Basal Xenobot transcriptomics reveals changes and novel control modality in cells freed from organismal influence

For the network analysis and comparisons with the thanatotranscritome, we mapped Xenopus genes to their human orthologs using HCOP189.

For controlling and providing acoustic vibration stimulus, we used a setup (Fig. 4A) containing a speaker (RECOIL RW8D2 Echo Series 2-ohms, 400 Watts) connected to a digital audio amplifier (Kinter K3118 Texas Instruments, 20 Watts output) operated using a laptop computer. An online tone generator (https://www.szynalski.com/tone-generator/) was used to set a sine-type wave with frequency of 300 Hz. The computer volume and online tone generator volumes were set to 100%. A P60 petridish coated at the bottom with 2–3 mm layer of 1% agarose made in 0.75X MMR was placed on top of the speaker. Fourteen milliliters of 0.75X MMR was added into the petridish. The volume of amplifier was set to 3.5 at which point no physical movement of media in the dish was observed in response to acoustic vibration stimulus. These settings were kept unchanged throughout all acoustic vibration stimulus experiments. An iPod camera mounted on a Stemi SV6 dissection microscope eyepiece was used to capture time-lapse imaging. Subjects (day 1 Xenobots, day 7 Xenobots, or age-matched stage 35 embryos) were placed in the center of petridish. Time-lapse recording was conducted for 10 min before initiation of vibration stimulus, followed by 10 min of vibration stimulus, and lastly 10 min after vibration stimulus was tuned off.

Movement behavior was recorded using an iPod camera mounted on a Stemi SV6 dissection microscope eyepiece. ProShot app was used to capture time-lapse imaging of one frame every 2 s. Time-lapse recordings were compressed to 30 frames per second movie which results in 30 s movies of 30 min of motion tracking (10 min before stimulation, 10 min during stimulation, and 10 min after stimulation). Thus 1 s of movie = 1 min of actual time. Motion tracking of behavior captured in time-lapse videos was done using Ethovision XT v.15 (Noldus Information Technology). A scaled background image was used to set scale for distance. Motion tracking was completed and recorded. Any tracking errors were manually corrected in the track editor. All X–Y coordinates of each subject for every frame across the time-lapse video were exported for data analysis.

Xenobots were manufactured from embryos obtained from multiple female frogs. On maturity (with autonomous movement) to day 7, Xenobots were fixed in MEMFA fixative (100 mM Mops pH 7.4, 2 mM EGTA, 1 mM MgSO4, and 3.7% (v/v) formaldehyde) for 4 hours at room temperature on a nutator, followed by 3 washes for 15 min each with PBST (phosphate-buffered saline + 0.1% Tween 20) and stored at 4 °C until ready for staining. Fixed Xenobots were first imaged using Nikon SMZ-1500 microscope at 6X with a Teledyne Infinity camera and Infinity Analyze software, and two axial radius’ a and b were measured. Xenobots were then cut through the middle into two halves and stained with nuclear stain NucBlue (Thermofisher R37605—2 drops/ml) for 30 min followed by 3 washes for 5 min with PBST. The interior cut surface of the Xenobot was imaged for detecting nuclei using Leica Stellaris Sp8 confocal microscope with 16X water objective and DAPI excitation and emission settings. A 7 µM depth was imaged. Using the image the third axial radius c of Xenobot was measured. Using Fiji/imageJ total number of nuclei in the Xenobot section was counted, followed by calculating the area of that Xenobot section, volume of the imaged Xenobot section (area X depth—7 µM), nuclei per unit volume (total number of nuclei/calculated section volume), total volume of that Xenobot (Total volume = (4/3) * (22/7) * radius-a * radius-b * radius-c), and finally total nuclei in that Xenobot (Nuclei per unit volume * Total volume).

Cilia were visualized by immunostaining with monoclonal anti-acetylated α-tubulin antibody (Sigma-Aldrich, T7451) using previously described protocol190. Xenopus embryos were obtained from multiple female frogs. A subset of the embryos was used to manufacture Xenobots, and rest were used as age-matched control embryos. On maturity (autonomous movement in Xenobots) at day 7, Xenobots and age-matched embryos were fixed in MEMFA fixative (100 mM Mops pH 7.4, 2 mM EGTA, 1 mM MgSO4, and 3.7% (v/v) formaldehyde) for 4 h at room temperature on a nutator, followed by 3 washes for 15 min each with PBST (phosphate-buffered saline + 0.1% Tween 20) and stored at 4 °C until ready for processing. Samples were first blocked with 10% goat serum in PBST at room temperature for 1 h. Samples were then incubated in monoclonal anti-acetylated α-tubulin antibody diluted at 1:1000 in the block (10% goat serum in PBST) at 4 °C overnight on a rocker. Samples were then washed 3 times for 15 min each in PBST followed by incubation in Alexa Fluor 555-conjugated goat anti-mouse secondary antibody at 1:500 dilution at 4 °C overnight. Finally, samples were washed 3 times for 15 min each in PBST. Cilia were imaged using Leica Stellaris Sp8 confocal microscope, 40X water objective with 4X zoom and Alexa-555 excitation and emission settings. Cilia length numbers were counted using Fiji/ImageJ software. Number of cilia per multiciliated cell and cilia length were counted from multiple multiciliated cells from each sample and more than 4 different samples in each treatment group.

Statistical analyses and visualizations were conducted using software appropriate for that experimental setup. The statistical methods, tests, and software employed for each experiment are varied and explained in detail in the methods, text, and figure legends, wherever applicable. Reproducibility was determined by conducting all experiments with a minimum of three independent biological replicates representing independent samples derived from independent batches. Details for each experiment are outlined in respective methods, text, and figure legends.

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

We thank Douglas Blackiston, Will Ratcliff, and all members of the Levin lab for helpful discussions, Erin Switzer for Xenopus husbandry and assistance, Rakela Colon for general laboratory supply and equipment management, Albert Tai from Tufts Genomic core for RNAseq setup consultation, Jonathan Dreyfuss and Hui Pan from Joslin Diabetes Center for RNAseq analysis consultation, Meghan Short from Tufts Clinical and Translational Science Institute for statistical analysis consultation, and Julia Poirier for manuscript preparation. This publication was made possible through the support of Grant 62212 from the John Templeton Foundation (awarded to M.L.). Research reported in this publication was supported by the National Institutes of Health S10 shared instrumentation grant (S10OD032203) awarded to Tufts University Core Facility Genomics Core for a NovaSeq facility. The opinions expressed in this publication are those of the authors and do not necessarily reflect the views of the funding agencies.

V.P.P., and M.L. conceived the ideas and specific experimental approaches. V.P.P. and P.T. carried out experiments and data collection. L.P-L., P.E., and M.S. performed the data analysis. V.P.P. and M.L. wrote the manuscript, with text contributions from all co-authors.

Communications Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editors: Aylin Bircan, Christina Karlsson Rosenthal.

RNA-sequencing data generated during the study are available in the NCBI GEO public repository with accession numbers (GSE275807 and GSE277182). Curated data are available in the Supplementary Data. Any other data are available from the corresponding author upon request.

The authors declare the following competing interests: M.L. is scientific co-founder of a company called Fauna Systems, which is involved in the space of AI-designed biorobotics.

The online version contains supplementary material available at 10.1038/s42003-025-08086-9.

RNA-sequencing data generated during the study are available in the NCBI GEO public repository with accession numbers (GSE275807 and GSE277182). Curated data are available in the Supplementary Data. Any other data are available from the corresponding author upon request.