dI2 interneurons (INs) were labeled as cells that expressed both the Foxd3 and Ngn1 enhancers (Avraham et al., 2009; see Figure 1—figure supplement 1A). (A–D) dI2 axonal projection during …
Localization of dI2 neurons at the sciatic level.
The X/Y coordinates of dI2, large-diameter dI2, and small-diameter dI2 neurons (Figure 1F).
Localization of excitatory and inhibitory dI2 neurons.
The X/Y coordinates of dI2 neurons expressing either vGlut2 or VIATT (Figure 1I).
(A) The different DNA constructs used in this study. For targeted expression in dI1 cells, the EdI1 enhancer was used (Avraham et al., 2009). To target dI2 cells, the intersection of the Ngn1 and Fox…
(A) Premigratory dI2 (yellow) and early ventral migrating dI2 (cyan) neurons are Lhx1+Pax2- (cyan arrows). Cross-section of an embryonic day (E) 5 chick neural tube, expressing GFP in dI2 neurons …
Pattern of expression of transcription factors (TFs) in dI2 neurons.
Expression of TFs in dI2 neurons (Figure 1—figure supplement 2E).
(A) Density plot and laminar distribution of dI2 somata in crural plexus segments (N = 551 cells) (left). Density plot of dI2large (magenta) and dI2small (yellow) neurons in the crural plexus …
Localization of dI2 neurons at the crural and brachial levels.
The X/Y coordinates of large diameter dI2 and small diameter dI2 neurons in the crural and brachial spinal levels (Figure 1—figure supplement 3A and B).
(A) Experimental setup for labeling dI2 neurons that project to the cerebellum. dI2 neurons were genetically targeted at HH18, and precerebellar neurons were labeled using intracerebellar injection …
Localization of precerebellar neurons and dI2 synapses at the sciatic level.
(A) dI2 synapses in the central cerebellar nuclei. A cross-section of an embryonic day (E) 17 chick cerebellum. dI2 synapses (magenta), synaptotagmin (cyan). The arrowhead points to the midline, the …
Localization of precerebellar neurons and dI2 synapses at the crural level.
The X/Y coordinates of precerebellar neurons (Figure 1—figure supplement 1B) and dI2 synapses (Figure 2—figure supplement 1C) at the crural level.
(A) Spinal cord scheme describing dI2 axonal projection along the rostrocaudal axis (caudal is to the left, and rostral is to the right). The full lines represent the lumbar and brachial levels …
(A) A longitudinal bifurcation of a rostrally projecting dI2 axon. 3D reconstructions of light-sheet microscopy images of a lumbar-level dI2 neuron projecting at the brachial level. The bifurcated …
Analysis if the dI2 and ventral spinocerebellar tract (VSCT) axons at the superior peduncle.
The number of axons expressing GFP or/and cherry at the superior peduncle (Figure 3—figure supplement 1D).
Schematic representations of the experimental design for labeling dI2::GFP or dI2::Cherry interneurons (INs; cyan) and potential sources of synaptic inputs (magenta). The soma densities of dI2 INs …
Localizations of pre-dI2 terminals and synapses at the sciatic level.
The X/Y coordinates of dorsal root ganglion (DRG) axons and premotor neurons (pre-MNs) axons (Figure 4A and B). The X/Y coordinates of dI1, V1, and synapses and 5HT terminals at the sciatic level (Figure 4C–E).
(A) Premotor neurons (red) contacted by dI2::SV2-GFP and colabeled with synaptotagmin (syn in blue) antibody. (B) A group of SV2-GFP-labeled boutons and the syn content (blue foci). Note that only …
Validation of the use of SV2-GFP reporter as an indicator for synapses.
The volume of GFP+/syn+ and GFP+/syn- dI2 synapses on premotor neurons (Figure 4—figure supplement 1C).
(A, B) Schematic illustration of the strategy for studying the potential innervation of motor neurons by dI1 neurons (A). dI1 neurons were targeted by a dI1-specific enhancer (Figure 1—figure …
Localization of dI1 synapses.
The X/Y coordinates of dI1 synapses at the sciatic level (Figure 4—figure supplement 2C).
(A, B) Sparse sensory innervation of ventrally located dI2 neurons. Cross-section of an embryonic day (E) 17 embryo at the lumbar spinal cord (crural plexus level). A ventrally located dI2 neuron is …
Distribution of dorsal root ganglion (DRG) terminals on dI2 neurons.
The ventral/dorsal location and the large/small somata of dI2 neurons that are contacted by DRG terminals (Figure 4—figure supplement 3E,F).
Localizations of pre-dI2 terminals and synapses at the crural level.
The X/Y coordinates of dI1 synapses and 5HT terminals at the crural level (Figure 4—figure supplement 3G,H).
(A) Whole-mount staining of the spinal cord (thoracic segments) expressing alkaline phosphatase (AP) in dI2 neurons. The lumbar dI2 neurons (not included in the image) were labeled with AP. dI2 axon …
Localization of dI2 synapses on post-dI2 neurons at the sciatic and brachial levels.
The X/Y coordinates of dI2 synapses, ipsi premotor neurons (pre-MNs), commissural pre-MNs, and contralateral dI2 at the sciatic level; dI2 synapses and ipsi pre-MNs at the brachial level (Figure 5).
Cell soma density of putative targets (magenta) of dI2 neurons and dI2 synaptic densities (dI2::SV2-GFP, yellow) are illustrated in (A–E). The laminar distribution of the somata and synapses is …
(A) Schematic illustration of chick hindlimb joints (bold) and bones (regular). The knee joint connects the femur and the tibiotarsus, and the ankle connects the tibiotarsus and the tarsometatarsus, …
Analysis of knee height trajectories during the swing phase.
The knee height trajectories during the normalized swing in all the analyzed steps of all chicks (Figure 6D).
Analysis of TMP angles during the swing phase.
The TMP angle trajectories during the normalized swing in all the analyzed steps of all chicks (Figure 6E).
Statistical analysis of knee height trajectories and TMP angles.
Statistical analysis for the data presented in Figure 6.
The mean left-right phase of the control and TeTX-treated chicks. The mean phase values are indicated by the r-vectors (arrows). The Rayleigh critical values (p=0.05) are indicated by blue circles.
Analysis of left-right phase.
The left-right phase of the control and TeTX-treated chicks (Figure 6—figure supplement 1).
(A) The percentage of steps with body collapses in the controls and TeTX-manipulated hatchlings (n = 4 and n = 5, respectively). p-value<0.0001 (Z-test). See Table 3 for the proportions of falls at …
Analysis of collapses.
The number of collapses of each chick (Figure 7A).
(A, B) The range of knee height and TMP angle (A and B, respectively, see text for details) for each chicken (four controls and five TeTX-treated chicks). Purple lines: averages of each group …
Statistical analysis of knee height trajectories and TMP angles of each chick.
Statistical analysis for the data presented in Figure 7—figure supplement 1.
Analyses of the landing angle. (A) Scheme of bones and joints of the chicken leg, indicating the landing angle. (B) A box plot showing the landing angles. The control and manipulated chickens did …
Analysis of the landing angles in all steps.
The landing angles of each chick (Figure 7—figure supplement 2B).
Analysis of the landing angles prior to a collapse.
The landing angles before and not before a collapse in each chick (Figure 7—figure supplement 2C).
Caudal to rostral transverse images of light-sheet microscopy images along the lumbar segments of an embryonic day (E) 13 spinal cord expressing Cherry in dI2 neurons (a reference to the location of …
3D reconstruction of light-sheet microscopy images of the lumbar spinal cord. In the transparent mode, dI2 axons are apparent on the contralateral side. The trajectory of two representative axons …
3D reconstruction of light-sheet microscopy images of dI2 axons entering and exiting the brachial spinal cord. Two representative axons (ventral and lateral projection axons in yellow and green, …
3D reconstruction of light-sheet microscopy images of dI2 axons projecting into the brain stem and the cerebellum (blue and red, respectively). Cerebellum midline crossing is demonstrated for two …
Chick | Mean swing velocity (cm/s) | Mean left-right phase (°) | # of steps |
---|---|---|---|
TeTX1 | 46.78 ± 22.13 | 184.679 ± 33.003 | 113 |
TeTX2 | 62.24 ± 20.17 | 182.293 ± 32.01 | 63 |
TeTX3 | 48.06 ± 20.04 | 180.784 ± 31.064 | 69 |
TeTX4 | 57.24 ± 24.35 | 180.502 ± 36.291 | 59 |
TeTX5 | 36.66 ± 17.61 | 181.97 ± 35.787 | 93 |
Control 1 (GFP) | 79.65 ± 37.77 | 182.369 ± 35.366 | 47 |
Control 2 (GFP) | 41.91 ± 20.41 | 182.384 ± 26.708 | 19 |
Control 3 (not electroporated) | 41.09 ± 16.59 | N.D. | 121 |
Control 4 (not electroporated) | 42.3 ± 30.91 | N.D. | 51 |
Chick | Maximum stride width (cm) | # of steps |
---|---|---|
TeTX1 | 5.11 ± 1.89 | 97 |
TeTX2 | 5.32 ± 1.38 | 36 |
TeTX3 | 4.5 ± 1.01 | 27 |
TeTX4 | 4.9 ± 1.16 | 49 |
TeTX5 | 5.82 ± 1.71 | 110 |
Control 8 | 4.15 ± 1.07 | 137 |
Control 9 | 4.32 ± 1.32 | 115 |
Chick | % of steps with collapse | TMP angle: mean range (°) | # of steps | % of steps with collapse | ||||||
---|---|---|---|---|---|---|---|---|---|---|
All steps, mean | Combined mean | Minus collapses, mean | Minus collapses, combined mean | All steps, mean | Combined mean | Minus collapses | Minus collapses, combined mean | |||
TeTX1 | 4.4 | 3.05 ± 0.49 | 3.11 ± 0.74 | 2.87 ± 0.38 | 2.83 ± 0.57 | 82.27 ± 22 | 72.71 ± 20.52 | 79.4 ± 24.5 | 68.97 ± 21.47 | 113 |
TeTX2 | 20.60 | 3.5 ± 0.3 | 3.35 ± 0.47 | 71.79 ± 25 | 71.1 ± 25.68 | 63 | ||||
TeTX3 | 18.8 | 2.57 ± 0.31 | 2.27 ± 0.26 | 64.17 ± 21 | 62.79 ± 20.9 | 69 | ||||
TeTX4 | 20.45 | 2.54 ± 0.55 | 2.477 ± 0.56 | 72.48 ± 17 | 66.22 ± 19.1 | 59 | ||||
TeTX5 | 29 | 3.86 ± 0.83 | 3.2 ± 0.2 | 72.86 ± 12 | 65.85 ± 13.13 | 93 | ||||
Control 1 (GFP) | 2.12 | 1.91 ± 0.22 | 1.98 ± 0.33 | 1.91 ± 0.22 | 1.98 ± 0.33 | 56.8 ± 16.4 | 49.34 ± 16.03 | 56.95 ± 16.6 | 49.4 ± 16.18 | 47 |
Control 2 (GFP) | 0 | 1.83 ± 0.25 | 1.83 ± 0.25 | 41.42 ± 18.4 | 41.42 ± 18.4 | 19 | ||||
Control 3 (not electroporated) | 0 | 2.42 ± 0.23 | 2.42 ± 0.23 | 54.86 ± 9.65 | 54.86 ± 9.65 | 121 | ||||
Control 4 (not electroporated) | 0 | 1.75 ± 0.1 | 1.75 ± 0.1 | 44.12 ± 12.51 | 44.12 ± 12.51 | 51 |
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (Chicken) | Gallus gallus | Gil-Guy Farm, Israel | NCBI Taxon: 9031 | |
Strain, strain background (Pseudorabies virus) | PRV152 | Enquist and Card, 2003 | NCBI Taxon: 10345 | |
Strain, strain background (Pseudorabies virus) | PRV614 | Enquist and Card, 2003 | NCBI Taxon: 10345 | |
Antibody | Rabbit anti-GFP(polyclonal) | Molecular Probes, Eugene, Oregon, USA | A-11122RRID:AB_221569 | Dilution(1:1000) |
Antibody | Mouse anti-GFP(monoclonal) | Abcam | Ab1218AB_298911 | Dilution(1:100) |
Antibody | Goat anti-GFP(polyclonal) | Abcam | Ab6673RRID:AB_305643 | Dilution(1:300) |
Antibody | Rabbit anti-RFP(polyclonal) | Acris | AP09229PU-NRRID:AB_2035909 | Dilution(1:1000) |
Antibody | Goat anti-ChAT(polyclonal) | Millipore, USA | AB144PRRID:AB_2079751 | Dilution(1:300) |
Antibody | Mouse anti-synaptotagmin(monoclonal) | Hybridoma Bank, University of Iowa, Iowa City, USA | ASV30RRID:AB_2295002 | Dilution(1:100) |
Antibody | Mouse anti-lhx1/5(monoclonal) | Hybridoma Bank, University of Iowa, Iowa City, USA | 4F2RRID: AB_531784 | Dilution(1:100) |
Antibody | Mouse anti-FoxP4(monoclonal) | Hybridoma Bank, University of Iowa, Iowa City, USA | PCRP-FOXP4-1G7RRID:AB_2618641 | Dilution(1:50) |
Antibody | Rabbit anti-Pax2(polyclonal) | Abcam | ab79389RRID:AB_1603338 | Dilution(1:50) |
Antibody | Chicken anti- lacZ(polyclonal) | Abcam | ab79389RRID:AB_307210 | Dilution(1:300) |
Antibody | Rabbit anti-calbindin(polyclonal) | Swant | D-28kRRID:AB_2314070 | Dilution(1:200) |
Antibody | Goat anti-FoxP2(polyclonal) | Abcam | ab1307RRID:AB_1268914 | Dilution(1:1000) |
Antibody | Rabbit anti-5-HT(polyclonal) | Abcam | ab140495 | Dilution(1:100) |
Recombinant DNA reagent | EdI1::Cre | Avraham et al., 2009 | N/A | |
Recombinant DNA reagent | Ngn1::Cre | Avraham et al., 2009 | N/A | |
Recombinant DNA reagent | Ngn1::FLPo | Hadas et al., 2014 | N/A | |
Recombinant DNA reagent | Foxd3::FLPo | Hadas et al., 2014 | N/A | |
Recombinant DNA reagent | Foxd3::Cre | Avraham et al., 2009 | N/A | |
Recombinant DNA reagent | Isl1::Cre | Avraham et al., 2010a | N/A | |
Recombinant DNA reagent | CAG-LSL-GFP | Hadas et al., 2014 | N/A | |
Recombinant DNA reagent | CAG-LSL-SV2-GFP | Hadas et al., 2014 | N/A | |
Recombinant DNA reagent | CAG-FSF-LSL-GFP | Hadas et al., 2014 | N/A | |
Recombinant DNA reagent | CAG-FSF-LSL-SV2-GFP | This paper | N/A | Figure 1—figure supplement 1;can be obtained from the Klar lab |
Recombinant DNA reagent | CAG-FSF-LSL-cherry | This paper | N/A | Figure 1—figure supplement 1;can be obtained from the Klar lab |
Recombinant DNA reagent | CAG-FSF-LSL-SV2-cherry | This paper | N/A | Figure 1—figure supplement 1;can be obtained from the Klar lab |
Recombinant DNA reagent | CAG-FSF-LSL-AP | This paper | N/A | Figure 1—figure supplement 1;can be obtained from the Klar lab |
Recombinant DNA reagent | CAG-LSL-TeXT | This paper | N/A | Figure 1—figure supplement 1;can be obtained from the Klar lab |
Recombinant DNA reagent | CAG-LSL-F_SV2-cherry_F-GFP | This paper | N/A | Figure 1—figure supplement 1;can be obtained from the Klar lab |
Recombinant DNA reagent | pGEMTEZ-TeTxLC | Addgene | #32640 | |
Sequence-based reagent | Foxd3-F | This paper | PCR primers | TCATCACCATGGCCATCCTG |
Sequence-based reagent | Foxd3-R | This paper | PCR primers | GCTGGGCTCGGATTTCACGAT |
Sequence-based reagent | vGlut2-F | This paper | PCR primers | GGAAGATGGGAAGCCCATGG |
Sequence-based reagent | vGlut2-R | This paper | PCR primers | GAAGTCGGCAATTTGTCCCC |
Sequence-based reagent | VIAAT-F | This paper | PCR primers | CTGAACGTCACCAACGCCATCC |
Sequence-based reagent | VIAAT-R | This paper | PCR primers | GGGTAGGAGAGCAAGGCTTTG |
Commercial assay or kit | NucleoBond Xtra Midi | Macherey-Nagel | Cat # 740410.50 | |
Chemical compound, drug | CTB conjugated to Alexa Fluor 647 | Thermo Fisher | C34778 | 0.3 M |
Software, algorithm | JMP | JMP | https://www.jmp.com/en_gb/home.html | |
Software, algorithm | Adobe Photoshop | Adobe | https://www.adobe.com/il_en/ | |
Software, algorithm | ImageJ | ImageJ | https://imagej.nih.gov/ij/ | |
Software, algorithm | IMARIS | Oxford Instruments | https://imaris.oxinst.com/ | |
Software, algorithm | MacVector | MacVector | https://macvector.com/index.html | |
Other(electroporator) | BTX Electroporator | BTX Harvard Apparatus | Cat#45-0662 | |
Other(confocal microscope) | FV1000; Olympus | Olympus | https://www.olympus-global.com/ | |
Other(microscope) | Eclipse Ni | Nikon | https://www.nikon.com/ | |
Other(light-sheet microscope) | LaVision Ultramicroscope II light-sheet microscope | LaVision BioTec | https://www.lavisionbiotec.com/ |
Weight, force, and number of electroporated cells.
See statistical tests in Supplementary Statistical analysis tables.