Project description:Comparison of the transcriptome of embryonic day 12 motor neurons and postnatal day 8 motor neurons. Comparison of the transcriptome of motor neurons without Hoxc8 protein and control littermates. Comparison of the transcriptome of postnatal day 8 motor neurons from the brachial, thoracic and lumbar regions of the spinal cord.
Project description:The motor neuron (MN)–hexamer complex consisting of LIM homeobox 3, Islet-1, and nuclear LIM interactor is a key determinant of motor neuron specification and differentiation. To gain insights into the transcriptional network in motor neuron development, we performed a genome-wide ChIP-sequencing analysis and found that the MN–hexamer directly regulates a wide array of motor neuron genes by binding to the HxRE (hexamer response element) shared among the target genes. Interestingly, STAT3-binding motif is highly enriched in the MN–hexamer–bound peaks in addition to the HxRE. We also found that a transcriptionally active form of STAT3 is expressed in embryonic motor neurons and that STAT3 associates with the MN–hexamer, enhancing the transcriptional activity of the MN–hexamer in an upstream signal-dependent manner. Correspondingly, STAT3 was needed for motor neuron differentiation in the developing spinal cord. Together, our studies uncover crucial gene regulatory mechanisms that couple MN–hexamer and STAT-activating extracellular signals to promote motor neuron differentiation in vertebrate spinal cord. To explain our experimental scheme briefly, we are interested in finding target sites for the dimer of transcription factors Isl1 and Lhx3. To mimic the biological activity of Isl1/Lhx3 dimer, we made Isl1-Lhx3 fusion and found that Isl1-Lhx3 has a potent biological activity in multiple systems (i.e. generation of ectopic motor neurons). Then we made ES cell line that induces Flag-tagged Isl1-Lhx3 expression upon Dox treatment. These *mouse* ES cells differentiate to motor neurons (iMN-ESCs) when treated with Dox following EB formation. To identify genomic binding sites of Isl1-Lhx3 (Flag-tagged), we performed ChIP with Flag antibody (pull down of Flag-Isl1-Lhx3) in ES cells treated with Dox. ChIP with Flag antibody in ES cells treated with vehicle (no Dox) was done as a negative control in parallel, and sequenced along with +Dox sample. We have done these experiments twice (two sets).
Project description:To determine what kind of genes are involved in vocal learning ability, we performed microarray experiments using 3 vocal learning species (zebra finch, budgerigar, Anna's hummingbird) and 2 non-vocal learning species(ring dive, and Japanese quail) from the bird group. All of the animals are male adults. They were isolated over night and had 1hour light exposure at morning. Birds who did not sing were used in this experiment. We used 2-3 animals each species. We used the 12th motor neuron for both vocal learners and non-vocal learners. We used the Supra Spinal motor neuron (ssp) as control area for both groups.
Project description:The motor neuron (MN)–hexamer complex consisting of LIM homeobox 3, Islet-1, and nuclear LIM interactor is a key determinant of motor neuron specification and differentiation. To gain insights into the transcriptional network in motor neuron development, we performed a genome-wide ChIP-sequencing analysis and found that the MN–hexamer directly regulates a wide array of motor neuron genes by binding to the HxRE (hexamer response element) shared among the target genes. Interestingly, STAT3-binding motif is highly enriched in the MN–hexamer–bound peaks in addition to the HxRE. We also found that a transcriptionally active form of STAT3 is expressed in embryonic motor neurons and that STAT3 associates with the MN–hexamer, enhancing the transcriptional activity of the MN–hexamer in an upstream signal-dependent manner. Correspondingly, STAT3 was needed for motor neuron differentiation in the developing spinal cord. Together, our studies uncover crucial gene regulatory mechanisms that couple MN–hexamer and STAT-activating extracellular signals to promote motor neuron differentiation in vertebrate spinal cord.
Project description:[original Title] Rapid and synchronous clearance of PcG histone modifications from Hox genes anticipates motor neuron differentiation. Hox genes are expressed in patterns that are spatially and temporally collinear with their chromosomal organization. This feature is an evolutionarily conserved hallmark of embryonic development, and in vertebrates it is critical, among others, for the specification of motor neuron subtypes and the wiring of sensory-motor circuits. We show here that the differentiation of motor neurons from stem cells is accompanied by a synchronous, domain-wide clearance of M-bM-^@M-^\repressiveM-bM-^@M-^] Polycomb (PcG)-dependent histone methylation from Hox gene chromatin domains. These findings argue against the idea, advanced recently, that the collinear dynamics of Hox gene expression invariably reflects the progressive clearance of repressive chromatin modifications. The rapid establishment of stable chromatin domains in response to a transient patterning signal likely serves as a molecular correlate of enduring rostro-caudal neural identity, which underlies the specification of postmitotic motor neuron subtype diversity and neuronal circuit assembly. The differentiation of ventral motor neurons is induced by treating embryonic stem cell cultures with retinoic acid and hedgehog agonist. Here, ChIP-chip using a custom Agilent array is used to profile the occupancy of H3K27me3, H3K4me3, and H3K79me2 at various defined stages during the differentiation process.
Project description:To investigate the role of motor neuron autophagy in ALS, we generated mice in which the critical autophagy gene Atg7 was specifically disrupted in motor neurons (Atg7 cKO). We also bred these mice to the SOD1G93A mouse model of ALS. Then we performed RNA sequencing on lumbar spinal cords from these mice to determine how motor neuron autophagy inhibition altered gene expression.
Project description:We profiled miRNAs from embryonic stem cells to motor neurons derived via RA (retinoic acid) and distinguished different developmental stages by days. Those developmental stages are ESC, Day2, Day3, Day4, and both motor neuron and interneuron with day7. We also provide the fractionated nuclear and cytoplasm miRNA profiling with motor neuron and interneuron respectivey.
Project description:[original Title] Rapid and synchronous clearance of PcG histone modifications from Hox genes anticipates motor neuron differentiation. Hox genes are expressed in patterns that are spatially and temporally collinear with their chromosomal organization. This feature is an evolutionarily conserved hallmark of embryonic development, and in vertebrates it is critical, among others, for the specification of motor neuron subtypes and the wiring of sensory-motor circuits. We show here that the differentiation of motor neurons from stem cells is accompanied by a synchronous, domain-wide clearance of “repressive” Polycomb (PcG)-dependent histone methylation from Hox gene chromatin domains. These findings argue against the idea, advanced recently, that the collinear dynamics of Hox gene expression invariably reflects the progressive clearance of repressive chromatin modifications. The rapid establishment of stable chromatin domains in response to a transient patterning signal likely serves as a molecular correlate of enduring rostro-caudal neural identity, which underlies the specification of postmitotic motor neuron subtype diversity and neuronal circuit assembly.