Loss of the transcription factor Meis1 prevents sympathetic neurons target-field innervation and increases susceptibility to sudden cardiac death
- Institut national de la santé et de la recherche médicale, France
- INSERM U1046, CNRS UMR 9214, University of Montpellier, France
- Gui-de-Chauliac hospital, France
- The Methodist Hospital Research Institute, United States
Figures
Figure 1 with 2 supplements
Specific Meis1 inactivation in the PNS results in sympatho-vagal deregulation of cardiac functions.
(A) Immunochemistry for Meis1 and the transcription factor Phox2b on sections of SCGs from E16.5 WT and PLATCRE::Meis1LoxP/LoxP mice, and western blot analysis for Meis1 from adult stellate ganglia. …
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Figure 1—source data 1
Morphologic and left ventricular function parameters assessed by Doppler echocardiography in WT and PLATCRE::Meis1LoxP/LoxP mice.
HR, heart rate; AWTd, anterior wall thickness during diastole; AWTs, anterior wall thickness during systole; LVIDd, left ventricular internal diameter during diastole; LVIDs, left ventricular internal diameter during systole; PWTd, posterior wall thickness during diastole; PWTs, posterior wall thickness during systole; EF, ejection fraction; FS, fractional shortening; FAC, fractional area change; AoVTI, aortic velocity time integral; E, mitral inflow early filling velocity; A, mitral inflow late filling velocity. Data are represented as mean ± SD. Abbreviations: HR: Heart Rate; AWTd: Anterior Wall Thickness during diastole; AWTs: Anterior Wall Thickness during systole; LVIDd: Left Ventricular Internal Diameter during diastole; LVIDs: Left Ventricular Internal Diameter during systole; PWTd: Posterior Wall Thickness during diastole; PWTs: Posterior Wall Thickness during systole; EF (%) M-mode: Ejection Fraction; FS (%) M-mode: Fractional Shortening; Ao VTI: Aortic flow Velocity Time Integral; E wave: Mitral inflow early filling velocity (passive inflow); A wave: Mitral inflow late filling velocity (active inflow); E/A: Ratio E wave / A wave FAC (%): Fractional area; EF (%) B-mode: Ejection Fraction measured on B-mode.
- https://doi.org/10.7554/eLife.11627.004
Figure 1—figure supplement 1
Sympatho-vagal deregulation of cardiac functions following Meis1 inactivation in the PNS.
(A) Targeting vector used for the generation of the Meis1 conditional knockout mouse strain (Meis1LoxP/LoxP). The exon 8 of the gene encoding the beginning of the homeodomain has been flanked with …
Figure 1—figure supplement 2
Cardiac echography analysis revealed no difference following Meis1 inactivation.
(A) Representative B-mode images of heart parasternal long axis view. (B) Representative B-mode images of heart short axis view. (C) Representative M-mode images of heart short axis view at the …
Figure 2 with 1 supplement
Meis1 expression in sympathetic neurons is incompatible with early sympathetic specification but coincides with target-field innervation onset.
(A) ISH for Meis1, Gata3 and Klf7, and immunochemistry for Phox2b and TH in WT E11.5 embryonic SCGs. (B) ISH for Meis1, Hand2, Gata3 and Klf7 and immunochemistry for Phox2b, TH, Ntrk1 and Ret in WT …
Figure 2—figure supplement 1
Meis1 is the only member of the Meis family to be expressed by embryonic sympathetic neurons and is indifferently expressed by noradrenergic and cholinergic sympathetic neurons.
(A) In situ hybridization (ISH) for Meis1, Meis2 and Meis3 on E11.5, E13.5 and E14.5 WT embryos showed that Meis1 is the only member of the family to be expressed in sympathetic ganglia. Black …
Figure 3 with 2 supplements
Exacerbated neuronal apoptosis in Meis1 mutant sympathetic neurons.
(A) ISH for Gata3 on SCGs of PLATCRE::Meis1LoxP/LoxP and WT embryos at E14.5, 16.5 and E18.5. (B) Quantification of the volume of the SCGs in WT vs PLATCRE::Meis1LoxP/LoxP mice at E14.5, E16.5 and …
Figure 3—figure supplement 1
Survival of glial progenitor cells is not affected following Meis1 inactivation.
(A) ISH for Sox10 and immunochemistry for Sox2 and Casp3 on E14.5 and E16.5 SCGs from WT and PLATCRE::Meis1LoxP/LoxP embryos. White arrows point at Casp3+ cells. (B) Immunochemistry for Sox2 and …
Figure 3—figure supplement 2
Meis1 inactivation does not affect early proliferation of sympathetic neurons.
(A) Pregnant mice were injected with 0.7 mg 5-ethynyl-2´-deoxyuridine (EdU) at 14 and 16 days post-coitum. 3 hr following injection, pregnant mice were sacrificed and embryos were dissected, fixed …
Figure 4 with 1 supplement
Lack of terminal sympathetic target-field innervation in Meis1 mutant embryos.
(A) Immunochemistry for TH on SCGs from E14.5 and E16.5 WT and PLATCRE::Meis1LoxP/LoxP embryos showing that proximal axonal growth of sympathetic neurons is not affected. Sy Nerve = sympathetic …
Figure 4—figure supplement 1
Sympathetic target-field innervation of peripheral organs is compromised following Meis1 loss of function.
(A and B) Visualization of TH+ sympathetic fibers (in red) and others PGP9.5+/TH- peripheral nervous projections (in green) by immunochemistry in the salivary glands (A) and the trachea (B) of PLATCR…
Figure 5 with 1 supplement
Target-field innervation signaling pathways in Meis1-inactivated sympathetic neurons.
(A) ISH for the neurotrophins high affinity receptor Ntrk1 in E14.5 and E16.5 WT and PLATCRE::Meis1LoxP/LoxP SCGs. (B) Quantification of Ntrk1 ISH signal intensity in E16.5 WT and PLATCRE::Meis1LoxP/…
Figure 5—figure supplement 1
Meis1 loss of function does not interfere with the expression of other neurotrophins receptors than Ntrk1 and its expression is not dependent on Ngf/Ntrk1 signaling.
ISH for Ntrk2 and Ntrk3, the high-affinity receptors for Ntf2 and Ntf3 respectively and for Ngfr, the low affinity receptor for all members of the neurotrophins family on WT and PLATCRE::Meis1LoxP/Lo…
Figure 6 with 2 supplements
Meis1 target genes encode proteins necessary for early endosomes formation.
(A) ISH for Syt1 on SCGs of E14.5 and E16.5 WT and PLATCRE::Meis1LoxP/LoxP embryos. (B) Immunochemistry for clathrin and TH on SCGs of E16.5 WT and PLATCRE::Meis1LoxP/LoxP embryos. (C) …
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Figure 6—source data 1
Meis1 target genes in embryonic sympathetic neurons.
To identify Meis1 target genes, a ChIP-seq assay was conducted on E16.5 WT SCGs. The table includes the precipitated sequence ID (column A), the chromosome where the precipitated sequences match (column B), the start (column C) and the end (column D) of the precipitated sequences, the strand that the precipitated sequences recognize (column E), the name of the gene located within the 20kb of the precipitated sequence (column F), the gene accession number (column G), the strand where the 5’–3’ genes is located (column H) and the fold enrichment vs control experiment (column I).
- https://doi.org/10.7554/eLife.11627.017
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Figure 6—source data 2
Bibliographical-based classification of Meis1 potential target genes.
The table indicates the precipitated sequence ID (column A) and the chromosome where the precipitated sequences match (column B). Bibliographical search for each identified Mei1 potential target genes revealed a strong enrichment in genes involved in synapse formation, function and architecture (column C), in target-field innervation (column D) and/or cardiac functions and/or pathologies (column E).
- https://doi.org/10.7554/eLife.11627.018
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Figure 6—source data 3
Bibliographical classification of Meis1 potential target genes according to their function in endocytosis, exocytosis and vesicles transport.
The table indicates the genes names with one major reference corresponding to their implication in synaptic machinery (column 1), their ability to bind the clathrin and/or the dynein complexes (column 2), their involvement in cargoes traffic (column 3), in microtubule-associated axonal growth (column 4), in the regulation of endocytosis (column 5) and others genes indirectly implicated in these functions (column 6). Importantly, because of their wide expression in different vesicles types, many of these genes such as Lnp, Syt1, Htt, Nav1 or Stab2 can be classified in more than one column. Genes highlighted in yellow were also identified by Wilson et al. (Wilson et al., 2010).
- https://doi.org/10.7554/eLife.11627.019
Figure 6—figure supplement 1
Bibliographical classification of Meis1 potential target genes.
(A) Diagram representing, among all identified Meis1 potential target genes, the percentage of genes with known functions in synapse machinery and target-field innervation. A significant proportion …
Figure 6—figure supplement 2
Analysis of endocytic ultrastructures in sympathetic mutant fibers.
(A) Representative electron microscopy images of sympathetic nerves exiting the SCG dissected from E16.5 WT and PLATCRE::Meis1LoxP/LoxP embryos. Note that WT axons contain more endocytic structures …
Figure 7
Meis1 is necessary for early endosomes trafficking.
(A) Camera lucida of TH-immunochemistry and quantification of neurites length on SCGs explants form WT and PLATCRE::Meis1LoxP/LoxP embryos. (B) Individual frames and corresponding kymograph of a 2 …
Videos
Video 1
Traffic of Rab5-GFP+ endosomes in WT neurons.
Representative videos showing 2 min acquisition of Rab5-GFP+ endosomes trafficking in the axons of cultured WT sympathetic neurons. See also Figure 7.
Video 2
Impaired traffic of Rab5-GFP+ endosomes in Meis1 inactivated neurons.
Representative videos showing 2 min acquisition of Rab5-GFP+ endosomes trafficking in the axons of cultured PLATCRE::Meis1LoxP/LoxP sympathetic neurons. See also Figure 7.
Video 3
Traffic of Rab5-GFP+ endosomes in WT neurons.
Representative videos showing 2 min acquisition of Rab5-GFP+ endosomes trafficking in the axons of cultured WT sympathetic neurons. See also Figure 7.
Video 4
Impaired traffic of Rab5-GFP+ endosomes in Meis1 inactivated neurons.
Representative videos showing 2 min acquisition of Rab5-GFP+ endosomes trafficking in the axons of cultured PLATCRE::Meis1LoxP/LoxP sympathetic neurons. See also Figure 7.
