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• W4246680377 abstract "Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Aberrant signaling through the Raf/MEK/ERK (ERK/MAPK) pathway causes pathology in a family of neurodevelopmental disorders known as 'RASopathies' and is implicated in autism pathogenesis. Here, we have determined the functions of ERK/MAPK signaling in developing neocortical excitatory neurons. Our data reveal a critical requirement for ERK/MAPK signaling in the morphological development and survival of large Ctip2+ neurons in layer 5. Loss of Map2k1/2 (Mek1/2) led to deficits in corticospinal tract formation and subsequent corticospinal neuron apoptosis. ERK/MAPK hyperactivation also led to reduced corticospinal axon elongation, but was associated with enhanced arborization. ERK/MAPK signaling was dispensable for axonal outgrowth of layer 2/3 callosal neurons. However, Map2k1/2 deletion led to reduced expression of Arc and enhanced intrinsic excitability in both layers 2/3 and 5, in addition to imbalanced synaptic excitation and inhibition. These data demonstrate selective requirements for ERK/MAPK signaling in layer 5 circuit development and general effects on cortical pyramidal neuron excitability. https://doi.org/10.7554/eLife.11123.001 eLife digest In the nervous system, cells called neurons form networks that relay information in the form of electrical signals around the brain and the rest of the body. Typically, an electrical signal travels from branch-like structures at one end of the cell, through the cell body and then along a long fiber called an axon to reach junctions with another neurons. The connections between neurons start to form as the nervous system develops in the embryo, and any errors or delays in this process can cause severe neurological disorders and intellectual disabilities. For example, genetic mutations affecting a communication system within cells known as the ERK/MAPK pathway can lead to a family of syndromes called the “RASopathies”. Abnormalities in this pathway may also contribute to certain types of autism. However, it is not clear how alterations to the ERK/MAPK pathway cause these conditions. Xing et al. investigated whether ERK/MAPK signaling regulates the formation of connections between neurons and the activity of neurons in mouse brains. The experiments showed that the growth of axons that extend from an area of the brain called the cerebral cortex towards the spinal cord are particularly sensitive to changes in the level of signaling through the ERK/MAPK pathway. On the other hand, inhibiting the pathway has relatively little effect on the growth of axons within the cerebral cortex. Further experiments showed that many neurons in the cerebral cortex require the ERK/MAPK pathway to activate genes that alter neuronal activity and the strength of the connections between neurons. Xing et al.’s findings suggest that defects in the connections between the cerebral cortex and different regions of the nervous system may contribute to the symptoms observed in patients with conditions linked to alterations in ERK/MAPK activity. Future studies will focus on understanding the molecular mechanisms by which ERK/MAPK pathway influences the organization and activity of neuron circuits during the development of the nervous system. https://doi.org/10.7554/eLife.11123.002 Introduction The canonical Ras/Raf/MEK/ERK (ERK/MAPK) signaling pathway is a key intracellular signaling cascade downstream of cell surface receptors critical for brain development (Samuels et al., 2009). In the developing cortex, the ERK/MAPK pathway is thought to be particularly important for neuronal responses to neurotransmitters and receptor tyrosine kinase (RTK) ligands, such as FGFs and neurotrophins. In the mature brain, it is well established that ERK/MAPK plays a central role in the activity-dependent plasticity of neural circuits (Shilyansky et al., 2010; Thomas and Huganir, 2004). Importantly, a number of human neurodevelopmental syndromes have been linked to aberrant ERK/MAPK activity. This related group of human syndromes, increasingly referred to as RASopathies, are caused by genetic mutations in core components or regulators of the ERK/MAPK signaling cascade (Rauen, 2013). Macrocephaly, neurodevelopmental delay, cognitive impairment, and epilepsy are frequently observed in RASopathy patients with clinical manifestations being dependent on the precise causative mutation (Rauen, 2013). RASopathies are most often associated with hyperactive ERK/MAPK signaling (eg. Neurofibromatosis type 1 (NF1), Noonan, Costello, and Cardiofaciocutaneous (CFC) syndromes) (Rauen, 2013). However, mutations that lead to diminished ERK/MAPK activation have been identified in a subset of LEOPARD and CFC syndrome patients (Kontaridis et al., 2006; Nowaczyk et al., 2014). Abnormal Ras/MAPK signaling has also been observed in models of other monogenic neurodevelopmental disorders including Fragile X syndrome and Tuberous Sclerosis (Chévere-Torres et al., 2012; Faridar et al., 2014; Osterweil et al., 2013; Zhang et al., 2014). Recent exciting work has shown that pharmacological normalization of pathological Ras/MAPK activity is sufficient for correcting select cellular and behavioral abnormalities in Fragile X, Tuberous Sclerosis, NF1, Noonan, and Costello syndrome mutant mice (Cui et al., 2008; Lee et al., 2014; Li et al., 2005; Osterweil et al., 2013; Wang et al., 2012; Zhang et al., 2014). However, our understanding of these disorders remains rudimentary as there is limited information on brain cell type-specific consequences of either loss- or gain-of-function through this pathway. Accumulating evidence also suggests that pathological ERK/MAPK signaling contributes to certain forms of autism. Altered Ras/MAPK signaling has been identified as a common downstream mediator of divergent genetic mutations linked to autism, and MAPK3/ERK1 is present in a region of 16p11.2 mutated in ~1% of cases of autism (Eichler and Zimmerman, 2008; Gilman et al., 2012; Gilman et al., 2011; Kumar et al., 2008; Pinto et al., 2010; Pucilowska et al., 2015; Weiss et al., 2008). Little is known about how ERK/MAPK signaling might relate to the pathogenesis of autism. An important current research theme is that the behavioral manifestations of autism spectrum disorders (ASDs) may be linked to both functional hypo- and hyper-connectivity between distinct brain regions (Geschwind and Levitt, 2007; Just et al., 2007; Keown et al., 2013; Supekar et al., 2013). Furthermore, recent work in postmortem brains of autistic patients suggests that local patches of disorganization, in which cortical layers 4–5 are particularly affected, play an important role in disease pathogenesis (Stoner et al., 2014). In one study, co-expression network analyses of autism-linked genetic mutations suggested that layer 5 in prefrontal and sensorimotor cortex is a key site of convergence for pathogenesis (Willsey et al., 2013). Whether aberrant ERK/MAPK signaling might result in cortical layer disorganization and defective long-range connectivity is unknown. To address questions of cell type specificity and consequences for circuit formation, we have defined the effects of ERK/MAPK loss- and gain-of-function on the development of cortical pyramidal neurons. Pyramidal neuron-specific functions of ERK/MAPK signaling were assessed by deleting the upstream kinases Map2k1/Mek1 and Map2k2/Mek2 (hereafter referred to as Map2k1/2) or overexpressing hyperactive Map2k1. Conditional deletion of Map2k1/2 led to major disruption of layer 5 with noticeably fewer CTIP2-expressing large neurons compared to controls. Further, long range axon extension of layer 5 corticospinal projection neurons during early development was markedly impaired. Subsequent to delayed entry of axons into the cervical spinal cord, many layer 5 projection neurons in sensorimotor cortices underwent apoptosis. Gain-of-function ERK/MAPK signaling also affected layer 5 CST neurons with a resultant decrease in axon elongation and associated increase in axon branching. The morphological requirement for ERK/MAPK signaling was specific for layer 5, as layer 2/3 was not disrupted and callosal projection neurons in upper cortical layers do not exhibit overt changes in axon extension or targeting following Map2k1/2 deletion. In contrast to the layer-specific functions of ERK/MAPK on axonal development, we found that ERK/MAPK was required for the expression of ARC and other plasticity-associated genes across all cortical lamina. Further, loss of ERK/MAPK signaling in pyramidal neurons disrupted excitatory and inhibitory neurotransmission and altered intrinsic excitability in both layers 2/3 and 5. Our data reveal unexpectedly specific requirements for ERK/MAPK signaling in layer 5 circuit development and general effects on the excitability of cortical pyramidal neurons in multiple layers. Results Excitatory neuron-specific modification of ERK/MAPK activity Previous work has shown that MAPK1/3 (aka ERK1/2) is activated in embryonic cortical neurons, albeit at much lower levels than in the ventricular zone (Faedo et al., 2010; Li et al., 2014; Pucilowska et al., 2012; Toyoda et al., 2010). In western blots of sensorimotor cortical lysates from P1, 2, 7, 14, and 21 day old mice, we found that the levels of pan-MAPK1/3(ERK1/2) and pan-MAP2K1/2 show a steady but evident increase from a relatively lower level at birth (Figure 1—figure supplement 1A). Phosphorylated-MAPK1/3(ERK1/2) and phosphorylated-MAP2K1/2 levels were also relatively low at birth but increased noticeably by P7 and peaked at P14 (Figure 1—figure supplement 1A) (Oliveira et al., 2008). The pattern of phosphorylated-MAPK1/3(ERK1/2) expression in P3 histological sections of cortex did not exhibit any clear-cut laminar specificity (Figure 1—figure supplement 1B). These findings indicate that ERK/MAPK signaling is activated in the developing cortex, peaking during the second postnatal week. We generated a mouse model to test the direct, neuron-autonomous role of ERK/MAPK signaling by inactivating Map2k1/2 specifically in immature mouse cortical excitatory neurons. We conditionally deleted Map2k1/2 with a Cre-dependent Map2k1 allele, a germ-line Map2k2 deletion allele, and Cre-recombinase under the control of the NeuroD6/Nex promoter (Map2k1loxp/loxp;Map2k2-/-;Neurod6-Cre,referred to hereafter as Map2k1/2;Neurod6-Cre) (Goebbels et al., 2006). As expected, Neurod6-Cre activated reporter-gene expression in the Cre-dependent EYFP mouse line, Ai3, in excitatory, but not inhibitory, neurons in the neocortex by mid-embryogenesis (Figure 1—figure supplement 1C–E) (Madisen et al., 2010). Western blotting of neocortical lysates and immunolabeling show that Map2k1/2;Neurod6-Cre mice exhibit significantly reduced MAP2K1 levels by birth and reduced phosphorylation of ERK/MAPK substrates, RSK, and MSK (Figure 1—figure supplement 1F–H). Complete loss of MAP2K1 protein would not be expected in whole cortical lysates due to MAP2K1 expression in inhibitory interneurons and non-neuronal cell types (Figure 1—figure supplement 1F). These data show that the Neurod6-Cre-mediated genetic targeting strategy is effective at inducing loss of ERK/MAPK signaling in developing cortical excitatory neurons. Map2k1/2;Neurod6-Cre pups were born at normal Mendelian ratios without overt differences from littermate controls. However, a delay in overall growth could be detected by the end of the first postnatal week and lethality was invariably observed in the third to fourth postnatal week. Behaviorally, P14 Map2k1/2;Neurod6-Cre mice exhibited spontaneous and persistent hindlimb clasping when lifted by the tail, an indicator of neurological impairment. A qualitatively similar effect on growth, neurological function, and viability was also observed in Mapk1loxp/loxp;Mapk3-/-;Neurod6-Cre mice (data not shown), demonstrating that phenotypes are conserved following deletion of different core components of the ERK/MAPK cascade. Map2k1/2;Neurod6-Cre mice exhibited an average reduction in body weight of 37.1 ± 11.3% at P14 and neocortical volume was reduced by 23.2 ± 6.4% (mean ± SEM, n=7, <0.0001). A significant increase in neuron (NEUN/RBFOX3+ cell) density was observed in Map2k1/2;Neurod6-Cre sensory cortices from 1636 ± 185 neurons/mm2 to 2168 ± 231 neurons/mm2 (mean ± SEM, n=5, p=0.002) (Figure 1A–B). Given the significant difference in cortical size, we extrapolated a relative estimate of neuronal number by multiplying the density measurement by the relative change in cortical volume between individual littermate control and mutant pairs. This estimate showed no significant difference in relative global neuron number between mutant and control cortices (98.0 ± 3.5%, mean ± SEM, n=5, p=0.61). We did, however, detect a significant reduction in the size of NEUN+ soma in P14 Map2k1/2;Neurod6-Cre sensory cortices in all cortical layers with the most pronounced effect on layer 5 neurons (Figure 1C). In contrast, no decrease in the size of parvalbumin-expressing, GABAergic neurons in the cortex was observed (data not shown). These data show that ERK/MAPK signaling is necessary for neurological function and the somal growth of excitatory neurons, but do not provide strong support for a widespread role in regulating global excitatory neuron number. Figure 1 with 1 supplement see all Download asset Open asset Loss of ERK/MAPK signaling leads to a reduction in the number of CTIP+ layer 5 neurons and reduced neuronal somal size. (A–B). Immunostaining of P14 control (A) and Map2k1/2;Neurod6-Cre (B) sagittal forebrain sections for all neurons, callosal projection neurons, and subcortical projection neurons with NEUN, SATB2, and CTIP2, respectively, revealed an aberrant pattern of CTIP2 expression in layer 5 of mutant sensorimotor cortices (yellow arrows) (n=6, scale bar=100 µm). (C) Quantification of somal size was performed by measuring the cross-sectional area of randomly selected, NEUN labeled soma in distinct cortical layers. A reduction in the size of NEUN labeled soma in P14 mutant sensory cortices was detected in all cortical layers, but was particularly pronounced in layer 5 (n= >300 total neurons per layer derived from five independent mice per condition, mean ± SEM, *p<0.001). (D–I) Representative confocal images of CTIP2 immunolabeling in radial columns of primary motor (D–E), sensory (F–G), and visual (H–I) cortex from P14 control (D, F, H) and Map2k1/2;Neurod6-Cre (E, G, I) brains (scale bar=30 µm). (J) Quantification of the relative number of layer 5 CTIP2+ neurons as a proportion of the total number of NEUN+ neurons in a cortical column revealed a substantial decrease in motor and sensory, but not visual, cortices in P14 mutant mice (n=4, mean ± SEM, *p<0.05). https://doi.org/10.7554/eLife.11123.003 Recombination in Neurod6-Cre mice is particularly strong in the cerebral cortex, however, sparse recombination has been detected outside of the cortex (Figure 1—figure supplement 1C) (Goebbels et al., 2006). To confirm the deficits in Map2k1/2;Neurod6-Cre mice are specifically due to cortical malfunction, we deleted Map2k1/2 using an independent Cre line, Emx1-Cre. Emx1-Cre induces recombination in dorsal telencephalic progenitors by E9.5 that generate excitatory neurons specifically within the cortex and hippocampus (Gorski et al., 2002). Map2k1loxp/loxp;Map2k2-/-;Emx1-Cre (Map2k1/2;Emx1-Cre) exhibit cortex specific MAP2K1 deletion and a pattern of growth delay, clasping behavior, and lethality similar to that observed in Map2k1/2;Neurod6-Cre mice (Figure 1—figure supplement 1I–J). We conclude that ERK/MAPK signaling in cortical excitatory neurons is necessary for appropriate nervous system function and mouse viability. Disruption of layer 5 and reduced number of CTIP2+ neurons after ERK/MAPK inactivation The profound decrease in layer 5 neuron soma size caused by Map2k1/2 deletion suggests that ERK/MAPK signaling is particularly important for the development of this layer. Comprehensive analyses of layer 5 neurons have identified specific gene expression patterns, morphologies, and electrophysiological characteristics for this neuronal subtype (Greig et al., 2013; Kwan et al., 2012; Leone et al., 2008). We analyzed the expression pattern of a critical transcription factor for layer 5 development, CTIP2/BCL11B, in control and mutant cortices (Figure 1A–B) (Arlotta et al., 2005). The appearance of co-labeled CTIP2+/NEUN+ neurons in layer 5 appeared disrupted in rostral P14 Map2k1/2;Neurod6-Cre cortices compared to controls (Figure 1B, yellow arrows). CTIP2+ layer 5 neurons are a small percentage of the total cortical neuron population. In the sensory cortex of control mice, we found that CTIP2+ layer 5 neurons represent only 9.61 ± 0.84% (mean ± SEM, n=5) of all NEUN+ neurons within a radial column. Thus, our previous global NEUN estimates across an entire cortical column were not sensitive enough for detecting changes confined to sparse neuronal subtypes. To quantify the number of CTIP2+ neurons, we determined the relative proportion of cells in layer 5 that express CTIP2+ as a percentage of NEUN+ cells across all lamina in a radial cortical column. This measurement was performed in motor, sensory, and visual cortices. Indeed, we found that P14 Map2k1/2;Neurod6-Cre primary motor and sensory cortices exhibit a clear decrease in the relative proportion of CTIP2+ neurons in layer 5 (Figure 1D–G, J). Qualitatively similar results were observed in P14 Map2k1/2;Emx1-Cre cortices (Figure 2—figure supplement 1A–B). The relative proportion of CTIP2+ neurons in visual cortices (Figure 1H–I, J) was not significantly diminished in Map2k1/2;Neurod6-Cre mutants, suggesting the effect in sensory and motor cortex was due to loss of the cell type and not a result of ERK/MAPK regulation of global CTIP2 expression levels. Failure of CST development and absence of large tufted neurons in layer 5 Layer 5 neurons are morphologically heterogeneous with distinct subpopulations that can be differentiated by cortico-cortical or subcortical axonal projections. We examined layer 5 projections in the hindbrain corticobulbar tract (CBT) and spinal cord corticospinal tract (CST) of P14 Map2k1/2 mutant mice. Immunostaining for a well-established marker of corticospinal projections, PKCγ, and genetic labeling with Ai3 revealed a profound decrease in the size of the CBT in P14 Map2k1/2;Neurod6-Cre hindbrains (Figure 2A–B, Figure 2—figure supplement 1E–F). CST labeling was also strikingly reduced in the cervical spinal cord and essentially absent in the lumbar spinal cord in both Map2k1/2;Neurod6-Cre (Figure 2C–D) and Map2k1/2;Emx1-Cre mutants (Figure 2—figure supplement 1C–D). Analysis of rare Map2k1/2 mutants that survived as late as P24 revealed that no CST axons were present in lumbar spinal cords (data not shown). These data provide further evidence of an overt loss of corticospinal neurons by the second postnatal week. Figure 2 with 1 supplement see all Download asset Open asset Corticospinal tract defects in Map2k1/2;Neurod6-Cre mice. (A–D) To further evaluate the loss of layer 5 projection neurons, we examined the expression of a well-established corticospinal tract marker, PKCγ. Compared to control hindbrains (A) and spinal cords (C), a profound decrease in corticospinal tract labeling was observed in the Map2k1/2;Neurod6-Cre hindbrain (B-yellow arrows, scale bar=200 µm) and spinal cord (D), consistent with the reduced number of CTIP2+ layer 5 neurons (df=dorsal funiculus, n=3, scale bar=50 µm). (E–F) The Thy1-YFP reporter line, YFP16, labels a small fraction of layer 5 neurons in sensorimotor cortex. Many layer 5 neurons labeled in this line have large complex apical dendritic arbors (yellow arrows) that are consistent with the known morphology of subcerebral projection neurons, including corticospinal neurons. EYFP expressing neurons heavily branch in layer 1 as shown in representative confocal images of sagittal sections from P18 control mice (E, yellow arrows). In Map2k1/2;Neurod6-Cre cortices, we noted a significant reduction in the number of EYFP labeled neurons in layer 5 with complex apical dendritic arbors (F) (n=3, scale bar=100 µm). https://doi.org/10.7554/eLife.11123.005 Figure 2—source data 1 Reduced expression of layer 5 neuron markers following loss of Map2k1/2. (A) Gene expression profiling of RNA extracts from the cortex of control and Map2k1/2;Neurod6-Cre mice collected at P9 (n=2) and P14 (n=3) revealed significantly diminished expression (fold change>1.5 and p<0.05) of many genes that are known to be expressed in layer 5 neurons (red filled boxes). https://doi.org/10.7554/eLife.11123.006 Download elife-11123-fig2-data1-v1.xlsx Corticospinal neurons represent a subset of the entire CTIP2+ population in sensorimotor layer 5 (Arlotta et al., 2005). The lack of layer 5 neuron loss in the Map2k1/2;Neurod6-Cre visual cortex suggests that ERK/MAPK signaling is dispensable for the development of projection neurons targeting structures other than the spinal cord. Past work has shown that callosally projecting layer 5 neurons have significantly shorter and less complex apical dendritic arbors than subcortical projection neurons (Larsen et al., 2007; Molnár and Cheung, 2006). To determine which of these classes was affected by Map2k1/2 deletion, a Thy1-based reporter, YFP-16, that fluorescently labels a small proportion of layer 5 neurons in sensorimotor cortex, was bred with Map2k1/2;Neurod6-Cre mice (Feng et al., 2000). These mutants clearly show that the fluorescently labeled layer 5 neurons in P14-P21 Map2k1/2;Neurod6-Cre;YFP-16 sensorimotor cortices have substantially shorter apical dendrites than in controls (Figure 2E–F). The reduction in large, tufted neurons in layer 5 of mutant mice provide further evidence for a deficit in the development of corticospinal projection neurons. Gene expression profiling of ERK/MAPK inactivated cortices To gain further insight into the cellular and molecular mechanisms utilized by ERK/MAPK signaling to regulate cortical development, we performed microarray profiling of P14 whole cortical lysates from control and Map2k1/2;Neurod6-Cre mutants. The validity of the screen was verified by a highly significant -2.76 ± 0.13 (p-val<0.001)-fold reduction in the expression of Map2k1-exon 3 at the probe-level in P14 mutant samples. We observed a pronounced decrease in a large number of genes that are known to be highly expressed in layer 5 projection neurons at P9 or P14, including Etv1/Er81, Adcyap1, Ldb2, Dkk3, Lmo4, and Opn3 (fold-change>1.5 p<0.05) (Arlotta et al., 2005; Chen et al., 2005; Chen et al., 2005; Molyneaux et al., 2007) (Figure 2—source data 1). Moreover, Fezf2, a well-known master transcription factor important for corticospinal neuron development, showed a strong trend toward diminished expression in Map2k1/2;Neurod6-Cre mutants (Chen et al., 2005; Chen et al., 2005; Molyneaux et al., 2005). The gene profiling results provide further support for a specific loss of layer 5 neurons possibly due to cell death or altered fate specification in Map2k1/2 mutant mice. ERK/MAPK signaling is necessary for corticospinal axon extension The loss of layer 5 neurons following deletion of Map2k1/2 could be due to an early disruption in the initial specification of this neuronal subtype. During layer 5 neuron development, Neurod6-Cre is not expressed until neurons are post-mitotic (Goebbels et al., 2006; Wu et al., 2005). However, it remained possible that post-mitotic stages of embryonic layer 5 neuron specification were altered during embryogenesis. In newborn mutant pups, we found that the expression and number of CTIP2+ neurons in presumptive layer 5 was not diminished following Map2k1/2 deletion (Figure 3A–C). Thus, ERK/MAPK signaling is not required for the initial establishment of the correct numbers of the CTIP2+ deep-layer neuron population. Figure 3 with 1 supplement see all Download asset Open asset Layer 5 neuron corticospinal axon outgrowth requires ERK/MAPK signaling in vivo. (A–C) Expression of a well-known master transcription factor for layer 5 neurons, CTIP2, was intact in cortical layer 5 as shown in representative confocal images of newborn control (A) and Map2k1/2;Neurod6-Cre (B) sensorimotor cortices (scale bar=50 µm). Quantitation of the number of CTIP2-expressing nuclei in cortical layer 5 (C) did not reveal a significant difference in CTIP2+ neuron density between control and mutant neonates (n=3, mean ± SEM, p=0.89). (D–E) In vivo DiI injections into the sensorimotor cortex of P0.5 control and Map2k1/2;Neurod6-Cre neonates were performed and mice were collected three days after injection. The extent of anterograde DiI labeling was analyzed in coronal sections through the spinal cord (D). We observed a significant decrease in the extent of corticospinal (cst) elongation in mutant mice, especially in the lower cervical/thoracic spinal cord segments (yellow arrows in D) (n=3, scale bar=100 µm). Quantitation of CST length relative to the medullary decussation revealed a significant decrease in corticospinal axon growth in mutant spinal cords (E) (n=3, mean ± SEM, *p=0.02). (F–I) The initial stages of corticospinal elongation in the dorsal spinal cord can be visualized in sagittal sections of the caudal medulla and rostral spinal cord from P2 Emx1-Cre;Ai3 mice. Immunoenhancement of the Ai3 reporter with an EGFP antibody clearly shows that the caudal extension of corticospinal axons (yellow arrowheads) is profoundly reduced in P2 Map2k1loxp/loxp;Map2k2-/-;Emx1-Cre;Ai3 mutant mice (H) when compared to Map2k1loxp/wt;Map2k2-/-;Emx1-Cre;Ai3 controls (F) (n=3, scale bar=200 µm). Whole mount visualization of the CST coursing through the ventral medulla in control (G) and mutant (I) hindbrains did not reveal an overt difference in corticospinal growth (n=3). (J–P) Compared to control mice (J–L), Neurod6-Cre mediated deletion of Igf1r did not alter the relative area of PKCγ labeling in the cervical (K, N, P) or lumbar (L, O, P) CST compared to the medullary CBT (J, M) in mutant mice (M–O) at P21 (n=3, scale bar=50 µm). https://doi.org/10.7554/eLife.11123.008 By P3 in wild-type neonates, corticospinal axons have projected through the ventral hindbrain, crossed the midline at the medullary/spinal cord boundary, and are extending into cervical spinal cord through the dorsal funiculus (Schreyer and Jones, 1982). We asked whether ERK/MAPK signaling was required for the initial outgrowth of corticospinal projections in vivo. In vivo DiI injections into the motor cortex of P0.5 neonates were performed to assess subcortical axon growth, especially into the spinal cord (Figure 3—figure supplement 1A). Strikingly, analysis of anterogradely labeled axonal projections at P3-4 revealed a highly significant decrease in the extension of corticospinal axons into the lower cervical/upper thoracic segments of spinal cord (Figure 3D–E). Moreover, a profound decrease in the caudal extension of descending corticospinal axons into the spinal cord of P2 Map2k1/2;Emx1-Cre;Ai3 mice was also observed (Figure 3F,H). DiI, PKCγ, and Ai3 labeling of subcortical projections in Map2k1/2;Neurod6-Cre and Map2k1/2;Emx1-Cre mice revealed that layer 5 neuron growth into the tectum and pons/medulla was not significantly decreased (Figure 3G,I; Figure 3—figure supplement 1A–D). These data demonstrate that the initial growth of corticospinal axons to the level of the hindbrain is not significantly disrupted in Map2k1/2 mutants, however, ERK/MAPK signaling is clearly necessary for corticospinal axon elongation into the spinal cord. In vitro studies have demonstrated that IGF1 signaling acting via PI3K and ERK/MAPK promotes corticospinal axon outgrowth in vitro (Özdinler and Macklis, 2006). IGF1 is also focally and intensely expressed in the medulla in the presumptive inferior olivary nucleus during the precise developmental time frame (P1 – P2) that CST axons normally enter the spinal cord (Figure 3—figure supplement 1E). IGF1 expression is temporally regulated, being present by E18 and much diminished by P14 (data not shown). Map2k1/2 deletion in CST axons does not affect IGF1 expression by these cells (Figure 3—figure supplement 1E–F). Thus, IGF1 may act via ERK/MAPK within corticospinal neurons to regulate corticospinal outgrowth. We functionally tested whether cortical neuron specific loss of IGF1R would disrupt the CST development by generating Igf1rloxp/loxp;Neurod6-Cre mutants (Liu et al., 2009). Total body weight and brain size were significantly reduced in Igf1rloxp/loxp;Neurod6-Cre mutants (Figure 3—figure supplement 1I) and a clear loss of IGF1R immunoreactivity was observed in P7 Igf1rloxp/loxp;Neurod6-Cre cortices (Figure 3—figure supplement 1G–H). However, the expression of CTIP2 in layer 5 was not significantly altered (Figure 3—figure supplement 1J–K). Further, PKCγ labeling of the CST in the cervical and lumbar spinal cord did not reveal a significant difference in rostrocaudal extension in Igf1rloxp/loxp;Neurod6-Cre mutants (Figure 3J–P). These data suggest that direct regulation of ERK/MAPK in layer 5 neurons by IGF1R signaling is not a significant regulator of neuronal survival and CST development in vivo. Caspase-3 activation in Layer 5 neurons after ERK/MAPK deletion By P3 we noted a dramatic increase in the number of activated caspase-3 labeled neurons" @default.
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• W4246680377 title "Decision letter: Layer specific and general requirements for ERK/MAPK signaling in the developing neocortex" @default.
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