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Abstract BackgroundV0v spinal interneurons are highly conserved, glutamatergic, commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However, we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells. MethodsTo identify candidate members of V0v gene regulatory networks, we FAC-sorted wild-type andevx1;evx2double mutant zebrafish V0v spinal interneurons and expression-profiled them using microarrays and single cell RNA-seq. We also used in situ hybridization to compare expression of a subset of candidate genes inevx1;evx2double mutants and wild-type siblings. ResultsOur data reveal two molecularly distinct subtypes of zebrafish V0v spinal interneurons at 48 h and suggest that, by this stage of development,evx1;evx2double mutant cells transfate into either inhibitory spinal interneurons, or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons, plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes arehmx2andhmx3a. Intriguingly, we show that Hmx2/3a, repress dI2 interneuron expression ofskor1aandnefma, two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulateskor1aandnefmaexpression in V0v interneurons by repressing Hmx2/3a expression. ConclusionsThis study identifies two molecularly distinct subsets of zebrafish V0v spinal interneurons, as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of these cells. Our data further suggest that in the absence of both Evx1 and Evx2, V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then, later, start to express markers of distinct types of inhibitory spinal interneurons, or motoneurons. Taken together, our findings significantly increase our knowledge of V0v and spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type.
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Evolution of Lbx spinal cord expression and function
Ladybird homeobox (Lbx) transcription factors have crucial functions in muscle and nervous system development in many animals. Amniotes have two Lbx genes, but only Lbx1 is expressed in spinal cord. In contrast, teleosts have three lbx genes and we show here that zebrafish lbx1a, lbx1b and lbx2 are expressed by distinct spinal cell types, and that lbx1a is expressed in dI4, dI5 and dI6 interneurons, as in amniotes. Our data examining lbx expression in Scyliorhinus canicula and Xenopus tropicalis suggest that the spinal interneuron expression of zebrafish lbx1a is ancestral, whereas lbx1b has acquired a new expression pattern in spinal cord progenitor cells. lbx2 spinal expression was probably acquired in the ray-finned lineage, as this gene is not expressed in the spinal cords of either amniotes or S. canicula. We also show that the spinal function of zebrafish lbx1a is conserved with mouse Lbx1. In zebrafish lbx1a mutants, there is a reduction in the number of inhibitory spinal interneurons and an increase in the number of excitatory spinal interneurons, similar to mouse Lbx1 mutants. Interestingly, the number of inhibitory spinal interneurons is also reduced in lbx1b mutants, although in this case the number of excitatory interneurons is not increased. lbx1a;lbx1b double mutants have a similar spinal interneuron phenotype to lbx1a single mutants. Taken together these data suggest that lbx1b and lbx1a may be required in succession for correct specification of dI4 and dI6 spinal interneurons, although only lbx1a is required for suppression of excitatory fates in these cells.
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- Award ID(s):
- 1755354
- PAR ID:
- 10276471
- Date Published:
- Journal Name:
- Evolution development
- ISSN:
- 1525-142X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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null (Ed.)Abstract Transcription factors that contain a homeodomain DNA-binding domain have crucial functions in most aspects of cellular function and embryonic development in both animals and plants. Hmx proteins are a subfamily of NK homeodomain-containing proteins that have fundamental roles in development of sensory structures such as the eye and the ear. However, Hmx functions in spinal cord development have not been analyzed. Here, we show that zebrafish (Danio rerio) hmx2 and hmx3a are coexpressed in spinal dI2 and V1 interneurons, whereas hmx3b, hmx1, and hmx4 are not expressed in spinal cord. Using mutational analyses, we demonstrate that, in addition to its previously reported role in ear development, hmx3a is required for correct specification of a subset of spinal interneuron neurotransmitter phenotypes, as well as correct lateral line progression and survival to adulthood. Surprisingly, despite similar expression patterns of hmx2 and hmx3a during embryonic development, zebrafish hmx2 mutants are viable and have no obviously abnormal phenotypes in sensory structures or neurons that require hmx3a. In addition, embryos homozygous for deletions of both hmx2 and hmx3a have identical phenotypes to severe hmx3a single mutants. However, mutating hmx2 in hypomorphic hmx3a mutants that usually develop normally, results in abnormal ear and lateral line phenotypes. This suggests that while hmx2 cannot compensate for loss of hmx3a, it does function in these developmental processes, although to a much lesser extent than hmx3a. More surprisingly, our mutational analyses suggest that Hmx3a may not require its homeodomain DNA-binding domain for its roles in viability or embryonic development.more » « less
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Abstract BackgroundThe spinal cord is a crucial part of the vertebrate CNS, controlling movements and receiving and processing sensory information from the trunk and limbs. However, there is much we do not know about how this essential organ develops. Here, we describe expression of 21 transcription factors and one transcriptional regulator in zebrafish spinal cord. ResultsWe analyzed the expression ofaurkb,foxb1a,foxb1b,her8a,homeza,ivns1abpb,mybl2b,myt1a,nr2f1b,onecut1,sall1a,sall3a,sall3b,sall4,sox2,sox19b,sp8b,tsc22d1,wdhd1,zfhx3b,znf804a, andznf1032in wild‐type andMIB E3 ubiquitin protein ligase 1zebrafish embryos. While all of these genes are broadly expressed in spinal cord, they have distinct expression patterns from one another. Some are predominantly expressed in progenitor domains, and others in subsets of post‐mitotic cells. Given the conservation of spinal cord development, and the transcription factors and transcriptional regulators that orchestrate it, we expect that these genes will have similar spinal cord expression patterns in other vertebrates, including mammals and humans. ConclusionsOur data identify 22 different transcriptional regulators that are strong candidates for playing different roles in spinal cord development. For several of these genes, this is the first published description of their spinal cord expression.more » « less
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INTRODUCTION Balance between excitatory and inhibitory neuron (interneuron) populations in the cortex promotes normal brain function. Interneurons are primarily generated in the medial, caudal, and lateral ganglionic eminences (MGE, CGE, and LGE) of the ventral embryonic forebrain; these subregions give rise to distinct interneuron subpopulations. In rodents, the MGE generates cortical interneurons, the parvalbumin + (PV + ) and somatostatin + (SST + ) subtypes that connect with excitatory neurons to regulate their activity. Defects in interneuron production have been implicated in neurodevelopmental and psychiatric disorders including autism, epilepsy, and schizophrenia. RATIONALE How does the human MGE (hMGE) produce the number of interneurons required to populate the forebrain? The hMGE contains progenitor clusters distinct from what has been observed in the rodent MGE and other germinal zones of the human brain. This cytoarchitecture could be the key to understanding interneuron neurogenesis. We investigated the cellular and molecular properties of different compartments within the developing hMGE, from 14 gestational weeks (GW) to 39 GW (term), to study their contribution to the production of inhibitory interneurons. We developed a xenotransplantation assay to follow the migration and maturation of the human interneurons derived from this germinal region. RESULTS Within the hMGE, densely packed aggregates (nests) of doublecortin + (DCX + ) and LHX6 + cells were surrounded by nestin + progenitor cells and their processes. These DCX + cell–enriched nests (DENs) were observed in the hMGE but not in the adjacent LGE. We found that cells within DENs expressed molecular markers associated with young neurons, such as DCX, and polysialylated neural cell adhesion molecule (PSA-NCAM). A subpopulation also expressed Ki-67, a marker of proliferation; therefore, we refer to these cells as neuroblasts. A fraction of DCX + cells inside DENs expressed SOX2 and E2F1, transcription factors associated with progenitor and proliferative properties. More than 20% of DCX + cells in the hMGE were dividing, specifically within DENs. Proliferating neuroblasts in DENs persisted in the hMGE throughout prenatal human brain development. The division of DCX + cells was confirmed by transmission electron microscopy and time-lapse microscopy. Electron microscopy revealed adhesion contacts between cells within DENs, providing multiple sites to anchor DEN cells together. Neuroblasts within DENs express PCDH19, and nestin + progenitors surrounding DENs express PCDH10; these findings suggest a role for differential cell adhesion in DEN formation and maintenance. When transplanted into the neonatal mouse brain, dissociated hMGE cells reformed DENs containing proliferative DCX + cells, similar to DENs observed in the prenatal human brain. This suggests that DENs are generated by cell-autonomous mechanisms. In addition to forming DENs, transplanted hMGE-derived neuroblasts generated young neurons that migrated extensively into cortical and subcortical regions in the host mouse brain. One year after transplantation, these neuroblasts had differentiated into distinct γ-aminobutyric acid–expressing (GABAergic) interneuron subtypes, including SST + and PV + cells, that showed morphological and functional maturation. CONCLUSION The hMGE harbors DENs, where cells expressing early neuronal markers continue to divide and produce GABAergic interneurons. This MGE-specific arrangement of neuroblasts in the human brain is present until birth, supporting expanded neurogenesis for inhibitory neurons. Given the robust neurogenic output from this region, knowledge of the mechanisms underlying cortical interneuron production in the hMGE will provide insights into the cell types and developmental periods that are most vulnerable to genetic or environmental insults. Nests of DCX + cells in the ventral prenatal brain. Schematic of a coronal view of the embryonic human forebrain showing the medial ganglionic eminence (MGE, green), with nests of DCX + cells (DENs, green). Nestin + progenitor cells (blue) are present within the VZ and iSVZ and are intercalated in the oSVZ (where DENs reside). The initial segment of the oSVZ contains palisades of nestin + progenitors referred to as type I clusters (light blue cells) around DENs. In the outer part of the oSVZ, DENs transition to chains of migrating DCX + cells; surrounding nestin + progenitors are arranged into groups of cells referred to as type II clusters (white cells). In addition to proliferation of nestin + progenitors, cell division is present among DCX + cells within DENs, suggesting multiple progenitor states for the generation of MGE-derived interneurons in the human forebrain. ILLUSTRATION: NOEL SIRIVANSANTImore » « less
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INTRODUCTION The analysis of the human brain is a central goal of neuroscience, but for methodological reasons, research has focused on model organisms, the mouse in particular. Because substantial homology was found at the level of ion channels, transcriptional programs, and basic neuronal types, a strong similarity of neuronal circuits across species has also been assumed. However, a rigorous test of the configuration of local neuronal circuitry in mouse versus human—in particular, in the gray matter of the cerebral cortex—is missing. The about 1000-fold increase in number of neurons is the most obvious evolutionary change of neuronal network properties from mouse to human. Whether the structure of the local cortical circuitry has changed as well is, however, unclear. Recent data from transcriptomic analyses has indicated an increase in the proportion of inhibitory interneurons from mouse to human. But what the effect of such a change is on the circuit configurations found in the human cerebral cortex is not known. This is, however, of particular interest also to the study of neuropsychiatric disorders because in these, the alteration of inhibitory-to-excitatory synaptic balance has been identified as one possible mechanistic underpinning. RATIONALE We used recent methodological improvements in connectomics to acquire data from one macaque and two human individuals, using biopsies of the temporal, parietal, and frontal cortex. Human tissue was obtained from neurosurgical interventions related to tumor removal, in which access path tissue was harvested that was not primarily affected by the underlying disease. A key concern in the analysis of human patient tissue has been the relation to epilepsy surgery, when the underlying disease has required often year-long treatment with pharmaceuticals, plausibly altering synaptic connectivity. Therefore, the analysis of nonepileptic surgery tissue seemed of particular importance. We also included data from one macaque individual, who was not known to have any brain-related pathology. RESULTS We acquired three-dimensional electron microscopy data from temporal and frontal cortex of human and temporal and parietal cortex of macaque. From these, we obtained connectomic reconstructions and compared these with five connectomes from mouse cortex. On the basis of these data, we were able to determine the effect of the about 2.5-fold expansion of the interneuron pool in macaque and human cortex compared with that of mouse. Contrary to expectation, the inhibitory-to-excitatory synaptic balance on pyramidal neurons in macaque and human cortex was not substantially altered. Rather, the interneuron pool was selectively expanded for bipolar-type interneurons, which prefer the innervation of other interneurons, and which further increased their preference for interneuron innervation from mouse to human. These changes were each multifold, yielding in effect an about 10-fold expanded interneuron-to-interneuron network in the human cortex that is only sparsely present in mouse. The total amount of synaptic input to pyramidal neurons, however, did not change according to the threefold thickening of the cortex; rather, a modest increase from about 12,000 synaptic inputs in mouse to about 15,000 in human was found. CONCLUSION The principal cells of the cerebral cortex, pyramidal neurons, maintain almost constant inhibitory-to-excitatory input balance and total synaptic input across 100 million years of evolutionary divergence, which is particularly noteworthy with the concomitant 1000-fold expansion of the neuronal network size and the 2.5-fold increase of inhibitory interneurons from mouse to human. Rather, the key network change from mouse to human is an expansion of almost an order of magnitude of an interneuron-to-interneuron network that is virtually absent in mouse but constitutes a substantial part of the human cortical network. Whether this new network is primarily created through the expansion of existing neuronal types, or is related to the creation of new interneuron subtypes, requires further study. The discovery of this network component in human cortex encourages detailed analysis of its function in health and disease. Connectomic screening across mammalian species: Comparison of five mouse, two macaque, and two human connectomic datasets from the cerebral cortex. ( A ) Automated reconstructions of all neurons with their cell bodies in the volume shown, using random colors. The analyzed connectomes comprised a total of ~1.6 million synapses. Arrows indicate evolutionary divergence: the last common ancestor between human and mouse, approximately 100 million years ago, and the last common ancestor between human and macaque, about 20 million years ago. ( B ) Illustration of the about 10-fold expansion of the interneuron-to-interneuron network from mouse to human.more » « less
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