Biology subjects

Christiansen, L.

Publications and source records attributed to Christiansen, L..

15 recordsLinked to original sources

bioRxiv2026

Immediate cortical responses to transcranial magnetic stimulation of the human motor leg area

Background: Immediate transcranial evoked potentials (iTEPs) are ultra-early electroencephalographic responses elicited by single-pulse transcranial magnetic stimulation (TMS) of the primary motor cortical hand area (M1-HAND). Their characteristic latency, waveform, and sensitivity to short-interval intracortical inhibition suggest a close relationship to the cortical generators of indirect corticospinal volleys (I-waves). Here, we tested the hypothesis that iTEPs constitute a fundamental physiological response of human motor cortex and therefore generalize across motor representations, including the leg representation (M1-LEG). Methods: Single-pulse TMS was delivered over the motor cortical representation of the right tibialis anterior (TA) in 16 healthy volunteers. In a subset of participants (n=10), stimulus-response curves and paired-pulse short-interval intracortical inhibition (SICI) measures were obtained. Results: TMS of M1-LEG elicited reproducible iTEPs consisting of a slow positive deflection with superimposed high-frequency peaks with topographic maxima in electrodes adjacent to the stimulation site. M1-LEG iTEPs emerged close to active motor threshold and increased in prevalence and amplitude with increasing stimulation intensity. Paired-pulse stimulation produced consistent SICI of the second and third iTEP peaks. M1-LEG stimulation also evoked conventional later TEPs with latencies and polarities closely resembling those recorded after M1-HAND stimulation. Conclusions: TMS of the human M1-LEG elicits robust iTEPs that closely resemble those recorded from M1-HAND in their timing, intensity dependence, and sensitivity to intracortical inhibition. These findings indicate that iTEPs are not specific to M1-HAND but reflect a general physiological response of human motor cortex closely linked to corticospinal I-wave generation. This broadens their utility as a non-invasive probe of excitatory and inhibitory circuit dynamics throughout the human motor system.

neuroscience↗

bioRxiv2026

Opposing modulation of cortical and corticospinal excitability across movement-related beta stages

Discrete voluntary movement depends on rapid changes in neural excitability across cortical and corticospinal circuits, yet how intrinsic movement-related neural states shape multi-level excitability remains unclear. Here, we combined individualized, state-targeted transcranial magnetic stimulation (TMS) with electroencephalography and electromyography recordings during visually cued finger movements to probe excitability across movement-related beta-band dynamics during two complementary experiments. Immediate transsynaptic cortical excitability closely tracked intrinsic beta dynamics, with attenuation of the second immediate TMS-evoked potential during beta desynchronization and recovery during the post-movement beta rebound. In contrast, corticospinal excitability showed the opposite pattern, with larger motor-evoked potentials during beta desynchronization and reduced responses during beta rebound. Together, these findings identify endogenous beta-state dynamics as a key regulator of movement-related cortical excitability and reveal a fundamental dissociation between how intrinsic brain activity tunes local cortical excitability and corticospinal output in humans.

neuroscience↗

bioRxiv2026

Direct Assessment of Short-Latency Intracortical Inhibition via Immediate TMS-Evoked Potentials

Short-interval intracortical inhibition (SICI) is the most widely used neurophysiological index of GABAergic inhibition in the human cortex. However, it is an indirect measure, inferring synaptic inhibition from suppression of peripherally recorded motor-evoked potentials (MEPs) elicited by transcranial magnetic stimulation (TMS). In the standard protocol, a subthreshold conditioning pulse suppresses the MEP evoked by a suprathreshold test pulse delivered 1-5 ms later. Interpretation is further complicated by temporal overlap with short-interval intracortical facilitation (SICF), reflecting excitatory interactions at interstimulus intervals of [~]1.5 and 2.7 ms. To overcome these limitations, we recorded immediate TMS-evoked EEG potentials (iTEPs; 1-10 ms post-stimulus) as a more direct measure of motor cortical activity in 16 healthy volunteers (20-35 years; 7 male). The conventional SICI protocol suppressed only later components of the iTEP, likely corresponding to late corticospinal volleys previously identified in epidural spinal recordings after suprathreshold TMS, while the earliest iTEP component was unaffected. Importantly, later iTEPs were suppressed to a similar extent whether conditioning-test intervals coincided with SICF peaks or troughs, and the magnitude of iTEP suppression correlated with concurrently recorded paired-pulse MEP suppression. SICI also reduced an early TEP component (N15; 10-20 ms), but paired-pulse N15 suppression showed a different dependence on stimulus intensity and did not correlate with MEP suppression. These findings demonstrate that SICI measured via MEPs does not reflect a global index of cortical GABAergic motor cortical inhibition but instead reflects inhibition within specific cortical circuits that can be investigated directly with iTEPs.

neuroscience↗

bioRxiv2026

Transcranial Direct Current Stimulation enhances long-term retention after 5 days of lower-limb motor skill learning

Transcranial direct current stimulation (tDCS) holds the potential to affect behavior by modulating ongoing neural activity, and tDCS paired with hand motor practice can enhance motor learning. While augmenting the behavioral benefits of motor practice is relevant for neurorehabilitation following central and peripheral lesions to the motor system, as well as in sports, the short- and long-term effects of tDCS targeting the mesial motor cortex (M1-Leg) during lower-limb motor skill practice remain unexplored. We tested whether five days of anodal tDCS over M1-Leg during training of a sequential visuomotor tracking task improves within- and between-session learning and one-week retention. Participants were randomized to skill practice with active tDCS, skill practice with sham stimulation, or volume-matched non-skilled ankle movements with sham stimulation. Changes in corticospinal excitability accompanying skill and nonskill motor practice with real and sham tDCS, were assessed as motor evoked potential amplitudes recorded from the tibialis anterior muscle at rest. Compared to non-skill practice, motor skill practice yielded robust sequence-specific performance gains, which were transferred to the untrained leg and persisted for at least one week after practice ended. Concurrent tDCS did not increase learning within or between training sessions, but it did lead to improved one-week retention compared to sham stimulation. Corticospinal excitability did not increase after practice and was unaffected by tDCS. These findings suggest that combining lower-limb motor skill practice with tDCS over M1-Leg can strengthen retention of skill learning without measurable changes in resting corticospinal excitability. This is relevant for motor practice scheduling in neurorehabilitation. Key pointsO_LITranscranial direct current stimulation (tDCS) is a weak electrical brain stimulation that may boost learning when paired with motor practice; however, its effects during lower-limb skill training are not well known. C_LIO_LIWe tested whether stimulation over the leg area of the motor cortex during 5 days of ankle skill training improves learning, consolidation, and delayed retention. C_LIO_LIAnkle skill training produced clear sequence-specific improvements that transferred to the untrained leg and were still present 1 week later. C_LIO_LIStimulation did not increase short-term learning, but it did improve 1-week retention compared to sham stimulation. C_LIO_LICorticospinal excitability assessed based on motor evoked potentials elicited by transcranial magnetic stimulation did not change with motor training or stimulation, suggesting that the observed positive effect of tDCS on delayed retention may arise from other brain network processes relevant to long-term motor learning and memory. C_LI

neuroscience↗

bioRxiv2026

No group-level changes in corticospinal excitability following low-intensity theta-burst ultrasound stimulation of the primary motor hand area.

BackgroundLow-intensity transcranial ultrasound stimulation (TUS) is a novel non-invasive brain-stimulation technique offering high spatial precision and depth penetrance. Theta burst TUS (tbTUS), featuring a temporal theta burst stimulation pattern, has been reported to facilitate corticomotor excitability, when applied to the human primary motor hand area (M1-HAND). However, replication attempts have yielded inconsistent results. MethodsFifteen healthy human participants underwent neuronavigated tbTUS targeting the left M1-HAND in the anterior wall of the precentral sulcus, as well as anterior and posterior active control sites. The three tbTUS conditions were applied in counterbalanced order on separate days. Corticospinal excitability was assessed via motor evoked potentials (MEPs) recorded before and 5, 15, 30, and 60 minutes after tbTUS, and analyzed using an rmANOVA. ResultsNone of the tbTUS conditions produced consistent group-level changes in MEP amplitude at any time point. Both intra- and inter-subject variability were high, and individual MEP changes following precentral tbTUS did not correlate with changes after control-site stimulation. ConclusionsWe did not observe reliable modulatory effects of neuronavigated tbTUS on corticospinal excitability. Methodological and hardware differences may account for discrepancies across studies. Our findings align with recent reports questioning the robustness of tbTUS-induced facilitation.

neuroscience↗

bioRxiv2025

Not two sides of the same coin: Divergent effects of motor state on corticospinal and cortical responses to TMS of motor cortex

BackgroundTranscranial magnetic stimulation (TMS) of the motor cortex can elicit motor evoked potentials (MEPs) in target muscles, reflecting corticospinal excitability. MEP amplitudes increase with TMS intensity and can be facilitated by tonic muscle pre-activation. Since conventional transcranial evoked potentials (TEPs) also grow with increasing TMS intensity, cortical and corticospinal responses are often considered two facets of the same process. If this were true, changes in physiological motor state should modulate TEPs and MEPs in a similar manner. MethodsTo compare the state-dependency of cortical and corticospinal responses to single-pulse TMS, we simultaneously recorded TEPs and MEPs in 16 healthy young adults during relaxation and isometric contraction of the right first dorsal interosseous (FDI) muscle. For each condition, 100 biphasic TMS pulses were delivered to the left primary motor hand area at five different intensities centered around the resting motor threshold. ResultsTEP and MEP amplitudes increased with stimulation intensity. As predicted, tonic muscle contraction consistently facilitated MEP. On the contrary, muscle contraction attenuated two key peaks of the TEP (N15 and N100). The state-dependent effects of corticospinal and cortical responses were not correlated. DiscussionBoth TEPs and MEPs are reliably modulated by motor state, yet they differ in direction and their magnitudes do not scale with each other. These findings challenge the assumption that cortical and corticospinal responses are two aspects of the same process. MEP facilitation during contraction likely reflects increased spinal excitability, whereas TEP attenuation may reflect reduced responsiveness of cortico-cortical or cortico-subcortical networks.

neuroscience↗

bioRxiv2025

Rostro-caudal TMS mapping of immediate transcranial evoked potentials reveals a pericentral crescendo-decrescendo pattern

BackgroundWe recently demonstrated that single-pulse TMS of the primary sensorimotor hand area (SM1HAND) elicits an immediate transcranial evoked potential (iTEP). This iTEP response appears within 2-7 ms post-TMS, featuring high-frequency peaks superimposed on a slow positive wave. Here, we used a linear TMS-EEG mapping approach to characterize the rostro-caudal iTEP expression and compared it to that of motor-evoked potentials (MEPs). MethodsIn 15 healthy young volunteers (9 females), we identified the iTEP hotspot in left SM1HAND. We applied single biphasic TMS pulses at an intensity of 110% of resting motor threshold over six cortical sites along a rostro-caudal axis (2 cm rostral to 3 cm caudal to the SM1HAND hotspot). We analyzed site-specific iTEP and MEP responses. ResultsiTEP magnitude decreased rostrally and caudally from the SM1HAND hotspot. MEPs exhibited a similar rostro-caudal crescendo-decrescendo pattern. While iTEP and MEP response profiles were similar, normalized iTEP amplitudes decayed less rapidly at the first postcentral site. DiscussionThese findings support the idea that pericentral iTEPs reflect a direct response signature of the pericentral cortex, possibly involving a synchronized TMS-induced excitation of cortical pyramidal tract neurons. Similar but non-identical rostro-caudal patterns suggest that iTEPs and MEPs may arise from overlapping but distinct neuronal populations.

neuroscience↗

bioRxiv2024

Single and paired TMS pulses engage spatially distinct corticomotor representations in human pericentral cortex

Single-pulse transcranial magnetic stimulation (TMS) of the primary motor hand area (M1-HAND) can assess corticomotor function in humans by evoking motor evoked potentials (MEP). Paired-pulse TMS at peri-threshold intensity elicits short-latency intracortical facilitation (SICF) with early peaks at inter-pulse intervals of 1.0-1.8ms (SICF1) and 2.4-3ms (SICF2). The similarity between the periodicity of SICF and indirect (I-)waves in the corticospinal volleys evoked by single-pulse TMS suggests that SICF originates from I-wave generating circuits. This study aimed to explore the mechanisms of MEP generation by mapping the corticomotor representations of single-pulse and paired-pulse TMS targeting SICF1 and SICF2 peaks in 14 participants (7 female). MEPs were recorded from two hand muscles and the spatial properties of each corticomotor map were analyzed. For both hand muscles, we found a consistent posterior shift of the center-of-gravity (CoG) for SICF maps compared to single-pulse maps, with a larger shift for SICF1. CoG displacement in the SICF1 map correlated with individual SICF1 latencies. Further, ADM maps consistently peaked more medially than FDI maps and paired-pulse TMS resulted in larger corticomotor maps than single-pulse TMS. This is the first study to show that circuits responsible for SICF have a more posterior representation in the precentral crown than those generating MEPs via single-pulse TMS. These findings indicate that paired-pulse TMS probing SICF1, SICF2, and single-pulse TMS engage overlapping but spatially distinct cortical circuits, adding further insights into the intricate organization of the human motor hand area. New & NoteworthySingle- and paired-pulse transcranial magnetic stimulation (TMS) is widely used to study corticomotor physiology in humans, but do they engage the same intracortical circuits? We compared the spatial properties of corticomotor maps elicited by single-pulse TMS to those elicited by paired-pulse short-latency intracortical facilitation (SICF). SICF maps consistently showed a posterior shift in center of gravity compared to single-pulse maps, suggesting that paired-pulse TMS engages cortical circuits that are spatially distinct from single-pulse TMS.

neuroscience↗

bioRxiv2024

Methodological choices matter: A systematic comparison of TMS-EEG studies targeting the primary motor cortex

BackgroundTranscranial magnetic stimulation (TMS) triggers time-locked cortical activity that can be recorded with electroencephalography (EEG). Transcranial evoked potentials (TEPs) are widely used to probe brain responses to TMS. MethodsHere, we systematically reviewed 137 published experiments that studied TEPs elicited from TMS to the human primary motor cortex (M1) in healthy individuals to investigate the impact of methodological choices. We scrutinized prevalent methodological choices and assessed how consistently they were reported in published papers. We extracted amplitudes and latencies from reported TEPs and compared total cortical activation and specific TEP peaks and components. ResultsReporting of methodological details was overall sufficient, but some relevant information regarding the TMS settings and the recording and pre-processing of EEG data were missing in more than 25% of the included experiments. The published TEP latencies and amplitudes confirm the prototypical TEP waveform of M1, comprising distinct N15, P30, N45, P60, N100, and P180 peaks. However, variations in amplitude and latencies were evident across studies. Higher stimulation intensities were associated with overall larger TEP amplitudes. Active noise masking during TMS generally resulted in lower TEP amplitudes compared to no or passive masking but did not specifically impact those TEP peaks linked to long-latency sensory processing. Studies implementing independent component analysis (ICA) for artifact removal generally reported lower TEP amplitudes. ConclusionSome aspects of reporting practices could be improved in TEP studies to enable replication. Methodological choices, including TMS intensity and the use of noise masking or ICA, introduce systematic differences in reported TEP amplitudes. Further investigation into the significance of these and other methodological factors and their interactions is warranted.

neuroscience↗

bioRxiv2023

Transcranial magnetic stimulation of primary motor cortex elicits a site-specific immediate transcranial evoked potential

BackgroundTranscranial evoked potentials (TEPs) measured via electroencephalography (EEG) are widely used to study the cortical responses to transcranial magnetic stimulation (TMS). Immediate transcranial evoked potentials (i-TEPs) have been obscured by pulse and muscular artifacts. Thus, the TEP peaks that are commonly reported have latencies that are too long to be caused by direct excitation of cortical neurons. MethodsIn 14 healthy individuals, we recorded i-TEPs evoked by a single biphasic TMS pulse targeting the primary motor hand area (M1HAND) or parietal or midline control sites. Sampling EEG at 50 kHz enabled us to reduce the duration of the TMS pulse artifact to a few milliseconds, while minor adjustments of the TMS coil tilt or position enabled us to avoid cranial muscular twitches during the experiment. ResultsWe observed an early positive EEG deflection starting after approx. 2 ms combined with a series of peaks with an inter-peak interval of [~]1.1-1.4 ms in electrodes overlying the stimulated sensorimotor region. This multi-peak i-TEP response was only evoked by TMS of the M1HAND region and was modified by changes in stimulation intensity and current direction. DiscussionSingle-pulse TMS of the M1HAND evokes an immediate local multi-peak response at the cortical site of stimulation. Our results suggest that the observed i-TEP patterns are genuine cortical responses evoked by TMS caused by synchronized excitation of pyramidal neurons in the targeted precentral cortex. This notion needs to be corroborated in future studies, including further investigations into the potential contribution of instrumental or physiological artifacts.

neuroscience↗

bioRxiv2023

Hebbian priming of human spinal motor learning

Learning or relearning of motor skills requires plasticity in relevant neural circuits. Motor recovery following lesions to the corticospinal system can be augmented through neuromodulation techniques targeting the affected or compensatory neural circuits. By repeatedly pairing transcranial magnetic stimulation of the primary motor cortex (M1) and motoneuronal electrical stimulation (i.e., paired corticomotoneuronal stimulation, PCMS) timed to arrive at the corticomotoneuronal (CM) synapses in close temporal proximity, spike-timing-dependent bidirectional changes in CM transmission can be induced in humans (Taylor & Martin, 2009). PCMS-induced increases in CM transmission have been demonstrated to transiently improve motor control in patients with spinal cord injury (Bunday & Perez 2012), whereas effects on the malleability of neural circuits are entirely unexplored. We hypothesized that PCMS can prime mechanisms of subsequent motor learning exclusively when directed to the neural circuitry underpinning the motor behavior. In three experiments, we provide the first evidence ( Experiment I) and a double-blinded, sham-controlled replication ( Experiment II) that PCMS targeting the spinal CM synapses can prime subsequent learning of rapid finger movements relying on spinal neuroplasticity. Finally, we demonstrate that the effects of PCMS are circuit-specific and bidirectional. When PCMS was timed to arrive at a facilitatory interval in M1 but an inhibitory interval at the CM synapses subsequent learning was transiently impeded ( Experiment III). Taken together, our results provide proof-of-principle that non-invasively induced plasticity governed by Hebbian learning rules interacts with experience-dependent plasticity in the spinal cord with positive implications for motor learning. Our results offer a mechanistic rationale for priming sensorimotor training with individualized PCMS to enhance the effects of motor practice in neurorehabilitation. HighlightsO_LIPaired corticomotoneuronal stimulations (PCMS) promote ballistic motor learning and facilitate corticospinal excitability compared to rest and sham protocols. C_LIO_LIA double-blinded sham experiment replicates priming effects of PCMS on ballistic motor learning and demonstrates long-term benefits of combined PCMS and motor practice. C_LIO_LIThe facilitating effect of PCMS on ballistic motor learning is circuit-specific with superior effects on ballistic motor learning after facilitating PCMS compared to control protocols. C_LI

neuroscience↗

bioRxiv2022

Multi-pulse transcranial magnetic stimulation of human motor cortex produces short-latency corticomotor facilitation via two distinct mechanisms

BackgroundSingle-pulse transcranial magnetic stimulation of the precentral hand representation (M1HAND) can elicit indirect waves in the corticospinal tract at a periodicity of ~660 Hz, called indirect or I-waves. These synchronized descending volleys are produced by transsynaptic excitation of fastconducting monosynaptic corticospinal axons in M1-HAND. Paired-pulse TMS can induce short-interval intracortical facilitation (SICF) of motor evoked potentials (MEPs) at inter-pulse intervals that match I-wave periodicity. ObjectiveTo examine whether short-latency corticospinal facilitation engages additional mechanisms independently of I-wave periodicity. MethodsIn 19 volunteers, one to four biphasic TMS pulses were applied to left M1-HAND with interpulse interval was adjusted to the first peak or first trough of the individual SICF curve. TMS was applied at different intensities to probe the intensity-response relationship. ResultsPairs, triplets, or quadruplets at individual peak-latency facilitated MEP amplitudes across a wide range of TMS intensities compared to single pulses. Multi-pulse TMSHAND at individual troughlatency also produced a consistent facilitation of MEP amplitude. Short-latency facilitation at trough-latency was less pronounced than short-latency facilitation at peak-latency, but the relative difference in facilitation decreased with increasing stimulus intensity. Increasing the number of pulses from two to four pulses had only a modest effect on MEP facilitation. ConclusionTwo mechanisms underly short-latency corticomotor facilitation caused by biphasic multi-pulse TMS. An intracortical mechanism is related to I-wave periodicity and engages fast-conducting direct projections to spinal motoneurons. A second corticospinal mechanism does not rely on I-wave rhythmicity and may be mediated by slower conducting indirect pyramidal tract projections from M1-HAND to spinal interneurons. The latter mechanism deserves more attention in TMS studies of the corticomotor system.

neuroscience↗

bioRxiv2022

Corticospinal excitability is influenced by the recent history of electrical digital stimulation: implications for the relative magnitude of short-latency afferent inhibition

BackgroundElectrical stimulation of the hand can suppress the motor evoked potential (MEP) elicited by transcranial magnetic stimulation (TMS) of the primary motor hand area (M1-HAND) when the afferent stimulus arrives in M1-HAND at the time of TMS. The magnitude of short-latency afferent inhibition (SAI) is expressed as the ratio between the conditioned and unconditioned MEP and is widely used to probe sensorimotor interactions in human physiological studies. Objective/HypothesisWe hypothesized that corticospinal excitability and SAI are influenced by the recent history of peripheral electrical stimulation. MethodsIn twenty healthy participants, we recorded MEPs from the right first dorsal interosseus muscle. MEPs were evoked by single-pulse TMS of the left M1-HAND alone (unconditioned TMS) or by TMS preceded by electrical stimulation of the right index finger ("homotopic" conditioning) or little finger ("heterotopic" conditioning). The three conditions were pseudo-randomly intermixed or delivered in blocks in which a single condition was repeated five or ten times. MEP amplitudes and SAI magnitudes were compared using linear mixed effect models. ResultsAll stimulation protocols consistently produced SAI, which was stronger after homotopic stimulation. Randomly intermingling the three stimulation conditions reduced the relative magnitude of homotopic and heterotopic SAI as opposed to blocked stimulation. The apparent attenuation of SAI was caused by a suppression of the unconditioned but not the conditioned MEP amplitude during the randomly intermixed condition. Conclusion(s)The recent history of afferent stimulation modulates corticospinal excitability. This "history effect" impacts on the relative magnitude of SAI depending on how conditioned and unconditioned responses are intermixed and needs to be taken into consideration when probing afferent inhibition and corticospinal excitability.

neuroscience↗

bioRxiv2019

A decade of epigenetic change in aging twins: genetic and environmental contributions to longitudinal DNA methylation

Summary/AbstractO_ST_ABSBackgroundC_ST_ABSEpigenetic changes may result from the interplay of environmental exposures and genetic influences and contribute to differences in age-related disease, disability and mortality risk. However, the etiologies contributing to stability and change in DNA methylation have rarely been examined longitudinally. MethodsWe considered DNA methylation in whole blood leukocyte DNA across a 10-year span in two samples of same-sex aging twins: (a) Swedish Adoption Twin Study of Aging (SATSA; N = 53 pairs, 53% female; 62.9 and 72.5 years, SD=7.2 years); (b) Longitudinal Study of Aging Danish Twins (LSADT; N = 43 pairs, 72% female, 76.2 and 86.1 years, SD=1.8 years). Joint biometrical analyses were conducted on 358,836 methylation probes in common. Bivariate twin models were fitted, adjusting for age, sex and country. ResultsOverall, results suggest genetic contributions to DNA methylation across 358,836 sites tended to be small and lessen across 10 years (broad heritability M=23.8% and 18.0%) but contributed to stability across time while person-specific factors explained emergent influences across the decade. Aging-specific sites identified from prior EWAS and methylation age clocks were more heritable than background sites. The 5,037 sites that showed the greatest heritable/familial-environmental influences (p<1E-07) were enriched for immune and inflammation pathways while 2,020 low stability sites showed enrichment in stress-related pathways. ConclusionsAcross time, stability in methylation is primarily due to genetic contributions, while novel experiences and exposures contribute to methylation differences. Elevated genetic contributions at age-related methylation sites suggest that adaptions to aging and senescence may be differentially impacted by genetic background.

genetics↗

bioRxiv2017

The cis-regulatory dynamics of embryonic development at single cell resolution

Single cell measurements of gene expression are providing new insights into lineage commitment, yet the regulatory changes underlying individual cell trajectories remain elusive. Here, we profiled chromatin accessibility in over 20,000 single nuclei across multiple stages of Drosophila embryogenesis. Our data reveal heterogeneity in the regulatory landscape prior to gastrulation that reflects anatomical position, a feature that aligns with future cell fate. During mid embryogenesis, tissue granularity emerges such that cell types can be inferred by their chromatin accessibility, while maintaining a signature of their germ layer of origin. We identify over 30,000 distal elements with tissue-specific accessibility. Using transgenic embryos, we tested the germ layer specificity of a subset of predicted enhancers, achieving near-perfect accuracy. Overall, these data demonstrate the power of shotgun single cell profiling of embryos to resolve dynamic changes in open chromatin during development, and to uncover the cis-regulatory programs of germ layers and cell types.

genomics↗