Figures and data
Neurogenesis order of V1 clades assayed by EdU birthdating.
A, Experimental design. Timed pregnant En1cre-Ai9:tdT females were EdU injected at one of seven time points between E9.5 and E12.5 and the spinal cords were harvested at P5. Tissue sections were processed for EdU (Click-iT™) and immunostained for transcription factors (TFs) representing major V1 clades. MafB was used to identify the MafA-Renshaw cell clade by location (“ventral MafB-V1 cells”). This is because MafA antibodies in our hands labeled few Renshaw cells. B, Patterns of EdU labeling at different embryonic times. Spinal neurons are born in a ventrolateral to dorsomedial sequence. Until E11, mostly ventral neurons are generated. C, Example of E11 EdU labeling in an En1-tdT spinal cord. EdU integrated in the DNA at the time of injection is diluted with subsequent cell divisions. To ensure we only counted V1 cells that incorporated EdU during S-phase after their final division, we only counted V1 cells with nuclei largely filled by EdU Click-iT™ reaction (arrows indicate four such strongly labeled V1 EdU+ cells). D, Percentages of V1 cells labeled with “strong” EdU in each mouse. The x-axis indicates individual animals (“<litter number>.<animal number>”). For the most part there was consistency in the percentage of EdU-labeled V1 interneurons at each time point, although we also noted variability between litters and among animals within a litter. One animal (459.2) showed the wrong EdU pattern for its injection age and was discarded (indicated by an X). E, EdU birthdating reveals a peak in V1 neurogenesis around E11 (error bars = SD; each dot represents one animal). At the time points flanking the peak there is a larger amount of variability, suggesting a fast-changing pace of V1 neurogenesis. F, Representative images of TF antibody staining combined with EdU labeling to determine birthdates of defined V1 clades. The time points represented were selected according to the maximal or near maximal generation of V1 interneurons in each clade. G-I, V1-clade neurogenesis quantification. Graphs represent the average ±SEM calculated from n = 3.9 ±0.3 mice per TF/date (not all TFs were tested in all mice). For each mouse average, we analyzed four ventral horns in Lumbar 4 or 5 segments. G, Percentage of V1s expressing each clade-specific TF labeled with EdU at each embryonic time point. Ventral MafB-V1s (Renshaw cells) and Pou6f2-V1s are mostly born before E11 (dorsal MafB-V1s are a subgroup of Pou6f2-V1s). Foxp2-V1 and Sp8-V1 interneurons have wider windows of neurogenesis, but most are generated after E11. H, Data normalized to the maximum percentage of V1s born in each group to compare peak generation in each clade. Ventral MafB-V1s, Pou6f2-V1s, Foxp2-V1s and Sp8-V1s have progressively later times of peak generation. I, Cum-sum graphs of V1-clades neurogenesis. Between 50% to 68% of all neurons in each V1 clade are labeled across all ages. By E11 almost all early-clade neurons are generated, while less than half of neurons in late-clades are.
V1 interneurons born at different embryonic times settle in different locations.
For each panel in the top row, blue open dots indicate all V1 interneurons positive for each TF at P5, and filled red dots indicate those that also have strong EdU. Each plot is from one representative animal injected at each of the indicated embryonic times (four ventral horns from each animal were analyzed with all cell locations superimposed in the diagram). The bottom rows show cellular density profiles of the V1 interneurons positive for each TF that have strong EdU from all animals EdU-injected at the indicated embryonic times; n indicates the number of animals in each plot. Four ventral horns were analyzed per animal. Contour plots are derived from 2D kernel density estimates of interneuron positions; lines encompass 10% increments. Only timepoints with representative numbers of TF+/EdU+ cells are shown. Blue directional arrows highlight the difference in settling locations for V1s in each clade born at this time point with respect to the previous time point. A, MafB-V1s are divided into two groups: ventral MafB-V1s corresponding to Renshaw cells and later-born dorsal MafB-V1s that correspond to a subgroup of Pou6f2-V1s. B, Pou6f2-V1s are born between E10 and E11.5 and always settle dorsally. C, Foxp2-V1s show large variations in location according to birthdate. Cells of increasingly older birthdates settle dorsally, laterally, ventro-laterally and then ventro-medially. D, A few Sp8-V1s are born early and are located ventrally, but the majority of Sp8-V1s are born later and locate dorsally before moving more medially those born the latest. The directional changes in settling positions according to birthdate is different for each V1 clade.
MafB-V1s visualized in a MafB:GFP mouse model.
A, In mature (>P15) mice, MafB-GFP+ cells in lamina VII are mostly contained in the V1 lineage (tdTomato) and distribute to the most dorsal and ventral regions of the distribution of V1s. Those that are also calbindin-IR fall in the typical ventral region occupied by Renshaw cells. In addition, there are many dorsal horn non-V1 MafB-GFP+ neurons. The small cells throughout white and gray matter are microglia. B, Compared to P15, a few more ventral horn neurons express MafB-GFP at P5, including some motoneurons and microglia whose MafB labeling is weaker. Within MafB-GFP+ V1 neurons the two groups located at the most dorsal and most ventral regions correspond to the V1 neurons that retain MafB-GFP at P15. Like P15, the ventrally located group expresses calbindin. The dorsal group expressed Pou6f2 at this age. Little-to-no Foxp2-IR or Sp8-IR is found in either dorsal or ventral groups of MafB-GFP+ V1s. C, Quantitation of MafB-V1 neurons in P5 mice. Around 13% of all V1s express MafB-GFP+, and the percentages located in the Renshaw cell area (“ventral”) or dorsal lamina VII (“dorsal”) are evenly split (n = 17 mice, 4 ventral horns each. Bars show SD). D, At P5, over half of the MafB-GFP+ V1 cells have detectable levels of MafB (Sigma) immunoreactivity in both the ventral and dorsal groups (n = 9 mice, 4 ventral horns each. Bars show SD). E, MafB-GFP+ / Pou6f2-IR V1 cells are exclusively in the dorsal MafB-GFP group, while MafB-GFP+ / calbindin-IR V1 cells are exclusively in the Renshaw cell area, Negligible number of MafB-GFP+ V1 cells express Foxp2 or Sp8. (n=4-5 mice, 4 ventral horns each. Bars show SD). F, EdU birthdating reveals similar proportions of EdU+ neurons at P5 for dorsal MafB-GFP vs MafB-IR neurons labeled at each embryonic time (n = 2 mice per timepoint per condition, 4 ventral horns each. Bars show SD). The mismatch in birthdates between MafB genetic and antibody labeling at E11 in the ventral group could arise because some E11-born ventral MafB-GFP V1s quickly downregulate MafB to levels undetectable with antibodies.
Genetic mouse models used to label the Foxp2-V1 lineage.
A, P5 mouse intersection of En1::Cre and Foxp2::Flpo with two reporter alleles (Ai9-tdTomato and RCE:dualEGFP) in the Rosa26 locus. Foxp2-V1s express GFP (green) and non-Foxp2-V1s tdTomato (red). A few “yellow cells” correspond with Foxp2-V1 neurons that failed to remove the tdTomato Ai9 reporter. B, Density contours demonstrate high spatial overlap between V1 neurons expressing Foxp2 (EGFP+) or not (tdT+ and EGFP-) (n=6 ventral horns). C, Expression of the lineage label is stable throughout postnatal development. This means that there is no additional foxp2 gene upregulation in new V1s after P0 (n=6 ventral horns per age in one animal, except for P5 in which 6 animals and 36 ventral horns are included; error bars show SEM). D, In P15 mice, expression of the lineage labels is also uniform across the lower spinal cord segments from thoracic 13 to sacral 1 (n = 6 ventral horns in each segment from 2 mice; error bars show SEM). E, The En1::Cre, Foxp2::Flpo, RCE:dualEGFP model detects more Foxp2-V1s in the postnatal spinal cord than Foxp2 antibody staining at P5. F, Foxp2-V1s with or without postnatal expression of Foxp2 occupy roughly similar areas in the P5 spinal cord (n=3 mice, 3 ventral horns per animal). G, Right, Foxp2-V1s make up roughly 60% of all V1s when measured with genetic labeling, but only about 30% of V1s maintain detectable Foxp2 expression at P5. Left, around half of Foxp2-V1s maintain detectable levels of Foxp2 at P5 (n is same as F; error bars show SD). H, FLTG reporter mice reveal lineage-labeled non-V1 Foxp2 cells in the spinal cord. eGFP is expressed in Foxp2-V1s and tdTomato in non-V1 Foxp2 cells. The zoomed images of the highlighted region with and without NeuN-IR demonstrate that non-V1 Foxp2 cells include non-neuronal cell types with astrocyte morphologies. Only neurons (NeuN-IR) are included in the contour plots which show non-V1 Foxp2 neurons located in the medial ventral horn and a few in the deep dorsal horn. (n = 2 animals at P5, 6 ventral horns in each). I, Most Foxp2-neurons (red, non-V1s or green, V1s) are in the ventral horn and their number in 30 μm thick L4-5 sections decreases with age as the neuropil matures and expands in size. Dorsal horn Foxp2-neurons maintain their numbers despite the growth of the spinal cord. Each point is one animal analyzed through 6 ventral horns (errors bars are SD). J, The percentage of Foxp2-lineage-labeled cells that are Foxp2-V1s remains constant throughout postnatal development (n=2 mice, 6 ventral horns each; error bars show SD).
The spatial distribution of Foxp2-V1 interneurons is closely associated with shifting motor columns throughout thoracic, lumbar and sacral levels of the spinal cord.
A, Foxp2-V1 lineage labeling and ChAT antibody staining for motoneuron identification in adult mouse spinal cords from thoracic to sacral levels. Foxp2-V1 neurons accumulate at the lateral edge of the ventral spinal cord but their locations shift as the LMC expands from L3 to L6. In addition, there is a distinct group of Foxp2-V1 interneurons dispersed at medial locations in all lumbar segments. B, Plots of Foxp2-V1 and ChAT-IR motoneuron cell body positions in x,y coordinates with 0,0 at the top of the central canal (n=4 ventral horns, 1 representative animal). C, Contour plots of kernel Foxp2-V1 cell density estimations. The highest density of Foxp2-V1 neurons cluster close to LMC motoneurons from L2 to L5 (contours enclose 10% increments, closer lines indicate higher density). Motoneuron numbers progressively increase from Th13 to L5 before dropping in number. D, The number of Foxp2-V1s per 50 μm thick section (ventral horn) significantly increases in lower lumbar segments from L3 to L6 compared to S1 (dots represent individual mice; n = 3-5 mice in different segments, each mouse estimate is from 6 ventral horns; bars show SEM; one-way ANOVA, post-hoc Bonferroni corrected t-tests: summary statistical results are in the right-hand table. See Table S3 for more details). E, The ratios of Foxp2-V1 neurons to MNs remain relatively constant at roughly 2.5:1 with no significant changes throughout the lumbar cord. Significance was only found for L3-L5 compared to S1 (*p<0.05; **p<0.01; post-hoc Bonferroni tests). High variability in S1 is likely due to the sharp rostro-caudal decrease in motoneuron numbers in S1 sections and possible sampling differences among animals.
Limb and axial motoneurons are densely innervated by Foxp2-V1s and Renshaw cells.
A, Motor column identifications in P20 mice following labeling with ChAT antibodies from lower thoracic to upper sacral spinal cord: PGC = preganglionic cell column; MMC = medial motor column; HMC = hypaxial motor column; LMCd/v = lateral motor column (dorsal/ventral). B, Schematic depiction of the rostro-caudal span of each motor column in the spinal cord segments studied. C, Synapse quantification. Axons of Foxp2 and non-Foxp2 V1 interneurons were respectively labeled with eGFP and tdT in en1Cre::foxp2flpo::R26 RCE:dualGFP/Ai9tdT mice. In en1Cre::R26 Ai9tdT mice we identified V1-Renshaw cell axons using calbindin antibodies. Synaptic locations were labeled with VGAT antibodies and the postsynaptic motoneurons with ChAT antibodies. Synapse densities were analyzed in a ribbon of membrane at mid-cell body level (7 optical planes, 1 µm z-step). C1, Single optical plane of a L4/5 LMCv motoneuron surrounded by genetically labeled Foxp2-V1 and non-Foxp2-V1 axons. Inhibitory synapses on ChAT-IR motoneurons are VGAT+. C2, Single optical image of a L4/5 LMCv ChAT-IR motoneuron receiving synapses from putative V1 Renshaw cells (genetically labeled V1 axons with calbindin-IR and VGAT). C3-4, Method for estimating synapse densities on motoneuron cell bodies using C2 as example. C3, V1-VGAT (red arrowheads) and V1-CB-VGAT synapses (yellow arrowhead) are marked (VGAT-IR is not shown for clarity), and the cell body contour annotated with regions corresponding to dendrite exits. This process was repeated in 7 consecutive mid-cell body optical planes (cross-sections with well-defined nucleus and nucleolus). C4, A membrane surface slab is reconstructed in 3D (two different rotations shown). The surface area corresponding to dendrite exits is subtracted from the total surface area of the slab to calculate the available surface area on the motoneuron cell body. V1-VGAT synapses (red), V1-CB-VGAT synapses (yellow), and CB-VGAT synapses (green) are marked. A similar process was followed for calculating Foxp2-V1 synapse density in dual labeled animals. D, Quantification of total V1-VGAT synapse densities on motoneuron cell bodies in different motor columns (n = 21-30 motoneurons per motor column, taken from 5 animals with 4-9 motoneurons per animal per motor column). Each data point is one motoneuron color coded by mouse origin. Average synaptic densities ±SD indicated to the right of scatter plots. A nested ANOVA indicated significant differences among motor column/segments (p < 0.0001) with no inter-animal variability (p = 0.4768). The table summarizes all post-hoc pairwise comparisons for average V1 synaptic densities of each motor column and segment (Bonferroni corrected t-tests) (Statistical details are in Supplementary Table S4). Colors indicate increased (>1, red) or decreased ratios (<1, blue) of column motoneurons vs row motoneurons. PGC neurons receive significantly fewer V1 synapses than MMC or LMC motoneurons. The LMC (ventral and dorsal) in lower lumbar (L4/L5) had significantly more V1 contacts than MMC motoneurons or L6 dorsal LMC. E, Comparison of synaptic densities from Foxp2-V1 and non-Foxp2-V1 neurons (top) or Renshaw cells (bottom). All motoneurons sampled in 2 to 3 animals for each comparison were pooled together. Densities of V1-VGAT synapses from Foxp2-V1s, non-Foxp2 V1s, or calbindin (CB)+ V1s (Renshaw cells) (n = 6-17 motoneurons sampled per motor column/segments, average = 12.1 ±2.9 SD) were compared using a two-way ANOVA for axon type vs motor column and segment. Foxp2-V1 vs non-Foxp2-V1 synapses: significant differences in density were found for type of synapse (p=0.001), motor column location (p<0.0001), and their interaction (p<0.0001). Significant differences after post-hoc Bonferroni tests are indicated (*p<0.05; ****p<0.0001). In general, synapses from Foxp2-V1 axons have higher density than non-Foxp2-V1 axons on HMC and LMC columns at all spinal segments except for L1/L2 LMC. MMC motoneurons receive similar synaptic densities from both types of V1 axons, except at the sacral level in which non-Foxp2 V1 synapses predominate. PGC neurons receive very low densities of V1 axons and there are no significant differences between either type in any region. Foxp2-V1 vs CB+V1 synapses: significant density differences were found for type of synapse (p<0.0001), motor column location (p<0.0001), and their interaction (p<0.0001). Significant differences between Foxp2-V1 and CB+V1 synapses after post-hoc Bonferroni tests are indicated (*p<0.05; ****p<0.0001). Synapses from Foxp2-V1 axons have higher density than CB+V1 axons in HMC and LMC columns at all spinal segments except for L1/L2 LMC. MMC motoneurons receive similar synaptic densities from both types of V1 axons in upper lumbar regions, but Foxp2-V1 synapse predominate in lower lumbar. In S1 the density of CB+/V1 synapses is significantly higher. The low synaptic densities estimated in PGC neurons for Foxp2-V1s and CB+ V1s are not significantly different. Details of all statistical comparisons are Supplementary tables S5 and S6. F, Comparing the numbers of Foxp2 and CB+ (Renshaw) V1 synapses to the total number of V1 synapses, we estimated their respective percentages. From these estimates we calculated that the remainder belongs to non-Foxp2 and non-CB+ Renshaw cells. The large majority of V1 synapses on the cell bodies of LMC, HMC and MMC motoneurons are either from Renshaw cells or Foxp2-V1s. Asterisks denote significant differences as found in E.
Subgroups of Foxp2-V1 interneurons defined by transcription factor expression at P5 and birthdate.
A, Otp (blue) and Foxp4 (white) expression in lineage-labeled Foxp2-V1s (eGFP, green) and non-Foxp2-V1s (tdT, red). The boxed area is shown at higher magnification with different color combinations for clarity. It shows that Foxp4-IR cells in the Foxp2-V1 population always also expressed Otp (arrows). Quantification: 49.8-51.2% of Foxp2-V1s express Otp and 20.7-21.7% Foxp4 (n=2 mice each examined in 3 ventral horns in L4/5). Very few non-Foxp2-V1 cells express either TF (Otp: 1.3-4.2% and Foxp2: 0.5-2.9%). B, Images of P5 spinal cords containing Foxp2-V1 lineage labeling (eGFP, omitted for clarity) and double immunolabeled for Otp/Foxp2, Foxp4/Foxp2 and Otp/Foxp4. C, Quantification of Foxp2-V1 interneurons with different combinations of TF expression at P5. For each combination, the left panels show cell distributions and the right graphs show the percentage of Foxp2-V1s with each combination (n = 3 mice each analyzed in 6 ventral horns). To discern groups with significantly different numbers of cells, the data was analyzed with one-way ANOVAS followed by Bonferroni corrected pair-wise comparisons (Statistical details in Supplemental Tables S7, S8 and S9. ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05). There are five main groups defined by TF expression patterns: Otp-Foxp2 (44%), Otp-Foxp2-Foxp4 (23%), Otp only (15%), Foxp2 only (9%) and no TF labeling (32% by subtracting all other groups). Some groups associate with specific locations: Otp-Foxp2 cells and the smaller proportion of Otp only cells are located laterally; Otp-Foxp4-Foxp2 cells are located latero-ventrally; medial cells either contain only Foxp2 or nothing; some dorsal cells are either Foxp2 only or do not express any of these TFs. D, Example image of EdU birthdating in en1Cre::foxp2flpo::R26 RCE:dualGFP/Ai9tdT dual-color mice pulse labeled at E11. Foxp2-V1s (green and yellow cells) are born between E10 and E12, with peak birthdate around E11 and after non-Foxp2 V1s (tdTomato only). The lower graph’s data is normalized to highlight the time of peak neurogenesis for each population (n=15.0 ± 4.1 ventral horns from one mouse per time point from E10 to E11 and 2 mice per time from E11.5 to E12.5; total 9 mice, bars show SEM). There is no difference between green (eGFP only) and the smaller population of yellow cells (eGFP and tdTomato). E, EdU labeling in the Foxp2-V1 dual-color genetic model combined with Otp and Foxp4 antibody staining. F, V1s expressing Foxp2, Otp, and Foxp4 at P5 are mostly born between E10.5 and E11.5 with neurogenesis time courses largely overlapping. The normalized plot indicates that peak neurogenesis for all three populations occurs at E11.5, although a marginally higher number of Otp V1 cells are born earlier (n=4 ventral horns from 3.27 ±1.34 mice per timepoint; error bars show SEM). Contour plots to the right show settling locations of Otp and Foxp4-IR populations born at each time point. G, Schematic of the L4-5 ventral horn summarizing Foxp2-V1 subgroups according to location and combinatorial expression of Otp, Foxp4, and Foxp2 at P5. LMCD: dorsal lateral motor column; LMCV: ventral lateral motor column; MMC: medial motor column.
Many Foxp2-V1 interneurons receive proprioceptive inputs, and some are reciprocal Ia inhibitory interneurons (IaINs).
A, Left, low magnification confocal image of Foxp2-V1 lineage-labeling (eGFP) combined with VGLUT1 antibody staining to identify primary proprioceptive afferent synapses. The boxed area is shown at high magnification (right) to demonstrate variability of VGLUT1 synapse density on Foxp2-V1 interneurons (I = absent, II = medium or low, III = high). For simplicity and rigor, we classified Foxp2-V1 interneurons as receiving or not receiving VGLUT1 synapses. B, Percentage of Foxp2-V1 interneurons with VGLUT1 synapses (both proximal and distal). Each dot is one animal estimate from 6 or 7 ventral horns with respectively 591 and 525 Foxp2-V1 interneurons sampled. Line represents the average of both animals, 69.6%. C, Distribution of Foxp2-V1 interneurons with and without VGLUT1 synapses (green and blue, respectively). The positioning of Foxp2-V1 interneurons receiving VGLUT1 synapses is biased laterally. D, Foxp2-V1 interneurons with (green) and without (blue) VGLUT1 synapses according to expression of Otp (left graph) or Foxp2 (right graph). Each dot represents one mouse, and the lines represent averages. The numbers of sections and genetically labeled Foxp2-V1 interneurons sampled in each mouse are as in B. In each mouse, this includes 237 and 236 Otp-IR cells and 256 and 225 Foxp2-IR cells. E, Experimental design to label spinal neurons that receive inputs from tibialis anterior (TA) muscle primary afferents and also connect to lateral gastrocnemius (LG) motoneurons, forming Ia reciprocal inhibitory connections from TA to LG. TA sensory afferents are labeled anterogradely with CTB followed by antibody detection of CTB and the presynaptic marker VGLUT1. Interneurons premotor to LG motoneurons are labeled by monosynaptic retrograde labeling with RVΔG-mCherry. F, Foxp2-V1 IaIN with the most TA/VGLUT1 contacts (31) in our sample (n = 5). Left, low magnification image of Foxp2-V1 interneurons (eGFP, green), RV-mCherry labeling (red) of LG muscle afferents in the dorsal horn and interneurons presynaptic to the LG and of TA afferents anterogradely labeled with CTB (white). The Foxp2-V1 interneuron contains mCherry (yellow cell, inside box). This cell is magnified in two panels to the right, one showing Foxp2-V1 and RV-mCherry and the other RV-mCherry and CTB labeling. Arrows in the zoomed image show examples of CTB synapses (confirmed with VGLUT1) on the dendrites of this neuron. Far right image is the 3D reconstructed cell (Neurolucida) with CTB/VGLUT1 synapses indicated on its dendrites by yellow stars. The axon initial trajectory is indicated (the axon is lost at the section cut surface). The blue area highlights lamina IX. G, Low magnification showing TA-CTB afferents (white) and LG-RV-mCherry labeled interneurons (red). Transsynaptically labeled interneurons are categorized according to position and Foxp2-V1 lineage labeling: images below show superimposed Foxp2-V1 eGFP (green) and additional VGLUT1 immunolabeling (blue). The location of the LMC is indicated. Contralateral interneurons were found in LX (as the one in this section) and in LVIII (the other two in this animal, not shown). H, High magnification images of two LVII LG-coupled interneurons (RV-mCherry, red) receiving synapses from TA afferents (dendrites in boxed regions are shown at high magnification demonstrating CTB-TA labeling and VGLUT1 content). The most medial interneuron belongs to the Foxp2-V1 lineage (see inset with +eGFP). I, Neurolucida neuronal reconstructions showed that the Foxp2-V1 interneuron contained a medium number of TA/VGLUT1 synapses (13 contacts) in our sample of putative IaINs derived from the Foxp2-V1 lineage (n=5), while the non-Foxp2-V1 interneuron contained the largest (115) of any LVII reconstructed interneuron with mCherry, including many proximal synapses. However, one reason for this difference is that the dendrites projecting toward regions with high densities of TA CTB/VGLUT1 synapses were out of this tissue section. J, Intersection of otp and en1 using the dual color strategy with simultaneous expression of the Ai9 tdT and RCE-DC eGFP reporters. V1 cells that expressed otp are labeled with eGFP (green). In these cells the Ai9-tdT reporter is effectively deleted by flpo recombination dependent on otp expression level: strong (eGFP only) and weak (eGFP and tdT). V1 cells that express tdT only (red) never express otp. In addition, the sections were labeled with antibodies for Otp (light blue) to reveal cells that retained expression of Otp at P5 and ChAT (deep blue) to localize the motor pools. Most V1s express eGFP, with tdT-only cells being a minority. Right, cell plot positions of some of the cell types identified in these sections (one mouse 6 ventral horns in L4/L5). Most cells are Otp-V1 and include green (eGFP only) and pink cells (eGFP and tdT). K, Quantification of cells with eGFP only (green), tdT only (red), or both (pink) (n=12 ventral horns from 2 mice, error bars show SD). 88.6% of V1s expressed eGFP, most without tdT (61.1% of V1s) and of these around half (53.3%) retained Otp expression by P5. No Otp expression (antibody staining) was detected in tdT cells, and very few cells co-expressing tdT and eGFP (“weak” Otp expression) were Otp-IR. L, Detection of Pou6f2, calbindin and Otp in genetically labeled Otp-V1 cells. Left confocal image and right cell plot (n=6 ventral horns from 1 mouse). Pou6f2-Otp-V1 cells concentrate in a dorsal band within the ventral horn. Calbindin-IR Otp1-V1 cells concentrate in the Renshaw region (ventral most region) and others are in the dorsal region of the ventral horn. Otp-V1 cells retaining Otp expression at P5 occupy all dorsoventral positions in the lateral spinal cord. M, Quantification of 6 ventral horns at L4/L5 from one animal; 397 Otp-V1 cells sampled. Most (72.5%) did not express Pou6f2 or calbindin, 12.8% expressed Pou6f2 and 14.4% calbindin. Only one Otp-V1 neuron in the whole sample co-expressed Pou6f2 and calbindin (0.25%).
Mouse models
