Neuronal specification of a motor neuron in C. elegans

The expression of specific transcription factors determines the differentiated features of postmitotic neurons. However, the mechanism by which specific molecules determine neuronal cell fate and the extent to which the functions of transcription factors are conserved in evolution are not fully understood. In C. elegans, the cholinergic and peptidergic SMB sensory/inter/motor neurons innervate muscle quadrants in the head and control the amplitude of sinusoidal movement. Here I showed that the LIM homeobox protein LIM-4 determines neuronal characteristics of the SMB neurons. In lim-4mutant animals, expression of terminal differentiation genes, such as the cholinergic gene battery and the flp-12 neuropeptide gene, is completely abolished and thus the function of the SMB neurons is compromised. LIM-4 activity promotes SMB identity by directly regulating the expression of the SMB marker genes via a distinct cis-regulatory motif. Two human LIM-4 orthologs, LHX6 and LHX8, functionally substitute for LIM-4 in C. elegans. Furthermore, C. elegans LIM-4 or human LHX6 can induce cholinergic and peptidergic characteristics in the human neuronal cell lines. My results indicate that the evolutionarily conserved LIM-4/LHX6 homeodomain proteins function in generation of precise neuronal subtypes.openI Introduction 1-- 1.1 Historical background 1-- 1.2 Terminal selector transcription factors coregulate termi- nal differentiation genes 11-- 1.3 Terminal selectors initiate and maintain the terminally differentiated state 15-- 1.4 Regulations of pan-neuronal features 17-- II Materials and Methods 20-- III Results 28-- 3.1 Expression of a neuropeptide gene in the SMB neurons is abolished in lim-4 mutants 28-- 3.2 Expression of terminally differentiated SMB markers including cholinergic genes is abolished in lim-4 mu- tants 39-- 3.3 The function of the SMB neurons is compromised in lim-4 mutants 47-- 3.4 lim-4 is expressed and functions in the SMB neurons to regulate their terminal specification 50-- 3.5 Postdevelopmental expression of LIM-4 is sufficient to restore the SMB-specific defects of lim-4 mutants 58-- 3.6 Expression of lim-4 is sufficient to induce the SMB identity in other cell-types 61-- 3.7 LIM-4 regulates gene expression via a cis-regulatory motif in the SMB markers 66-- 3.8 The function of lim-4 is conserved in human 76-- IV. Discussions 81-- V. References 89-- VI. Summary in Koreans 97-- VII. Appendix 99유사 분열 후의 신경 (post-mitoticneuron)들은 특정 전사 인자 (tran-scription factor)들의 발현에 따라 분화된 후의 특징들이 나타나게 된다. 그러나 신경 세포의 운명 (neuronal cell fate)을 결정하는데 관여하는 특정한 분자들과 이와 관련하여 진화 과정에서 보존된 전사인자들의 기능들에 대해 아직 잘 알려지지 않았다. 예쁜꼬마선충 (C. elegans)의 경우, 사분면에 걸쳐 발달된 머리 근육에 콜린성 (cholinergic)이자 펩티드성 (pepetidergic)인 감각/ 개재/ 운동신경 (sensory/inter/motor neuron)인 SMB의 신경이 연결되어 있어서 이를 통해 사인 곡선 모양의 움직임의 폭 (wave width)을 조절한다. 이 논문에서는 SMB가 신경으로 제대로 분화되고 신경세포로써의 특징을 가지게 하는데 LIM 호메오박스 (Homeobox) 단백질인 LIM-4가 중요하다는 것을 밝혀냈다. lim-4 돌연변이에서 SMB의 단말 분화 유전자들인 콜린성 유전자 배터리 (cholinergic gene battery)와 신경펩티드 유전자인 flp-12의 발현이 억제되었으며, 또한 SMB의 기능도 제대로 작동되지 않는 것을 확인하였다. LIM-4 단백질의 활성 정도에 따라 SMB에 발현하는 유전자들의 발현이 조절된다는 것을 알아냈으며, 이는 cis-작용부분 (cis-regulatory motif)을 통해 이루어지는 것을 밝혀냈다. 사람의 경우, LIM-4의 상동유전자 (orthologue)인 LHX6와 LHX8를 가지고 있으며, 예쁜꼬마선충에서 LIM-4 단백질을 대신하여 기능적으로 대체할 수 있는 것을 확인하였다. 이와 더불어, 예쁜꼬마선충 LIM-4 단백질과 인간 LHX6 단백질이 human 신경 세포들을 콜린성과 펩티드성 특징을 가진 신경으로 분화를 유도할 수 있다는 것을 밝혀냈다. 이 논문에서는 특정 신경으로 분화하기 위한 LIM-4/LHX6 호메오박스 단백질의 정밀한 조절 기능이 진화적으로 보존이 되었다는 것을 보여주었다.DoctordCollectio

Food-derived sensory cues modulate longevity via distinct neuroendocrine insulin-like peptides

Environmental fluctuations influence organismal aging by affecting various regulatory systems. One such system involves sensory neurons, which affect life span in many species. However, how sensory neurons coordinate organismal aging in response to changes in environmental signals remains elusive. Here, we found that a subset of sensory neurons shortens Caenorhabditis elegans' life span by differentially regulating the expression of a specific insulin-like peptide (ILP), INS-6. Notably, treatment with food-derived cues or optogenetic activation of sensory neurons significantly increases ins-6 expression and decreases life span. INS-6 in turn relays the longevity signals to nonneuronal tissues by decreasing the activity of the transcription factor DAF-16/FOXO. Together, our study delineates a mechanism through which environmental sensory cues regulate aging rates by modulating the activities of specific sensory neurons and ILPs.1186Ysciescopu

The Evolutionarily Conserved LIM Homeodomain Protein LIM-4/LHX6 Specifies the Terminal Identity of a Cholinergic and Peptidergic C. elegans Sensory/Inter/Motor Neuron-Type

The expression of specific transcription factors determines the differentiated features of postmitotic neurons. However, the mechanism by which specific molecules determine neuronal cell fate and the extent to which the functions of transcription factors are conserved in evolution are not fully understood. In C. elegans, the cholinergic and peptidergic SMB sensory/inter/motor neurons innervate muscle quadrants in the head and control the amplitude of sinusoidal movement. Here we show that the LIM homeobox protein LIM-4 determines neuronal characteristics of the SMB neurons. In lim-4 mutant animals, expression of terminal differentiation genes, such as the cholinergic gene battery and the flp-12 neuropeptide gene, is completely abolished and thus the function of the SMB neurons is compromised. LIM-4 activity promotes SMB identity by directly regulating the expression of the SMB marker genes via a distinct cis-regulatory motif. Two human LIM-4 orthologs, LHX6 and LHX8, functionally substitute for LIM-4 in C. elegans. Furthermore, C. elegans LIM-4 or human LHX6 can induce cholinergic and peptidergic characteristics in the human neuronal cell lines. Our results indicate that the evolutionarily conserved LIM-4/LHX6 homeodomain proteins function in generation of precise neuronal subtypes

Syndecan transmembrane domain specifically regulates downstream signaling events of the transmembrane receptor cytoplasmic domain

Despite the known importance of the transmembrane domain (TMD) of syndecan receptors in cell adhesion and signaling, the molecular basis for syndecan TMD function remains un-known. Using in vivo invertebrate models, we found that mammalian syndecan-2 rescued both the guidance defects in C. elegans hermaphrodite-specific neurons and the impaired development of the midline axons of Drosophila caused by the loss of endogenous syndecan. These compensatory ef-fects, however, were reduced significantly when syndecan-2 dimerization-defective TMD mutants were introduced. To further investigate the role of the TMD, we generated a chimera, 2eTPC, com-prising the TMD of syndecan-2 linked to the cytoplasmic domain of platelet-derived growth factor receptor (PDGFR). This chimera exhibited SDS-resistant dimer formation that was lost in the corre-sponding dimerization-defective syndecan-2 TMD mutant, 2eT(GL)PC. Moreover, 2eTPC specifically enhanced Tyr 579 and Tyr 857 phosphorylation in the PDGFR cytoplasmic domain, while the TMD mutant failed to support such phosphorylation. Finally, 2eTPC, but not 2eT(GL)PC, induced phosphorylation of Src and PI3 kinase (known downstream effectors of Tyr 579 phosphorylation) and promoted Src-mediated migration of NIH3T3 cells. Taken together, these data suggest that the TMD of a syndecan-2 specifically regulates receptor cytoplasmic domain function and subsequent downstream signaling events controlling cell behavior. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.1

Stretch sensitive neurons generate rhythmic motor patterns during forward movemnt in C. elegans

Coordinated voluntary and involuntary movements produce rhythmic motor activities to drive locomotory behaviors of animals. In C. elegans, forward locomotion, which is initiated by contraction and relaxation of head muscles, exhibits the rhythmic motor pattern. However, the neuronal and molecular mechanisms to generate rhythmic motor pattern are not fully understood. Previously, it was shown that ablation of the SMB or SMD neurons caused increased reversal and in addition, SMB ablation increased wave width of sinusoidal movement (Gray et al., 2005). To confirm whether the SMB or SMD neurons function in forward movement, we genetically ablated the SMB or SMD neurons. Either SMB or SMD ablated worms exhibited increased reversal rate compared to wild-type animals. We next found that SMB or SMD exhibited rhythmic calcium transients that were induced by the head bending. To gain further insight into which molecules generate rhythmic calcium transient in SMB, we examined SMB neuronal calcium transients in synaptic transmission mutants including unc-13 (neurotransmitter release regulator), unc-31 (dense-core vesicle fusion activator), or unc-9 (innexin). All three mutants showed normal calcium dynamic in SMB, indicating that the rhythmic calcium influx of SMB is independent of synaptic transmission, and SMB may act as a stretch sensitive/proprioceptive receptor neuron to sense head muscle contraction. To identify the stretch receptor(s) in SMB, we examined expression pattern of 29 TRP and DEG/ENaC channels and found that few genes including unc-8 (DEG/ENaC channel) were expressed in SMB. UNC-8 protein is localized to cell bodies and processes around nerve ring. We also observed that SMB specific unc-8 RNAi causes increased wave width. We are currently analyzing unc-8 mutant phenotypes

TRPC channels modulate locomotive behavior of C. elegans

Locomotion is mediated by coordinated processes between sensory and motor system in animals. C. elegansgenerates sinusoidal locomotion via periodic bending of its head and body. The putative proprioceptive SMD motor neurons, that innervate to head muscles and project posterior processes to the tail, have been proposed to sense the body stretch and regulate head locomotion (White et al., 1986; Hendricks et al., 2012; Shen et al., 2016). However, the molecular mechanisms by which SMD regulates head movementare still unclear. To identify factors that mediate SMD-mediated head bending, we performed candidate gene search and found that TRPC channels, trp-1and trp-2, are co-expressed in SMD (Feng et al., 2006). Since we did not observed altered locomotion defects in single mutants of either trp-1 or trp-2, we next generated trp-1 trp-2doublemutants and found that these animals exhibit ventral-directed circles during forward movement; we name this phenotype as ventralcircling. Expression of either TRP-1 or TRP-2 by using a SMDD specific promoter rescued ventral circling phenotype of trp-1 trp-2mutants, and SMDD axonal morphological defect mutants also showed the ventral circling. These results indicate that the ventralcircling phenotype of trp-1 trp-2 mutants is due to the functional defects of SMDD. Ca2+activity of SMD is correlated with head bending direction in wild-type animals, whereas Ca2+activity SMDD but not SMDV is not correlated with head bending in trp-1 trp-2 mutants. These impaired correlation Ca2+dynamic of SMDD with head bending in trp-1 trp-2double mutants was restored by expressing trp-1cDNA using the SMDD specific promoter. Furthermore, ectopic expression of the known stretch receptors, C. elegans trp-4 orDrosophila TRPγ in SMDD were sufficient to rescue ventral circling locomotion of trp-1 trp-2 double mutants. Currently, we are investigating stretch-activation of TRP-1 or TRP-2 by performing electrophysiology in heterologous systems. Taken together, we propose that trp-1and trp-2act as stretch receptors in the SMD motor neurons to sense the dorsal head movement and to correlate SMDD motor neuronal activity with head bendin

Sensory food cue shorten C. elegans lifespan via inducing neuroendocrine INS-6/insulin-like peptide

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A sensory-motor neuron type mediates proprioceptive coordination of steering in <i>C</i>. <i>elegans</i> via two TRPC channels

<div><p>Animal locomotion is mediated by a sensory system referred to as proprioception. Defects in the proprioceptive coordination of locomotion result in uncontrolled and inefficient movements. However, the molecular mechanisms underlying proprioception are not fully understood. Here, we identify two transient receptor potential cation (TRPC) channels, <i>trp-1</i> and <i>trp-2</i>, as necessary and sufficient for proprioceptive responses in <i>C</i>. <i>elegans</i> head steering locomotion. Both channels are expressed in the SMDD neurons, which are required and sufficient for head bending, and mediate coordinated head steering by sensing mechanical stretches due to the contraction of head muscle and orchestrating dorsal head muscle contractions. Moreover, the SMDD neurons play dual roles to sense muscle stretch as well as to control muscle contractions. These results demonstrate that distinct locomotion patterns require dynamic and homeostatic modulation of feedback signals between neurons and muscles.</p></div

The SMD neurons are stretch-sensitive proprioceptive neurons.

<p><b>(A-D)</b> Heat maps (A, B, D) and the average turning angle (C) for the SMDD- or SMDV-ablated animals or indicated genotypes. <i>n</i> = 8 for each. <b>(E)</b> Cross-correlations and peak correlations of the indicated genotypes. Peak correlation value is obtained from lag 0 of cross-correlation. <i>n</i> = 10 for each. <b>(F)</b> Schematic for inducing head bending using a platinum wire (left) and the percentage of worms with increased GCaMP3 intensity in SMD soma of <i>unc-54 (e1092)</i> mutant animals induced by head bending (right). D: dorsal; V: ventral. <i>n</i> = 30 for each. <b>(G)</b> Representative images of Ex[<i>myo-3</i>p::ReaChR; <i>lad-2</i>p-<i>Δ</i>1::GCaMP] transgenic animals upon green light stimulation in the presence and absence of retinal (ATR; left) and the percentage of animals that exhibit GCaMP3 signals in the SMD neurons (right). White rectangles indicate soma of SMDD/V. <i>n</i> = 40. <b>(H)</b> Model for the functions of <i>trp-1</i> and <i>trp-2</i> in the SMD neurons coordinating neuronal activity with the motor system to regulate turning angle during forward movement. Numerical values that underlie the graph are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004929#pbio.2004929.s020" target="_blank">S1 Data</a>. Error bars indicate SEM. ** and *** indicate significant differences from wild type at <i>p</i> < 0.01 and <i>p</i> < 0.001, respectively (one-way ANOVA test followed by the Tukey post hoc test). ATR, all-trans-retinal</p

TRP-1 and TPR-2 coordinate SMD neuronal activities with head bending and mediate head muscle contractions.

<p><b>(A, C)</b> Representative images showing GCaMP3 fluorescence in the SMD soma of a <i>flp-22</i>p<i>-Δ</i>4::GCaMP3 transgenic worm during dorsal and ventral head bending of the indicated genotypes. The head region (left) and a higher magnification of the boxed area (right) are shown. Red and blue boxes indicate the cell bodies of SMDV and SMDD, respectively. Anterior is to the left. Scale bar: 25 μm. <b>(B, D, E)</b> Calcium dynamics (top left) in the SMD cell bodies and the corresponding head bending (bottom left) in the same animal and cross-correlations (right) between SMDD or SMDV calcium responses and head bending <b>(B, D)</b> and cross-correlations of the indicated genotype <b>(E)</b>. Gray bars indicate the duration of the dorsal head bending. <i>n</i> = 10 for each. <b>(F)</b> Peak correlations between SMD calcium activity and head bending of the indicated genotypes. Peak correlation value is obtained from lag 0 of cross-correlation. <b>(G)</b> Representative images showing a <i>lad-2</i>p-<i>Δ</i>1::ReaChR::mKate2 transgenic animal upon green light stimulation (left) and the percentage of animals that exhibit circling locomotion in response to light stimulation in the presence and absence of retinal (ATR; right). Arrowheads indicate the ventral side of the body. <i>n</i> = 40. <b>(H)</b> Schematic of the head muscles innervated by SMDD and SMDV (left), single frame images of head muscles of <i>myo-3</i>p::GCaMP3.35; <i>flp-22</i>p-<i>Δ</i>4::GCaMP3 transgenic worms during head bending (middle), and the GCaMP fluorescent ratio of dorsal muscles to ventral muscles (right). D: dorsal; V: ventral. Anterior is to the left. Scale bar: 25 μm. <i>n</i> = 10 for each genotype. Numerical values that underlie the graph are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004929#pbio.2004929.s020" target="_blank">S1 Data</a>. Error bars indicate SEM. ** and *** indicate significant differences from wild type at <i>p</i> < 0.01 and <i>p</i> < 0.001, respectively (one-way ANOVA test followed by the Tukey post hoc test). ATR, all-trans-retinal; TRP, transient receptor potential</p