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craft-ca-core-ex-dev

2_test

{"project":"2_test","denotations":[{"id":"15836427-10850491-84719644","span":{"begin":1251,"end":1253},"obj":"10850491"},{"id":"15836427-2081815-84719645","span":{"begin":1593,"end":1595},"obj":"2081815"},{"id":"15836427-7812779-84719646","span":{"begin":1596,"end":1598},"obj":"7812779"},{"id":"15836427-11395002-84719647","span":{"begin":2126,"end":2128},"obj":"11395002"},{"id":"T86888","span":{"begin":1251,"end":1253},"obj":"10850491"},{"id":"T13726","span":{"begin":1593,"end":1595},"obj":"2081815"},{"id":"T49503","span":{"begin":1596,"end":1598},"obj":"7812779"},{"id":"T58140","span":{"begin":2126,"end":2128},"obj":"11395002"}],"text":"Only Precocious but Not Isochronic ETS Signaling in DRG Neurons Interferes with Neuronal Fate Acquisition\nThe observed differences in neuronal survival and neurite outgrowth between precocious and isochronic expression of EWS-Pea3 prompted us to perform a direct comparative analysis of gene expression between mice with precocious EWS-Pea3 expression and mice in which the expression of EWS-Pea3 is initiated in DRG sensory neurons from the time of normal onset of Er81 expression. Moreover, to rule out the possibility that a differential effect may be due to the different genetic strategies by which expression of EWS-Pea3 in proprioceptive afferents is achieved, we performed this analysis both in Er81EWS-Pea3/− and TauEWS-Pea3/+ PVCre/+ embryos.\nWe first analyzed expression of TrkC, a gene downregulated in DRG neurons of TauEWS-Pea3/+ Isl1Cre/+ embryos (Figure 7A and 7B). The level of expression of TrkC was indistinguishable from wild-type in DRG neurons of Er81EWS-Pea3/− and TauEWS-Pea3/+ PVCre/+ embryos (Figure 7A, 7C, and 7D). Moreover, PV was not expressed in DRG neurons of TauEWS-Pea3/+ Isl1Cre/+ embryos (Figure S5) but was expressed by proprioceptive afferents in both wild-type and Er81EWS-Pea3/− embryos (see Figures 1 and S5) [14]. We also found several genes that were ectopically upregulated in DRG neurons of TauEWS-Pea3/+ Isl1Cre/+ embryos (Figure 7). Calretinin and Calbindin, two different calcium-binding proteins expressed by subpopulations of DRG neurons in wild-type, Er81EWS-Pea3/−, and TauEWS-Pea3/+ PVCre/+ embryos (Figure 7E, 7G, and 7H; data not shown) [31,32], were induced in more than 95% of all DRG neurons of TauEWS-Pea3/+ Isl1Cre/+ embryos (Figures 7F and S5). These findings suggest that DRG neurons in TauEWS-Pea3/+ Isl1Cre/+ embryos fail to differentiate to a normal fate and instead acquire an aberrant identity distinct from any subpopulation of wild-type DRG neurons. Finally, to assess whether EWS-Pea3 expressed precociously acts exclusively cell-autonomously or whether it may also influence neighboring DRG neurons, we activated expression of EWS-Pea3 using Hb9Cre mice [33]. Due to a transient and rostro-caudally graded expression of Hb9 at neural plate stages, very few DRG neurons at brachial levels and increasingly more neurons progressing caudally undergo recombination in TauEWS-Pea3/+ Hb9Cre/+ and TaumGFP/+ Hb9Cre/+ embryos (Figure 8). Nevertheless, downregulation of Trk receptor expression or upregulation of Calretinin is restricted exclusively to neurons that have undergone recombination and cannot be observed in TaumGFP/+ Hb9Cre/+ embryos (Figure 8). Together, these results and the findings obtained from in vitro culture experiments (see Figures 4 and 5) demonstrate that precocious or isochronic expression of EWS-Pea3 in the same neurons leads to significantly different cell-autonomous cellular responses with respect to gene expression, neuronal survival, and neurite outgrowth (Figure 9).\nFigure 7 Gene Expression Analysis upon Induction of Precocious or Isochronic ETS Signaling\n(A–H) Analysis of TrkC expression by in situ hybridization (A–D), or Calretinin (red) and LacZ (green) expression by immunohistochemistry (E–H), on E16.5 lumbar DRG of wild-type (A and E), TauEWS-Pea3/+ Isl1Cre/+ (B and F), Er81EWS-Pea3/− (C and G), and TauEWS-Pea3/+ PVCre/+ (D and H) embryos.\n(I) Summary diagram illustrating deregulation of TrkC (red arrows, downregulation) and Calretinin (green arrows, upregulation) expression upon precocious (B and F) induction of EWS-Pea3 expression in DRG neurons (B and F; E10–E11, i.e., shortly after cell cycle exit, E9.5–E10). In contrast, activation of EWS-Pea3 from the endogenous Er81 locus (C and G; E12.5–E13) or via Cre recombinase expression from the PV locus activating late expression from the Tau locus (D and H; E14.5) does not interfere with the normal expression of TrkC and Calretinin (shown in grey).\nScale bar: (A–D), 65 μm; (E–H), 80 μm.\nFigure 8 Precocious ETS Signaling Induces Gene Expression Changes Cell-Autonomously\n(A–D) Expression of TrkA (A and C; green) or TrkC (B and D; green), and LacZ (red), in E16.5 lumbar DRG of TaumGFP/+ Hb9Cre/+ (A and B) and TauEWS-Pea3/+ Hb9Cre/+ (C and D) embryos.\n(E–L) Expression of Calretinin (green), LacZ (red), and Isl1 (F, J, H, and L; blue) in E16.5 brachial (E–H) and lumbar (I–L) DRG of TaumGFP/+ Hb9Cre/+ (E, F, I, and J) and TauEWS-Pea3/+ Hb9Cre/+ (G, H, K, and L) embryos.\nScale bar: (A–D), 80 μm; (E–L), 70 μm.\nFigure 9 Progressive Neuronal Specification Is Paralleled by a Developmental Shift in Response to ETS Transcription Factor Signaling\nSchematic summary diagram illustrating the importance of temporally appropriate upregulation of transcription factor expression during specification of DRG neurons for late aspects of neuronal differentiation and circuit assembly.\n(A–D) Expression of EWS-Pea3 from the endogenous Er81 locus can rescue anatomical defects observed in Er81−/− mice, and no change in expression of TrkC (green) or Calretinin (CR; grey) is observed in proprioceptive afferents (A, B, and D). In contrast, precocious ETS signaling leads to severe defects in the establishment of DRG neuronal projections accompanied by inappropriate gene expression changes (C; upregulation of CR (red) and downregulation of TrkC [grey]).\n(E) Precocious ETS signaling (red) during progressive specification of proprioceptive sensory neurons leads to aberrant neuronal differentiation (red dashed line). In contrast, the isochronic, target-induced (green; peripheral signal) onset of ETS transcription factor signaling (black) induces appropriate terminal neuronal differentiation (blue)."}

biosemtest

craft-ca-core-dev

PMC:1084331 / 26976-32647 JSONTXT

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craft-sa-dev

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2_test

biosemtest

craft-ca-core-dev

PMC:1084331 / 26976-32647 JSON TXT