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Journal of Molecular and Cellular... May 2021The intracardiac nervous system (ICNS) is composed of neurons, in association with Schwann cells (SC) and endoneurial cardiac fibroblasts (ECF). Besides heart rhythm...
BACKGROUND
The intracardiac nervous system (ICNS) is composed of neurons, in association with Schwann cells (SC) and endoneurial cardiac fibroblasts (ECF). Besides heart rhythm control, recent studies have implicated cardiac nerves in postnatal cardiac regeneration and cardiomyocyte size regulation, but cardiac SC and ECF remain understudied. During the postnatal period, the ICNS undergoes intense remodeling with nerve fasciculation and elongation throughout the myocardium, partially guided by the extracellular matrix (ECM). Here we report the origins, heterogeneity, and functions of SC and ECF that develop in proximity to neurons during postnatal ICNS maturation.
METHODS AND RESULTS
Periostin lineage (Postn+) cells include cardiac Remak SC and ECF during the postnatal period in mice. The developmental origins of cardiac SC and ECF were examined using Rosa26 reporter mice bred with Wnt1Cre, expressed in Neural crest (NC)-derived lineages, or tamoxifen-inducible Tcf21MerCreMer, expressed predominantly in epicardial-derived fibroblast lineages. ICNS components are NC-derived with the exceptions of the myelinating Plp1+ SC and the Tcf21+ lineage-derived intramural ventricular ECF. In addition, Postn+ lineage GFAP- Remak SC and ECF are present around the fasciculating cardiac nerves. Whole mount studies of the NC-derived cells confirmed postnatal maturation of the complex ICNS network from P0 to P30. Sympathetic, parasympathetic, and sensory neurons fasciculate from P0 to P7 indicated by co-staining with PSA-NCAM. Ablation of Postn+ cells from P0 to P6 or loss of Periostin leads to reduced fasciculation of cardiac sympathetic nerves. In addition, collagen remodeling surrounding maturing nerves of the postnatal heart is reduced in Postn-null mice.
CONCLUSIONS
Postn+ cells include cardiac SC and ECF during postnatal nerve maturation, and these cells have different embryonic origins. At P7, the Postn+ cells associated with cardiac nerves are mainly Remak SC and ECF. Ablation of the Postn+ cells from P0 to P6 and also loss of Postn in Postn-null mice leads to reduced fasciculation of cardiac nerves at P7.
Topics: Animals; Axon Fasciculation; Cell Adhesion Molecules; Fibroblasts; Gene Expression; Mice; Schwann Cells; Sympathetic Nervous System
PubMed: 33582160
DOI: 10.1016/j.yjmcc.2021.02.001 -
Frontiers in Neural Circuits 2021Precise positioning of neurons resulting from cell division and migration during development is critical for normal brain function. Disruption of neuronal migration can...
Precise positioning of neurons resulting from cell division and migration during development is critical for normal brain function. Disruption of neuronal migration can cause a myriad of neurological disorders. To investigate the functional consequences of defective neuronal positioning on circuit function, we studied a zebrafish () loss-of-function mutant () where the facial branchiomotor (FBM) neurons fail to migrate out of their birthplace. A jaw movement assay, which measures the opening of the zebrafish jaw (gape), showed that the frequency of gape events, but not their amplitude, was decreased in mutants. Consistent with this, a larval feeding assay revealed decreased food intake in mutants, indicating that the FBM circuit in mutants generates defective functional outputs. We tested various mechanisms that could generate defective functional outputs in mutants. While is ubiquitously expressed in neural and non-neural tissues, jaw cartilage and muscle developed normally in mutants, and muscle function also appeared to be unaffected. Although FBM neurons were mispositioned in mutants, axon pathfinding to jaw muscles was unaffected. Moreover, neuromuscular junctions established by FBM neurons on jaw muscles were similar between wildtype siblings and mutants. Interestingly, motor axons innervating the interhyoideus jaw muscle were frequently defasciculated in mutants. Furthermore, GCaMP imaging revealed that mutant FBM neurons were less active than their wildtype counterparts. These data show that aberrant positioning of FBM neurons in mutants is correlated with subtle defects in fasciculation and neuronal activity, potentially generating defective functional outputs.
Topics: Animals; Axons; Cell Movement; Motor Neurons; Neurogenesis; Zebrafish; Zebrafish Proteins
PubMed: 34248505
DOI: 10.3389/fncir.2021.690475 -
Nature Communications Nov 2020In the developing nervous system, axons navigate through complex terrains that change depending on when and where outgrowth begins. For instance, in the developing...
In the developing nervous system, axons navigate through complex terrains that change depending on when and where outgrowth begins. For instance, in the developing cochlea, spiral ganglion neurons extend their peripheral processes through a growing and heterogeneous environment en route to their final targets, the hair cells. Although the basic principles of axon guidance are well established, it remains unclear how axons adjust strategies over time and space. Here, we show that neurons with different positions in the spiral ganglion employ different guidance mechanisms, with evidence for both glia-guided growth and fasciculation along a neuronal scaffold. Processes from neurons in the rear of the ganglion are more directed and grow faster than those from neurons at the border of the ganglion. Further, processes at the wavefront grow more efficiently when in contact with glial precursors growing ahead of them. These findings suggest a tiered mechanism for reliable axon guidance.
Topics: Animals; Axon Guidance; Basic Helix-Loop-Helix Transcription Factors; Cell Movement; Cochlea; Female; Mice, Transgenic; Nerve Tissue Proteins; Neurites; Neuroglia; Neurons; Organ Culture Techniques; Pregnancy; Spiral Ganglion; Time-Lapse Imaging
PubMed: 33203842
DOI: 10.1038/s41467-020-19521-2 -
Development (Cambridge, England) Jun 2020Thalamocortical axons (TCAs) cross several tissues on their journey to the cortex. Mechanisms must be in place along the route to ensure they connect with their targets...
Thalamocortical axons (TCAs) cross several tissues on their journey to the cortex. Mechanisms must be in place along the route to ensure they connect with their targets in an orderly fashion. The ventral telencephalon acts as an instructive tissue, but the importance of the diencephalon in TCA mapping is unknown. We report that disruption of diencephalic development by Pax6 deletion results in a thalamocortical projection containing mapping errors. We used conditional mutagenesis to test whether these errors are due to the disruption of pioneer projections from prethalamus to thalamus and found that, although this correlates with abnormal TCA fasciculation, it does not induce topographical errors. To test whether the thalamus contains navigational cues for TCAs, we used slice culture transplants and gene expression studies. We found the thalamic environment is instructive for TCA navigation and that the molecular cues netrin 1 and semaphorin 3a are likely to be involved. Our findings indicate that the correct topographic mapping of TCAs onto the cortex requires the order to be established from the earliest stages of their growth by molecular cues in the thalamus itself.
Topics: Animals; Axons; Diencephalon; Embryo, Mammalian; Gene Expression Regulation, Developmental; Homeodomain Proteins; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Knockout; Mutagenesis; Netrin-1; Organ Culture Techniques; PAX6 Transcription Factor; Semaphorin-3A; Thalamus
PubMed: 32541009
DOI: 10.1242/dev.184523 -
Development (Cambridge, England) Aug 2020Fragile X mental retardation protein (FMRP) is an RNA-binding protein abundant in the nervous system. Functional loss of FMRP leads to sensory dysfunction and severe...
Fragile X mental retardation protein (FMRP) is an RNA-binding protein abundant in the nervous system. Functional loss of FMRP leads to sensory dysfunction and severe intellectual disabilities. In the auditory system, FMRP deficiency alters neuronal function and synaptic connectivity and results in perturbed processing of sound information. Nevertheless, roles of FMRP in embryonic development of the auditory hindbrain have not been identified. Here, we developed high-specificity approaches to genetically track and manipulate throughout development of the Atoh1 neuronal cell type, which is highly conserved in vertebrates, in the cochlear nucleus of chicken embryos. We identified distinct FMRP-containing granules in the growing axons of Atoh1 neurons and post-migrating NM cells. FMRP downregulation induced by CRISPR/Cas9 and shRNA techniques resulted in perturbed axonal pathfinding, delay in midline crossing, excess branching of neurites, and axonal targeting errors during the period of circuit development. Together, these results provide the first identification of FMRP localization and actions in developing axons of auditory neurons, and demonstrate the importance of investigating early embryonic alterations toward understanding the pathogenesis of neurodevelopmental disorders.
Topics: Animals; Auditory Pathways; Axons; Base Sequence; CRISPR-Cas Systems; Chick Embryo; Chickens; Dendrites; Fragile X Mental Retardation Protein; Neural Stem Cells; Presynaptic Terminals; RNA, Small Interfering; Rhombencephalon; Synapses; Time Factors
PubMed: 32747436
DOI: 10.1242/dev.188797 -
Frontiers in Neuroscience 2023The protein fasciculation and elongation zeta-1 (FEZ1) is involved in axon outgrowth but potentially interacts with various proteins with roles ranging from...
INTRODUCTION
The protein fasciculation and elongation zeta-1 (FEZ1) is involved in axon outgrowth but potentially interacts with various proteins with roles ranging from intracellular transport to transcription regulation. Gene association and other studies have identified as being directly, or indirectly, implicated in schizophrenia susceptibility. To explore potential roles in normal early human forebrain neurodevelopment, we mapped expression by region and cell type.
METHODS
All tissues were provided with maternal consent and ethical approval by the Human Developmental Biology Resource. RNAseq data were obtained from previously published sources. Thin paraffin sections from 8 to 21 post-conceptional weeks (PCW) samples were used for RNAScope hybridization and immunohistochemistry against mRNA and protein, and other marker proteins.
RESULTS
Tissue RNAseq revealed that is highly expressed in the human cerebral cortex between 7.5-17 PCW and single cell RNAseq at 17-18 PCW confirmed its expression in all neuroectoderm derived cells. The highest levels were found in more mature glutamatergic neurons, the lowest in GABAergic neurons and dividing progenitors. In the thalamus, single cell RNAseq similarly confirmed expression in multiple cell types. In cerebral cortex sections at 8-10 PCW, strong expression of mRNA and protein appeared confined to post-mitotic neurons, with low expression seen in progenitor zones. Protein expression was observed in some axon tracts by 16-19 PCW. However, in sub-cortical regions, was highly expressed in progenitor zones at early developmental stages, showing lower expression in post-mitotic cells.
DISCUSSION
FEZ1 has different expression patterns and potentially diverse functions in discrete forebrain regions during prenatal human development.
PubMed: 37746155
DOI: 10.3389/fnins.2023.1249973 -
International Journal of Molecular... Dec 2019The G protein-coupled cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), and their endocannabinoid (eCBs) ligands, have been implicated in several aspects of brain...
The G protein-coupled cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), and their endocannabinoid (eCBs) ligands, have been implicated in several aspects of brain wiring during development. Here we aim to assess whether interfering with CB1R affects development, neuritogenesis and pathfinding of GnRH and AgRP neurons, forebrain neurons that control respectively reproduction and appetite. We pharmacologically and genetically interfered with CB1R in zebrafish strains with fluorescently labeled GnRH3 and the AgRP1 neurons. By applying CB1R antagonists we observed a reduced number of GnRH3 neurons, fiber misrouting and altered fasciculation. Similar phenotypes were observed by CB1R knockdown. Interfering with CB1R also resulted in a reduced number, misrouting and poor fasciculation of the AgRP1 neuron's axonal projections. Using a bioinformatic approach followed by qPCR validation, we have attempted to link CB1R functions with known guidance and fasciculation proteins. The search identified stathmin-2, a protein controlling microtubule dynamics, previously demonstrated to be coexpressed with CB1R and now shown to be downregulated upon interference with CB1R in zebrafish. Together, these results raise the likely possibility that embryonic exposure to low doses of CB1R-interfering compounds could impact on the development of the neuroendocrine systems controlling sexual maturation, reproduction and food intake.
Topics: Agouti-Related Protein; Animals; Axons; Benzoxazines; Embryo, Nonmammalian; Embryonic Development; Gonadotropin-Releasing Hormone; Morpholines; Morpholinos; Naphthalenes; Neurons; Pyrrolidonecarboxylic Acid; Receptor, Cannabinoid, CB1; Zebrafish; Zebrafish Proteins
PubMed: 31881740
DOI: 10.3390/ijms21010168 -
BioRxiv : the Preprint Server For... Mar 2024Specific and highly diverse connectivity between functionally specialized regions of the nervous system is controlled at multiple scales, from anatomically organized...
Specific and highly diverse connectivity between functionally specialized regions of the nervous system is controlled at multiple scales, from anatomically organized connectivity following macroscopic axon tracts to individual axon target-finding and synapse formation. Identifying mechanisms that enable entire subpopulations of related neurons to project their axons with regional specificity within stereotyped tracts to form appropriate long-range connectivity is key to understanding brain development, organization, and function. Here, we investigate how axons of the cerebral cortex form precise connections between the two cortical hemispheres via the corpus callosum. We identify topographic principles of the developing trans-hemispheric callosal tract that emerge through intrinsic guidance executed by growing axons in the corpus callosum within the first postnatal week in mice. Using micro-transplantation of regionally distinct neurons, subtype-specific growth cone purification, subcellular proteomics, and in utero gene manipulation, we investigate guidance mechanisms of transhemispheric axons. We find that adhesion molecule levels instruct tract topography and target field guidance. We propose a model in which transcallosal axons in the developing brain perform a "handshake" that is guided through co-fasciculation with symmetric contralateral axons, resulting in the stereotyped homotopic connectivity between the brain's hemispheres.
PubMed: 38585721
DOI: 10.1101/2024.03.28.587108 -
BioRxiv : the Preprint Server For... Feb 2024The chromodomain helicase binding protein 4 (CHD4) is an ATP-dependent chromatin remodeler. pathogenic variants of cause Sifrim-Hitz-Weiss syndrome (SIHIWES). Patients...
The chromodomain helicase binding protein 4 (CHD4) is an ATP-dependent chromatin remodeler. pathogenic variants of cause Sifrim-Hitz-Weiss syndrome (SIHIWES). Patients with SIHIWES show delayed development, intellectual disability, facial dysmorphism, and hearing loss. Many cochlear cell types, including spiral ganglion neurons (SGNs), express CHD4. SGNs are the primary afferent neurons that convey sound information from the cochlea, but the function of CHD4 in SGNs is unknown. We employed the Neurog1(Ngn1) CreERT conditional knockout animals to delete in SGNs. SGNs are classified as type I and type II neurons. SGNs lacking CHD4 showed abnormal fasciculation of type I neurons along with improper pathfinding of type II fibers. CHD4 binding to chromatin from immortalized multipotent otic progenitor-derived neurons was used to identify candidate target genes in SGNs. Gene ontology analysis of CHD4 target genes revealed cellular processes involved in axon guidance, axonal fasciculation, and ephrin receptor signaling pathway. We validated increased transcripts in SGNs from conditional knockout cochleae. The results suggest that CHD4 attenuates the transcription of axon guidance genes to form the stereotypic pattern of SGN peripheral projections. The results implicate epigenetic changes in circuit wiring by modulating axon guidance molecule expression and provide insights into neurodevelopmental diseases.
PubMed: 38352369
DOI: 10.1101/2024.01.31.578202 -
Frontiers in Bioengineering and... 2024Motor nerve organoids could be generated by culturing a spheroid of motor neurons differentiated from human induced pluripotent stem (iPS) cells within a...
Motor nerve organoids could be generated by culturing a spheroid of motor neurons differentiated from human induced pluripotent stem (iPS) cells within a polydimethylsiloxane (PDMS) chip which guides direction and fasciculation of axons extended from the spheroid. To isolate axon bundles from motor nerve organoids, we developed a rapid laser dissection method based on localized photothermal combustion. By illuminating a blue laser on a black mark on the culture device using a dry-erase marker, we induced highly localized heating near the axon bundles. Moving the laser enabled spatial control over the local heating and severing of axon bundles. This laser dissection requires a black mark, as other colors did not produce the same localized heating effect. A CO laser destroyed the tissue and the device and could not be used. With this simple, economical laser dissection technique, we could rapidly collect abundant pure axon samples from motor nerve organoids for biochemical analysis. Extracted axonal proteins and RNA were indistinguishable from manual dissection. This method facilitates efficient axon isolation for further analyses.
PubMed: 38347914
DOI: 10.3389/fbioe.2024.1259138