense and antisense transcripts. Our findings on DPR synthesis from the C9ORF72 locus may be broadly applicable to many other nucleotide repeat expansion disorders.

A molecular atlas of adult C. elegans motor neurons reveals ancient diversity delineated by conserved transcription factor codes.

Motor neurons (MNs) constitute an ancient cell type targeted by multiple adult-onset diseases. It is therefore important to define the molecular makeup of adult MNs in animal models and extract organizing principles. Here, we generated a comprehensive molecular atlas of adult Caenorhabditis elegans MNs and a searchable database (http://celegans.spinalcordatlas.org). Single-cell RNA-sequencing of 13,200 cells revealed that ventral nerve cord MNs cluster into 29 molecularly distinct subclasses. All subclasses are delineated by unique expression codes of either neuropeptide or transcription factor gene families. Strikingly, we found that combinatorial codes of homeodomain transcription factor genes define adult MN diversity both in C. elegans and mice. Further, molecularly defined MN subclasses in C. elegans display distinct patterns of connectivity. Hence, our study couples the connectivity map of the C. elegans motor circuit with a molecular atlas of its constituent MNs, and uncovers organizing principles and conserved molecular codes of adult MN diversity.

A DNA nanodevice for mapping sodium at single-organelle resolution.

Cellular sodium ion (Na+) homeostasis is integral to organism physiology. Our current understanding of Na+ homeostasis is largely limited to Na+ transport at the plasma membrane. Organelles may also contribute to Na+ homeostasis; however, the direction of Na+ flow across organelle membranes is unknown because organellar Na+ cannot be imaged. Here we report a pH-independent, organelle-targetable, ratiometric probe that reports lumenal Na+. It is a DNA nanodevice containing a Na+-sensitive fluorophore, a reference dye and an organelle-targeting domain. By measuring Na+ at single endosome resolution in mammalian cells and Caenorhabditis elegans, we discovered that lumenal Na+ levels in each stage of the endolysosomal pathway exceed cytosolic levels and decrease as endosomes mature. Further, we find that lysosomal Na+ levels in nematodes are modulated by the Na+/H+ exchanger NHX-5 in response to salt stress. The ability to image subcellular Na+ will unveil mechanisms of Na+ homeostasis at an increased level of cellular detail.

Distinct roles for cell-autonomous Notch signaling in cardiomyocytes of the embryonic and adult heart.

RATIONALE: The Notch signaling pathway is important for cell-cell communication that controls tissue formation and homeostasis during embryonic and adult life, but the precise cell targets of Notch signaling in the mammalian heart remain poorly defined. OBJECTIVE: To investigate the functional role of Notch signaling in the cardiomyocyte compartment of the embryonic and adult heart. METHODS AND RESULTS: Here, we report that either conditional overexpression of Notch1 intracellular domain (NICD1) or selective silencing of Notch signaling in the embryonic cardiomyocyte compartment results in developmental defects and perinatal lethality. In contrast, augmentation of endogenous Notch reactivation after myocardial infarction in the adult, either by inducing cardiomyocyte-specific Notch1 transgene expression or by intramyocardial delivery of a Notch1 pseudoligand, increases survival rate, improves cardiac functional performance, and minimizes fibrosis, promoting antiapoptotic and angiogenic mechanisms. CONCLUSIONS: These results reveal a strict requirement for cell-autonomous modulation of Notch signaling during heart morphogenesis, and illustrate how the same signaling pathway that promotes congenital heart defects when perturbed in the embryo can be therapeutically redeployed for the treatment of adult myocardial damage.

Maintenance of neurotransmitter identity by Hox proteins through a homeostatic mechanism.

Hox transcription factors play fundamental roles during early patterning, but they are also expressed continuously, from embryonic stages through adulthood, in the nervous system. However, the functional significance of their sustained expression remains unclear. In C. elegans motor neurons (MNs), we find that LIN-39 (Scr/Dfd/Hox4-5) is continuously required during post-embryonic life to maintain neurotransmitter identity, a core element of neuronal function. LIN-39 acts directly to co-regulate genes that define cholinergic identity (e.g., unc-17/VAChT, cho-1/ChT). We further show that LIN-39, MAB-5 (Antp/Hox6-8) and the transcription factor UNC-3 (Collier/Ebf) operate in a positive feedforward loop to ensure continuous and robust expression of cholinergic identity genes. Finally, we identify a two-component design principle for homeostatic control of Hox gene expression in adult MNs: Hox transcriptional autoregulation is counterbalanced by negative UNC-3 feedback. These findings uncover a noncanonical role for Hox proteins during post-embryonic life, critically broadening their functional repertoire from early patterning to the control of neurotransmitter identity.

Control of spinal motor neuron terminal differentiation through sustained Hoxc8 gene activity.

Spinal motor neurons (MNs) constitute cellular substrates for several movement disorders. Although their early development has received much attention, how spinal MNs become and remain terminally differentiated is poorly understood. Here, we determined the transcriptome of mouse MNs located at the brachial domain of the spinal cord at embryonic and postnatal stages. We identified novel transcription factors (TFs) and terminal differentiation genes (e.g. ion channels, neurotransmitter receptors, adhesion molecules) with continuous expression in MNs. Interestingly, genes encoding homeodomain TFs (e.g. HOX, LIM), previously implicated in early MN development, continue to be expressed postnatally, suggesting later functions. To test this idea, we inactivated Hoxc8 at successive stages of mouse MN development and observed motor deficits. Our in vivo findings suggest that Hoxc8 is not only required to establish, but also maintain expression of several MN terminal differentiation markers. Data from in vitro generated MNs indicate Hoxc8 acts directly and is sufficient to induce expression of terminal differentiation genes. Our findings dovetail recent observations in Caenorhabditis elegans MNs, pointing toward an evolutionarily conserved role for Hox in neuronal terminal differentiation.

Serum and glucocorticoid-inducible kinase 1 (SGK1) is necessary for vascular remodeling during angiogenesis.

The unquestionable importance of the cardiovascular system for pre- and postnatal life has prompted dissection of the molecular mechanisms underlying its development. Serum and glucocorticoid-inducible kinase 1 (SGK1) is a serine/threonine kinase lying downstream of the phosphoinositide 3 (PI3) kinase pathway, whose embryonic function remains unknown. Here, we show that disruption of Sgk1 in the mouse C57BL/6J genetic background leads to embryonic lethality at embryonic day 10.5-11.5 due to severe embryonic and extraembryonic angiogenic defects and to impaired myocardial trabeculation. Absence of SGK1 results in increased apoptosis of endothelial cells, and of vascular smooth muscle cells highlighting a prosurvival role for SGK1 during angiogenesis. Sgk1 null embryos also display reduced expression levels of Notch signaling genes and decreased expression of the arterial markers Efnb2 and Nrp1. These findings uncover a novel and essential function for SGK1 in cardiovascular development contributing to a better understanding of mammalian angiogenesis.

Transgenic reporter analysis of ChIP-Seq-defined enhancers identifies novel target genes for the terminal selector UNC-3/Collier/Ebf.

Terminal selector-type transcription factors are key regulators of neuronal identity and function (Hobert and Kratsios, 2019; Kratsios and Hobert, 2018). Mechanistically, terminal selectors are thought to act directly through binding at the cis-regulatory region of genes (termed "terminal identity genes") that encode, among others, neurotransmitter [NT] synthesis proteins, ion channels, neuropeptides, and cell adhesion molecules (Hobert and Kratsios, 2019; Kratsios and Hobert, 2018). Although dozens of terminal selectors have been described thus far for individual neuron types of the nematode C. elegans (Hobert, 2016), the identification of their target genes has primarily relied on candidate approaches and availability of markers for neuronal terminal identity. Hence, unbiased methods are needed to identify the full spectrum of terminal selector target genes in individual neuron types. This study focuses on the phylogenetically conserved terminal selector UNC-3/Ebf (member of the Collier/Olf/Ebf family), which controls cholinergic motor neuron (MN) identity in the ventral nerve cord of the nematode C. elegans. To identify novel UNC-3 target genes, we took advantage of the genome-wide binding map of UNC-3 from our previous Chromatin Immunoprecipitation followed by Sequencing (ChIP-Seq) analysis (Li et al., 2020). We generated transgenic reporter lines for ten putative terminal identity genes (pxd-1, cal-2, lgc-4, ldb-1, nep-21, D2007.2, dmsr-2, ncs-2, npr-29, drn-1), whose expression patterns were largely unknown in C. elegans. Six of these reporter lines showed expression in ventral nerve cord MNs (nep-21, D2007.2, dmsr-2, ncs-2, npr-29, drn-1), whereas the remaining four (pxd-1, cal-2, lgc-4, ldb-1) showed expression in head and tail neurons, as well as some non-neuronal cells. Importantly, the number of ventral nerve cord MNs showing expression of the nep-21, D2007.2, and dmsr-2 reporters was significantly reduced in unc-3 null mutant animals, thereby expanding the repertoire of known UNC-3 target genes in these cells. Altogether, this study demonstrates that transgenic reporter analysis guided by ChIP-Seq results is a relatively efficient approach for the identification and validation of transcription factor target genes.