Mice
All experiments with animals were performed according to the National Institutes of Health Guide for Care and Use of Laboratory Animals and were approved by the Montana State University Institutional Animal Care and Use Committee under Protocol number 2023-65-1A. As such, we have complied with all relevant ethical regulations for animal use. All mice were maintained in individually ventilated caging systems. Non-recirculated, HEPA-filtered air was provided to the units in a positive mode. Autoclaved pine or sani-chip was used as direct bedding. A commercial diet was fed ad lib and mice were provided with chlorinated, reverse osmosis water. At four weeks of age, gel packs were placed on the floor of all cages containing CKOs to ensure hydration without needing to climb. For enrichment, mice were provided with an autoclaved cotton nestlet, autoclaved Enviro-DriTM, plastic shelters, and mice were group housed whenever possible. Cages were opened in a biological safety cabinet. All mice were euthanized via CO2 inhalation followed by cervical dislocation as a secondary measure. Elp1tm1a(KOMP)Wtsi “knockout first” mice containing a frt-flanked LacZ Elp1 (previously known as Ikbkap) reporter that disrupts Elp1 expression before the LoxP flanked 4th exon were obtained from the International Knockout Mouse Consortium (MGI ID: 1914544). This strain was generated on a C57Bl/6N background and has been previously described27. Following Flippase-mediated removal of the LacZ cassette, the resulting Elp1LoxP strain (Fig. S1a) was backcrossed onto a C57Bl/6J background more than 10 times. Elp1 CKO mice (Chat-Cre; Elp1LoxP/LoxP) were generated by crosses between Chat-Cre; Elp1+/LoxP × Elp1LoxP/LoxP; Chat-GFP/GFP mice. A humane endpoint for Elp1 CKO males was defined by a mass ≤ 55% of their control counterparts (Chat-Cre; Elp1+/LoxP; Chat-GFP) combined with a hindlimb clasping score of three and constant fasciculations when moving about the home cage. A humane endpoint for Elp1 CKO females was defined by a weight ≤ 60% of their control counterparts (Chat-Cre; Elp1+/LoxP; Chat-GFP) combined with a hindlimb clasping score of three and constant fasciculations when moving about the home cage. Gel packs were placed on the floor of all cages containing CKOs at four weeks of age to ensure hydration without needing to climb. The following strains were purchased from the Jackson Laboratory: ROSAmT-mG, stock no. 00757629; Chat-Cre, stock no. 00641026, Chat-GFP, stock no. 00790230. Elp3LoxP mice were derived from Elp3tm1.1Tac mice (MGI ID: 5704311) on a C57Bl/6 background37. Elp3 CKO mice were generated through the same genetic strategy as Elp1 CKO mice (Chat-Cre; Elp3+/LoxP × Elp3LoxP/LoxP) except that the Chat-GFP allele was not incorporated into the Elp3LoxP/LoxP strain. To visualize cholinergic neurons in Elp3 CKO mice and controls, an anti-ChAT antibody was used (see below). In all experiments utilizing Chat-Cre-positive mice, the Chat-Cre allele was hemizygous.
Randomization
Environmental factors, including lighting, temperature, and humidity, were controlled and consistent for all animals, and animal cages were placed randomly within the rack. For weight, PaGE, and hindlimb clasping studies, the order in which the animals were selected for measurement was randomized using an open-access randomization tool. For all IF studies, a random number generator was used to select control sections and cells. All sections and cells from CKOs were used, negating the need for randomization. For the NMJ analysis, muscle sections and individual NMJs were selected randomly using a random number generator (see below).
Blinding
All weight, PaGE, and hindlimb clasping analyses were performed blind. L.G. allocated the mice into control and CKO groups for data analysis after the data had been collected. All images of motor neurons were captured blind and measurements were allocated into control and CKO groups by L.G. after all measurements and quantification had been completed.
Inclusion and exclusion criteria
Genotype and sex were the only selection criteria used and were established a priori. For IF studies, tissue sections that were torn, stretched, or otherwise damaged were excluded.
Hindlimb clasping scoring
Hindlimb clasping was scored using a previously published scoring system31. Briefly, the mouse is suspended by the tail and hindlimb position is observed for 10 s. If hindlimbs are consistently splayed outward, away from the abdomen, the mouse is assigned a score of 0. If one hindlimb is retracted toward the abdomen for more than 50% of the time suspended, it receives a score of 1. If both hindlimbs are partially retracted toward the abdomen for more than 50% of the time suspended, it receives a score of 2. If both hindlimbs are entirely retracted and touching the abdomen for more than 50% of the time suspended, it receives a score of 3.
PaGE testing
Paw grip endurance (PaGE) testing as a measure of motor function was performed as described in Weydt et al. (2003)50, and was modified slightly. Briefly, each mouse was placed on a wire lid from a conventional rodent housing cage; the lid was gently shaken to induce gripping and turned upside down (180°). The latency until the mouse released both hind limbs was measured in seconds. Each mouse was tested five times with an arbitrary maximum of 60 s, and the longest latency to fall or release both hind limbs was recorded.
Immunofluorescence (IF)
All washing, blocking, secondary antibody, and post-fixation steps were performed at room temperature. All other steps were performed at 4 °C unless stated otherwise. Following fixation, tissues were rinsed in PBS, cryoprotected through a series of sucrose solutions in PBS (15%, 30%), incubated for 2 h in a 1:1 mixture of 30% sucrose and optimal cutting temperature (OCT) compound (Tissue-Tek, Torrance, CA), followed by 2 h in OCT. Tissues were then embedded in OCT and frozen in a dry ice ethanol bath. For immunostaining, slides were bathed in tris-buffered saline (TBS) for 10 min, followed by NGS block (10% normal goat serum, 1% glycine, 0.4% Triton ×-100 in 30 mM Tris, 150 mM NaCl) for 1 h, and overnight incubation in primary antibody (in NGS block). Slides were then rinsed in NGS block, incubated in Alexa Fluor secondary antibody (1:2000 in NGS block) for 1 h, rinsed in 3:1 TBS:NGS block, and mounted in Prolong Antifade Diamond (Invitrogen, La Jolla, CA). Control and experimental embryos were cryosectioned on the same day and sections were incubated in primary antibody on the same day that they were sectioned. For antigen retrieval, slides were incubated in citrate buffer at 95 °C for 10 min following sectioning.
Antibodies
Primary antibodies included the following: GFP (Abcam, ab13970, 1:1000), ELP1 (Abnova PAB12857, 1:800, knockout validated27,51. ELP3 (Proteintech, 17016-1-AP, 1:200, knockout validated here using Chat-Cre; Elp3LoxP/LoxP mice), NeuN (Proteintech, 26975-1-AP, 1:4000, see the manufacturer’s website for validation details), TDP-43 RRM2 (Proteintech, 10782-2-AP, 2 μg/ml, knockout validated). *Note: although the manufacturer’s website describes this antibody as recognizing the TDP-43 N-terminus, Tsuji et al. (2012)39, showed the antibody recognizes amino acids 203–209 of human TDP-43, which corresponds to the second RNA recognition motif. TDP-43 C-terminus (Proteintech, 12892-1-AP, 2 μg/ml, knockout validated), Fibrillarin (Novus Biologicals, NB300-269, 1:500, knockdown validated), ChAT (Millipore Sigma AB144P, 1:100, see the manufacturer’s website for validation details). Secondary antibodies used were Alexa Fluor goat anti-rabbit 488, goat anti-mouse 568, donkey anti-chick 488, donkey anti-goat 568 (Invitrogen, 1:2000).
Immunohistochemistry
LacZ staining was performed as previously described52. Briefly, slides with tissue sections (20 μm) were incubated for three hours in 1 ml of ×-gal solution (1 mg/ml ×-gal, 5 mM potassiμmferricyanide, 5 mM potassiμmforrocyanide, 2 mM MgCl2, 0.25% Triton ×-100 in PBS) at 30 °C. Tissue sections were then fixed in 1 ml of 4% paraformaldehyde for 10 min, followed by three washes in TBS. To combine with IF, tissue sections were then incubated in NGS block followed by the application of primary antibody as above.
Alpha motor neuron quantification
The Chat-GFP allele was included in all Elp1 CKO experiments where alpha motor neurons were quantified or measured. Only cells in the ventral horn (Fig. S6) that were GFP-positive and NeuN-positive were selected for further analysis35. Spinal cords were removed from both control (Chat-Cre; Elp1+/LoxP) and CKO (Chat-Cre; Elp1LoxP/LoxP) animals via hydraulic extrusion53. The lumbar enlargement was isolated and fixed for 2.5 h at 4 °C in 4% paraformaldehyde in PBS. Sixty 16 μm sections were trimmed to reach the L5/L4 level. Every other 16 μm section was then collected, filling one slide (approximately 48 sections). IF was performed as described above using anti-GFP and anti-NeuN antibodies, and the field of view (40×) containing the highest number of GFP-positive neurons in the ventral horn (lamina IX, Fig. S6) was photographed. For control mice, sections to be photographed were selected randomly using a random number generator. For the CKO, given the reduced number of large motor neurons, all sections were photographed, and the field of view (40×) was positioned to include the largest possible number of large motor neurons. The number of alpha motor neurons (GFP-positive and NeuN-positive) per ventral horn were counted and their areas measured using ImageJ. Averages were then calculated for the 10 sections with the largest number of alpha motor neurons (GFP-positive, NeuN-positive, and an area ≥ 440 μm2) present per section. The same approach was used to quantify the number of GFP-positive, NeuN-positive neurons with an area less than 440 μm2. For E18.5, embryos were decapitated and fixed in 4% paraformaldehyde in PBS for 2.25 h. Sections were cut at 16 μm and every other section was collected from the mid to upper lumbar axial level, filling two slides. Every other section was photographed, and the number of alpha motor neurons in a single hemisphere (the hemisphere with the highest number of alpha motor neurons) counted per section and an average calculated for the 12 sections with the largest number of alpha motor neurons present per section.
Neuromuscular junction analysis
The Chat-GFP allele was included in both control (Chat-Cre; Elp1+/LoxP) and CKO (Chat-Cre; Elp1LoxP/LoxP) animals to visualize innervation of the NMJ. Anterior tibialis muscles were dissected from female controls and CKOs and fixed for 20 min in 4% paraformaldehyde in PBS at 4 °C. The muscles were then worked up for cryosectioning as described above for spinal cords. Sections were cut at a thickness of 25 μm and every other section collected, filling two to three slides for a total of 60 sections. IF was performed as described above using an anti-GFP antibody to visualize innervation of the NMJ. Alpha bungarotoxin 555 (Molecular Probes, #B160; 1 μg/ml) was included at the secondary antibody step. Sections to be analyzed were selected randomly using a random number generator. NMJs were also randomly selected by generating a number between 1 and 10 and counting to that NMJ starting from the top right corner of the section. 50 NMJs were analyzed per animal and tallied as being either fully innervated (GFP pattern matches the pattern of alpha bungarotoxin), or not fully innervated (GFP either partially or totally missing). Anterior tibialis muscles from three control and three experimental animals were analyzed per time point.
TDP-43 and Fibrillarin corrected fluorescence and area analysis
Nu area in Fig. 4j corresponds to the entire nucleus (including the nucleolus). Fluorescence levels per unit area were calculated as follows: No = (No corrected total fluorescence (CTF)/No area); Nu = (Nu CTF–No CTF)/(Nu area–No area); Cytoplasm = (total cell CTF–Nu CTF)/(total cell area–Nu area). For TDP-43 and fibrillarin fluorescence measurements, spinal cords from control (Chat-Cre; Elp1+/LoxP) and CKO (Chat-Cre; Elp1LoxP/LoxP) were collected, processed for cryosectioning, and sectioned as described above (alpha motor neuron quantification). IF was performed as described above using either anti-TDP-43 and anti-GFP primary antibodies, or anti-fibrillarin and anti-GFP antibodies. Antigen retrieval was included for fibrillarin. For controls, a random number generator was used to randomly select sections. The field of view in the spinal cord ventral horn (lamina IX, Fig. S6) (40×) with the highest density of GFP-positive neurons was photographed. For CKOs, given the reduced number of surviving motor neurons, all GFP+ cells in the ventral horn were photographed. Identical exposure, gain, and offset settings were used for both control and experimental images. Next, for both controls and CKOs, ImageJ was used to measure the area of photographed, GFP+ cells. In cells with a soma area ≥ 440 μm2 and that contained a whole intact nucleus via DAPI staining, TDP-43 or fibrillarin areas and fluorescence levels were measured using Image J processing and analysis software (https://imagej.nih.gov/ij/). A minimum of 20 cells were analyzed per spinal cord for controls and between 8 and 20 for the CKOs given the reduced number of alpha motor neurons with soma areas ≥ 440 μm2 in the CKO spinal cords. A minimum of three spinal cords were analyzed per genotype and the exact number of spinal cords for each analysis is shown on the individual bar graphs in Fig. 4.
Statistics and reproducibility
Statistical analyses were performed using Excel, Prism, example and open-access one-way ANOVA and Tukey HSD software. Data are presented as mean ± standard deviation (SD), unless stated otherwise. For comparisons between two groups, a two-tailed unpaired Student’s t-test was used. For multiple group comparisons, one-way ANOVA was applied, followed by Tukey’s post hoc test.
Sample sizes were based on pilot studies and were calculated using a statistical power of 80% and a significance level of 0.05. Randomization was employed in all studies except in the selection of microscopic fields of view and large diameter (≥ 440 μm2) alpha motor neurons for CKOs. Since the number of large diameter alpha motor neurons is drastically reduced in Elp CKO mice, all lumbar enlargement ventral horn fields of view were captured and analyzed and all large diameter alpha motor neurons were included in our analyses. All experiments were independently replicated at least three times to ensure reproducibility and the number of biological replicates (n) are indicated in the figure legends and/or methods. All raw source data are available in the Supplementary Data files, or at Dryad (https://doi.org/10.5061/dryad.x0k6djhvb).
Microscopy
Images were captured using a Nikon TE200 inverted microscope equipped with a QImaging QICAM 12-bit Mono Fast 1394 Cooled camera and SPOT software. Identical exposure times, gain, and offset settings were used to capture control and experimental images.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
