bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1 Species-specific oscillation periods of human and mouse segmentation clocks 2 are due to cell autonomous differences in biochemical reaction parameters 3 4 Mitsuhiro Matsuda1, 2, Hanako Hayashi1, Jordi Garcia-Ojalvo3, Kumiko Yoshioka- 5 Kobayashi4, Ryoichiro Kageyama4, Yoshihiro Yamanaka5, Makoto Ikeya5, Junya 6 Toguchida4, 5, Cantas Alev5, Miki Ebisuya1, 2* 7 8 1RIKEN Center for Biosystems Dynamics Research (RIKEN BDR) 9 2-2-3 Minatojima-minamimachi, Chuo-ku, 650-0047 Kobe, Japan 10 2European Molecular Biology Laboratory (EMBL) Barcelona 11 Dr. Aiguader 88, 08003 Barcelona, Spain 12 3Department of Experimental and Health Sciences, Universitat Pompeu Fabra 13 Dr. Aiguader 88, 08003 Barcelona, Spain 14 4Institute for Frontier Life and Medical Sciences, Kyoto University 15 Shogoin-Kawahara-cho, Sakyo-ku, 606-8507 Kyoto, Japan 16 5Center for iPS Cell Research and Application (CiRA), Kyoto University 17 53 Shogoin-Kawahara-cho, Sakyo-ku, 606-8507 Kyoto, Japan 18 19 20 *Correspondence to M Ebisuya ([email protected]) 21

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1 Abstract 2 While the mechanisms of embryonic development are similar between mouse and human, 3 the tempo is in general slower in human. The cause of interspecies differences in 4 developmental time remains a mystery partly due to lack of an appropriate model system1. 5 Since murine and human embryos differ in their sizes, geometries, and nutrients, we use 6 in vitro differentiation of pluripotent stem cells (PSCs) to compare the same type of cells 7 between the species in similar culture conditions. As an example of well-defined 8 developmental time, we focus on the segmentation clock, oscillatory gene expression that 9 regulates the timing of sequential formation of body segments2-4. In this way we 10 recapitulate the murine and human segmentation clocks in vitro, showing that the species- 11 specific oscillation periods are derived from cell autonomous differences in the speeds of 12 biochemical reactions. Presomitic mesoderm (PSM)-like cells induced from murine and 13 human PSCs displayed the oscillatory expression of HES7, the core gene of the 14 segmentation clock5,6, with oscillation periods of 2-3 hours (mouse PSM) and 5-6 hours 15 (human PSM). Swapping HES7 loci between murine and human genomes did not change 16 the oscillation periods dramatically, denying the possibility that interspecies differences 17 in the sequences of HES7 loci might be the cause of the observed period difference. 18 Instead, we found that the biochemical reactions that determine the oscillation period, 19 such as the degradation of HES7 and delays in its expression, are slower in human PSM 20 compared with those in mouse PSM. With the measured biochemical parameters, our 21 mathematical model successfully accounted for the 2-3-fold period difference between 22 mouse and human. We further demonstrate that the concept of slower biochemical 23 reactions in human cells is generalizable to several other genes, as well as to another cell 24 type. These results collectively indicate that differences in the speeds of biochemical 25 reactions between murine and human cells give rise to the interspecies period difference 26 of the segmentation clock and may contribute to other interspecies differences in 27 developmental time. 28 29 Main 30 To compare murine and human segmentation clocks in vitro, we induced PSM-like cells 31 from mouse embryonic stem cells (ESCs) and human induced pluripotent stem cells 32 (iPSCs) (Fig. 1a), as other groups have recently reported7-12. In essence, our PSM 33 induction protocol is based on activation of WNT and FGF signaling as well as inhibition 34 of TGFβ and BMP signaling9,12. Prior to the PSM induction, mouse ESCs, which are in 35 the naïve pluripotent state, were pretreated with ACTIVIN A and bFGF and converted to 36 mouse epiblast-like cells (EpiLCs) that possess primed pluripotency as human iPSCs do.

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1 To visualize the segmentation clock in the induced PSM, we introduced a HES7 promoter- 2 luciferase reporter13,14, detecting clear synchronized oscillations of HES7 expression in 3 both murine and human PSM (Fig. 1b; Supplementary Video 1). Interestingly, the 4 oscillation periods, i.e., the durations for one cycle, were different between the species: 5 mouse PSM oscillated with a period of 122 ± 2 min (mean ± sd) whereas human PSM 6 exhibited a 322 ± 6 min period (Fig. 1c-e). These numbers are consistent with the 7 literature: The period of the murine segmentation clock in vivo is 2-3 hours13,15,16. While 8 visualizing the segmentation clock in a human embryo is ethically difficult, the human 9 period has been roughly estimated to be 4-6 hours with fixed samples through counting 10 the number of somites, which are periodically formed according to the segmentation 11 clock17,18. Thus, we concluded that our induced PSM recapitulates the species-specific 12 periods of the segmentation clock and serves as an ideal in vitro platform to investigate 13 the cause of the 2-3-fold period difference between mouse and human. 14 The gene regulatory network of the segmentation clock consists of two parts: the 15 intracellular network that gives rise to a cell autonomous oscillation in each cell and the 16 intercellular network that synchronizes the oscillations among neighboring cells 17 (Supplementary Fig. 1a)19-21. We therefore first attempted to clarify whether the 18 interspecies period difference stems from the single-cell oscillator or the multicellular 19 synchronized oscillations. Because cell-cell communication through NOTCH-DELTA 20 signaling has been reported to synchronize oscillations among cells by regulating HES7 21 expression20,22-24, we treated both murine and human PSM with a NOTCH inhibitor, 22 DAPT. While the expression level of the HES7 reporter decreased upon administration of 23 DAPT (Fig. 1f, Original), the oscillation period did not change significantly in either 24 species (Fig. 1f, g; Supplementary Fig. 1b). Although WNT and FGF signaling pathways 25 have also been reported to modulate the segmentation clock25-28, the existence of high 26 dosages of a WNT agonist, CHIR, and bFGF in the culture medium suggests that cell-cell 27 communication through these pathways should not be crucial for the interspecies period 28 difference. Furthermore, we measured the oscillation period in a sparse cell culture, where 29 cells do not contact each other (Fig. 1h; Supplementary Fig. 2; Supplementary Video 2). 30 Those isolated PSM cells still displayed the 2-3-fold period difference between the 31 species (mouse: 160 ± 9 min; human: 376 ± 51 min) (Fig. 1i), even though the oscillations 32 at the single-cell level were noisier and slower than the population level oscillations (see 33 Supplementary Fig. 2b, c). These results indicate that the period difference of HES7 34 oscillation between mouse and human is cell autonomous, and that the cause of the 35 interspecies difference should lie in the oscillation mechanism at the single-cell level.

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1 HES7 oscillations have been proposed to arise from a delayed autoregulatory 2 negative feedback loop: HES7, a transcription repressor, directly binds to and inhibits its 3 own promoter with time delays, resulting in an oscillatory expression of HES7 4 (Supplementary Fig. 1a)6,14,29,30. Knocking out other HES family members, such as HES1 5 and HES5, does not disrupt segmentation in mouse embryos31. Since HES7 itself is 6 considered the most critical gene for HES7 oscillation, we first hypothesized that 7 differences in the sequences of HES7 loci between murine and human genomes might 8 lead to the observed oscillation period difference. To test this hypothesis, we swapped 9 HES7 loci between mouse and human (Fig. 2a): the human HES7 locus, which was 10 defined as the sequence including a promoter, exons, introns, and UTRs of HES7, was 11 knocked into the mouse HES7 locus in mouse ESCs (Fig. 2b; Supplementary Fig. 3), and 12 the resulting cells were induced to differentiate into the PSM fate. The homozygous 13 knock-in (i.e., human HES7/human HES7, hereafter referred to as ‘homo swap’) PSM 14 and the heterozygous knock-in (human HES7/mouse HES7, hereafter referred to as 15 ‘hetero swap’) PSM showed slightly longer oscillation periods of 146 ± 7 min and 133 ± 16 4 min, respectively, as compared with the 124 ± 3 min period of wild-type (mouse 17 HES7/mouse HES7) mouse PSM (Fig. 2c, d; Supplementary Fig. 4a). Considering that 18 the period of wild-type human PSM is 322 min (see Fig. 2d, Wt Human PSM), however, 19 the period extension in homo swap mouse PSM is minor. To confirm this finding, we 20 created knock-in mice containing the human HES7 locus. The homo swap mice appeared 21 largely normal, even though their vertebrae, which are derivatives of somites and 22 therefore subject to the influence of the segmentation clock, displayed minor defects (Fig. 23 2e; Supplementary Fig. 5). The ex vivo measurements of the segmentation clock in homo 24 swap embryos showed ~30 min longer oscillation period as compared with wild-type 25 embryos (Fig. 2f-h; Supplementary Fig. 4b), consistent with the ~20 min period extension 26 in the homo swap samples of induced PSM (see Fig. 2d). However, the 20-30 min period 27 extension in homo swap PSM/embryos is far from the ~200 min period difference 28 between mouse and human, so these results suggest that human HES7 locus in mouse 29 PSM gives rise to an essentially mouse-like oscillation period. 30 One potential defect in our experimental design of interspecies genome 31 swapping is, however, that the swapped HES7 region might not be long enough, and that 32 a crucial sequence for the oscillation period might exist upstream of the HES7 promoter 33 we defined, for instance. To rule out this possibility, we performed ‘knock-out and rescue’ 34 assays (Fig. 2i): The endogenous mouse HES7 gene was first knocked out in mouse ESCs, 35 leading to disruption of the HES7 oscillation in the induced PSM (Fig. 2j). Then the 36 disrupted oscillation was rescued by introducing an exogenous construct containing a

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1 promoter, exons, introns, and UTRs of murine or human HES7 locus (Fig. 2k; 2 Supplementary Fig. 4c). Note that these exogenous constructs were integrated into 3 random positions of the genome by transposon vectors, implying that the HES7 regions 4 used for the constructs should be sufficiently long to restore the oscillations. Importantly, 5 both murine and human HES7 constructs restored mouse-like oscillation periods in the 6 mouse PSM (Fig. 2l). We further attempted a ‘complementary’ experiment: we knocked 7 out the endogenous human HES7 gene and rescued the disrupted oscillation with the 8 murine or human HES7 construct in human PSM (Fig. 2m, n). Again, murine and human 9 HES7 constructs were indistinguishable in terms of the restored oscillation period (Fig. 10 2o). These results collectively indicate that the 2-3-fold period difference between murine 11 and human segmentation clocks is not caused by the sequence differences between 12 murine and human HES7 loci. 13 We then hypothesized that differences not in the sequences but in the 14 biochemical reaction speeds of HES7 between murine and human cells might lead to the 15 observed oscillation period difference. Since the most important biochemical parameters

16 that affect the oscillation period of HES7 are the degradation rates of HES7 (i.e., δm and

17 δp in Fig. 3a) and the delays in the feedback loop of HES7 (τTx, τIn, τTl, and τRp in Fig. 18 3a)14,20,29,30,32, we measured those parameters in both murine and human PSM, exploring 19 which parameter(s) are different between the species. To measure the degradation rate of

20 HES7 protein (δp), we overexpressed either the murine or human HES7 sequence and 21 then halted its expression (Fig. 3b). Interestingly, both murine and human HES7 proteins 22 were degraded more slowly in human PSM as compared with mouse PSM (Fig. 3b, c; 23 Supplementary Fig. 6a), meaning that the changes in the degradation rate depend on the 24 differences not in the HES7 sequences but in the cellular environments (i.e., whether 25 HES7 is hosted in a murine or human cell). The half-life of HES7 protein in mouse was 26 previously reported to be 22 min29, consistent with our measurements where half-lives in 27 murine and human PSM were estimated to be 21 ± 0.8 min and 40 ± 4 min, respectively. 28 To measure the delay caused by the transcription and translation of HES7, we 29 induced the expression of HES7 and estimated the onset time by fitting the results to a 30 standard gene expression model in which transcription and translation are assumed to 31 occur in a linear manner with the corresponding delays (Fig. 3d, e; Supplementary Fig.

32 6b). The transcription and translation delay (τTxTl) of HES7 was estimated to be longer in 33 human PSM (30 ± 1 min) as compared with mouse PSM (17 ± 2 min) (Fig. 3f, top). The

34 fitting also allowed us to estimate the degradation rate of HES7 mRNA (δm), showing 35 slower degradation in human PSM (half-life in mouse: 10 ± 0.3 min; in human: 16 ± 0.3 36 min) (Fig. 3f, bottom). Note that the HES7 gene used in these measurements did not

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1 include the introns (see Fig. 3b, d). Since introns affect mRNA splicing and therefore 2 serve as another source of delays14,30,32, we measured the delay caused by HES7 introns 3 by creating HES7 promoter-luciferase reporters with (w/) and without (w/o) HES7 introns 4 (Fig. 3g, h) and estimating the phase difference between the oscillations of the two

5 reporters (Fig. 3g; Supplementary Fig. 7). Again, the HES7 intron delay (τIn) was longer 6 in human PSM (37 ± 3 min) compared with mouse PSM (13 ± 3 min) (Fig. 3i). Roughly 7 consistent with our measurements, the intron delay or splicing delay in mouse embryos 8 was previously reported to be 12-19 min14,32. Finally, to measure the delay for HES7 to 9 start repressing its own promoter, we induced the expression of HES7 and estimated the 10 onset of decline in the HES7 promoter activity (Fig. 3j; Supplementary Fig. 8). Fitting 11 the results to an open loop repression model in which the induced HES7 protein represses 12 the expression of HES7 promoter-luciferase reporter showed that the HES7 repression

13 delay (τRp) is negligible in both murine and human PSM. 14 To confirm that the degradation rates and delays measured in both murine and 15 human PSM can indeed explain the interspecies period difference in the segmentation 16 clock, we built a simple mathematical model of HES7 oscillation20 based directly on the

17 following parameters: δp, δm, τTxTl, τIn, and τRp (Fig. 3k; see Methods). Note that our 18 mathematical analyses of the model showed that the oscillation period depends on these 19 measured parameters (i.e., degradation rates and total delays), and that other parameters, 20 such as the transcription and translation rates and the repression threshold, essentially do 21 not affect the period (Supplementary Text 1)20. Even though one unmeasured parameter, 22 the repression Hill coefficient, potentially affects the oscillation period (Supplementary 23 Text 1), varying this parameter within a realistic range did not change the period 24 dramatically (Fig. 3l). Remarkably, our simulation of oscillations with the murine 25 parameters showed periods of ~150 min whereas that with human parameters showed 26 ~300 min periods (Fig. 3l), reproducing the 2-3-fold period difference between actual 27 murine and human PSM (see Fig. 1e). These results mean that the slower biochemical 28 reactions of HES7 (i.e., slower degradations and longer delays) in human PSM as 29 compared with those in mouse PSM are sufficient to quantitatively account for the longer 30 oscillation period of the human segmentation clock. 31 Next, we explored how universal our finding of slower biochemical reactions in 32 human cells is. To test whether it is specific to the HES7 gene or generalizable to other 33 genes, we measured the degradation rates of six other genes, transcription factors 34 expressed at the PSM stage7 (Fig. 4a, b; Supplementary Fig. 9). GBX2, MSGN1, and 35 TBX6 proteins showed slower degradation rates in human PSM than in mouse PSM, 36 whereas CDX2, EVX1, and Brachyury T did not (Fig. 4c). We also measured the

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1 transcription and intron delays (τTx, τIn) (Fig. 4d, e; Supplementary Fig. 10). TBX6, GBX2, 2 and MSGN1 showed longer delays in human PSM than in mouse PSM whereas EVX1 3 did not show a significant interspecies difference (Fig. 4f). These results suggest that the 4 slower biochemical reactions in human PSM with respect to mouse PSM can extend to 5 several other genes, but not to all genes. 6 Finally, to test whether the slower biochemical reactions in human cells are 7 specific to PSM or generalizable to other cell types, we induced neural progenitor cells 8 (NPCs) from mouse ESCs and human iPSCs (Fig. 4g)33,34. We measured the degradation 9 rates of neural marker genes34 in both murine and human induced NPCs (Fig. 4h; 10 Supplementary Fig. 11). All the four proteins tested, OTX2, FOXG1, PAX6, and SOX1, 11 showed slower degradation rates in human NPCs as compared with mouse NPCs (Fig. 12 4i). We also measured the transcription and intron delays of OTX2, FOXG1, and SOX1 13 (Fig. 4j; Supplementary Fig. 12), demonstrating slightly longer delays in human NPCs 14 for all three genes (Fig. 4k). These results suggest that slower biochemical reactions in 15 human cells can be applicable not only to the PSM fate but also to other cell types, even 16 though more systematic measurements will be necessary in the future. We propose that 17 murine and human cells possess species-specific cellular environments that affect speeds 18 of several biochemical reactions including degradations and delays (Fig. 4l), potentially 19 causing other interspecies differences in developmental time. The cellular environments 20 can mean any gene set or any cellular characteristic, such as the metabolic rate and cell 21 size. 22 In summary, we have shown that the human segmentation clock exhibits 2-3- 23 times slower oscillations in comparison with mouse, because of slower degradation rates 24 of HES7 and longer delays in its expression in a human PSM cell. An obvious next 25 challenge is to reveal the mechanism by which human cells display slower biochemical 26 reactions. Since our results have revealed the existence of several other genes that show 27 different reaction speeds between murine and human cells, it may be interesting to 28 classify the genes that show such an interspecies difference and to find commonalities 29 among them. Another future challenge is to investigate developmental time of other 30 species than mouse and human. Interestingly, delays due to splicing and export of mRNAs 31 of HES-related genes have previously been reported to be different among mouse, chick, 32 and zebrafish32. Because ESCs and iPSCs of diverse mammals are now available35,36, 33 their in vitro differentiation will enable to compare the same cell types among different 34 mammalian species and to study their different tempos of development. 35 36

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1 Methods 2 3 Pluripotent stem cell cultures 4 Mouse ESCs (EB5, a gift from H. Niwa) were maintained on gelatin coated dish with 5 GMEM containing 10% KSR, 1% FBS, nonessential amino acids (1 mM), β- 6 mercaptoethanol (0.1 mM), sodium pyruvate (1 mM), LIF (2000 U/ml), CHIR99021 (3 7 µM), and PD0325901 (1 µM). Human iPSCs (201B7, feederless) were maintained on 8 iMatrix-511 silk (Nippi) coated dishes or plates with StemFit AK02N medium 9 (Ajinomoto). 10 11 DNA constructs 12 The genetic constructs are listed in Supplementary Table 2. The promoters or genes were 13 subcloned into pDONR vector to create entry clones. These entry clones were recombined 14 with piggyBac vector (a gift from K. Woltjen)37 by using the Multisite Gateway 15 technology (Invitrogen). The DNA constructs were introduced into the cells with Amaxa 16 Nucleofector (Lonza). 17 18 Induction of murine and human PSM 19 Mouse ESCs were first cultured in N2B27 medium containing 1% KSR, ACTIVIN A (20 20 ng/ml), and bFGF (10 ng/ml) for 4 days and converted to mouse EpiLCs38,39. The induced 21 mouse EpiLCs were further cultured in CDMi40 containing SB431542 (10 µM), DMH1 22 (2 µM), CHIR99021 (10 µM), and bFGF (20 ng/ml) for 2 days to induce mouse PSM 23 cells. To induce human PSM cells, our 1 step induction protocol9 was mainly used. 24 Human iPSCs were seeded on a 35 mm dishes coated with iMatrix-511 silk or matrigel 25 and cultured for 4 days. Then the cells were cultured in CDMi containing SB431542 (10 26 µM), DMH1 (2 µM), CHIR99021 (10 µM), and bFGF (20 ng/ml) for another 3.5 days to 27 induce human PSM cells. For the degradation assay, our 2 step induction protocol12 was 28 used. Human iPSCs were seeded on a 35 mm dish coated with iMatrix-511 silk and 29 cultured for 5 days. Then the medium was changed into CDMi containing bFGF (20 30 ng/ml), CHIR99021 (10 µM), and ACTIVIN A (50 ng/ml) for 24 hours to induce 31 primitive streak (PS) cells. The induced PS cells were further cultured in CDMi 32 containing SB431542 (10 µM), CHIR99021 (3 µM), LDN-193189 (250 nM), and bFGF 33 (20 ng/ml) for 24 hours to induce human PSM cells. 34 35 Induction of murine and human NPCs

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1 To induce mouse NPCs, mouse ESCs were seeded on a gelatin coated dish and cultured 2 in the NDiff 227 medium (TAKARA) for 5 days33. To induce human NPCs, human iPSCs 3 were seeded on a matrigel coated dish and cultured in the STEMdiff SMADi Neural 4 Induction medium (STEMCELL Technologies) for 7 days34. NPC differentiation was 5 checked by immunostaining with an anti-PAX6 antibody (BioLegend). 6 7 Oscillation analyses 8 After the induction of murine or human PSM cells, the medium was changed into CDMi 9 containing SB431542 (10 µM), DMH1 (2 µM), CHIR99021 (1 µM), bFGF (20 ng/ml), 10 and D-luciferin (200 µM or 1mM) to monitor oscillations of the HES7 reporter signal. 11 For the single cell imaging, the induced PSM cells were re-seeded on iMatrix-511 silk 12 coated dish. After 6 hours, the medium was changed into CDMi containing SB431542 13 (10 µM), DMH1 (2 µM), CHIR99021 (1 µM), bFGF (20 ng/ml), Latrunculin A (0.5 14 µM)41, and D-luciferin (1 mM). Bioluminescence was measured with Kronos Dio 15 Luminometer (Atto) or LCV110 microscope (Olympus). The obtained signal was 16 detrended by using a 60 min (mouse), 90 min (human), or 100 min (human single cell) 17 moving average-subtraction method. The data displayed were normalized to the 1st peak 18 of oscillations. The oscillation period was defined as the time interval between the 1st and 19 4th peaks divided by 3 cycles. For ex vivo measurements, the period was defined with the 20 1st and 3rd peaks. 21 22 HES7 loci swapping 23 The CRISPR guide sequences for HES7 swapping were cloned into pSpCas9(BB)-2A- 24 GFP vector (addgene #48138)42 (see Supplementary Table 2). As the template for 25 homologous recombination, a bacterial artificial chromosome (BAC) containing human 26 HES7 locus (RP11-769H22)43 was obtained from BACPAC resources center (Children’s 27 Hospital & Research Center at Oakland). After homology arms were inserted (see 28 Supplementary Table 2), the purified BAC was introduced into mouse ESCs with 29 CIRSPR/Cas9 guides to swap the HES7 loci between mouse and human. 30 31 Southern blotting 32 Southern blotting was performed according to the DIG Application Manual for Filter 33 Hybridization (Roche) using the PCR DIG Probe Synthesis kit (Roche). The probe 34 sequences are available in Supplementary Table 2. 35 36 HES7 knockout

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1 The CRISPR guide sequences for HES7 knockout were cloned into pSpCas9(BB)-2A- 2 GFP (see Supplementary Table 2). HES7 knockout was performed using transient 3 transfection of multiple CRISPR guide constructs. The deletion of HES7 was confirmed 4 by PCR. 5 6 Transgenic mice and ex vivo tissue culture 7 HES7 swapping was performed in mouse ESCs (TT2)44. Two hetero swap ESC clones 8 were isolated, and chimeric mice were generated according to standard procedures. The 9 body segments of transgenic mice were imaged at 4 weeks with in vivo micro X-ray CT 10 System R_mCT (RIGAKU). Mice carrying the HES7 reporter pH7-UbLuc-In (−) were 11 previously described14. Time-lapse imaging of ex vivo culture was performed as described 12 previously13 with several modifications. Mouse embryos were collected at 10.5 dpc and 13 dissected in PBS containing 0.2% BSA. Tail portions from Wt and Homo swap embryos 14 were embedded in 0.2% low-melting point agarose in a silicon ring set onto a 35 mm 15 glass-bottom dish, and then cultured in DMEM/F12 containing 1% BSA, L-Glutamine (2 16 mM), D-Glucose (1 g/L), HEPES (15 mM), and D-luciferin (1 mM) with 5% CO2 and 17 80% O2. Imaging was performed on IX81 microscope (Olympus) equipped with 18 VersArray CCD camera (Princeton Instruments). All animal experiments were approved 19 by the Institutional Animal Care and Use Committee of RIKEN Kobe Branch or Kyoto 20 University, and performed according to animal experimentation guidelines of RIKEN and 21 Kyoto University. 22 23 Models and parameter measurements 24 1. HES7 oscillation model 25 To simulate the oscillation of HES7, previously proposed delay differential equations of 26 HES feedback loop were used20.

27 = ( ) m (1) 𝑑𝑑𝑑𝑑 𝛽𝛽 𝑝𝑝 𝑡𝑡 − τ 𝑛𝑛 𝑑𝑑𝑑𝑑 𝑚𝑚 𝑚𝑚 1 + � 𝐾𝐾 � − δ 28 = p (2) 𝑑𝑑𝑑𝑑 𝑝𝑝 𝑝𝑝 29 where𝑑𝑑𝑑𝑑 m𝛼𝛼𝛼𝛼�𝑡𝑡 and p −are τthe� concentrations− δ of mRNA and protein, respectively. δm and δp are 30 the degradation rates of mRNA and protein, α and β are the translation and transcription

31 rates, K is the repression threshold, and n is the repression Hill coefficient. τm and τp are 32 the mRNA and protein delays, and they have the following relationships with the 33 experimentally measured delays: 34 = + + , =

τ𝑚𝑚 τ𝑅𝑅𝑅𝑅 τ𝑇𝑇𝑇𝑇 τ𝐼𝐼𝐼𝐼 τ𝑝𝑝 τ𝑇𝑇𝑇𝑇 10 bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1 where τRp, τTx, τIn, and τTl are the repression, transcription, intron, and translation delays, 2 respectively. The parameter values are summarized in Supplementary Table 1. The 3 numerical calculation was performed with dde23 of MATLAB (Mathworks), and the 4 oscillation periods were estimated by computing the power spectra of the time series. 5 6 2. Degradation assay of HES7 7 The overexpression of a fusion construct of HES7 and NLuc was regulated by the rTetOne 8 system (reverse TetOne system; see Supplementary Table 2). The construct was 9 introduced into mouse ESCs or human iPSCs where the endogenous HES7 was knocked 10 out. After PSM cells were induced in the presence of Doxycycline (Dox; 100 ng/m), the 11 expression of the fusion protein was initiated by washing out Dox and changing the 12 medium into CDMi containing protected furimazine (Promega; 1 µM). After the NLuc 13 signal was confirmed 5-8 hours later, the expression of the fusion protein was halted by 14 Dox (300 ng/ml) addition, and the decay of NLuc signal was monitored with Kronos Dio 15 luminometer. To exclude the influence of residual mRNAs, only the later time points 16 where the NLuc signal displayed a single exponential decay curve were used. To estimate

17 the protein degradation rate (δp) of HES7, the slope of log-transformed data was 18 calculated with the least square method of R. 19 20 3. Expression delay assay of HES7 21 The overexpression of a fusion construct of HES7 (w/o intron) and NLuc was regulated 22 by the TetOne system. The construct was introduced into mouse ESCs or human iPSCs 23 where the endogenous HES7 was knocked out. After PSM cells were induced in the 24 absence of Dox, the medium was changed into CDMi containing protected furimazine (1 25 µM). Six hours after the medium change, the expression of the fusion protein was initiated 26 by Dox (300 ng/ml), and the onset of NLuc signal was monitored with Kronos Dio

27 luminometer. To estimate the sum of the transcription delay and translation delay (τTxTl)

28 of HES7 as well as the mRNA degradation rate (δm) of HES7, models for the expression 29 delay assay and degradation assay were constructed. 30 Expression delay model:

31 = m (t < τTx) 𝑑𝑑𝑑𝑑 𝑑𝑑𝑑𝑑 − δ𝑚𝑚 32 = m (t ≥ τTx) (3) 𝑑𝑑𝑑𝑑 𝑑𝑑𝑑𝑑 β𝑇𝑇 − δ𝑚𝑚 33 = ( ) p (4) 𝑑𝑑𝑑𝑑 𝑑𝑑𝑑𝑑 𝛼𝛼𝛼𝛼 𝑡𝑡 − τ𝑇𝑇𝑇𝑇 − δ𝑝𝑝 11 bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1 where βT is the transcription rate of the TetOne promoter. 2 The solution of this is

3 p(t) = 0 (t < τTxTl)

4 p(t) = ( ) ( ) + (t ≥ τTxTl) (5) 𝑎𝑎 −δ𝑚𝑚 𝑡𝑡 − τ𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 δ𝑚𝑚 −δ𝑝𝑝 𝑡𝑡 − τ𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 𝑎𝑎 5 where δ𝑚𝑚 =− δ𝑝𝑝 �𝑒𝑒+ , and =− δ𝑝𝑝 𝑒𝑒/ . � δ𝑝𝑝 6 Degradation model: τ𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 τ𝑇𝑇𝑇𝑇 τ𝑇𝑇𝑇𝑇 𝑎𝑎 𝛼𝛼β𝑇𝑇 δ𝑚𝑚

7 = m (t < τTx) 𝑑𝑑𝑑𝑑 𝑑𝑑𝑑𝑑 β𝑟𝑟𝑟𝑟 − δ𝑚𝑚 8 = m (t ≥ τTx) (6) 𝑑𝑑𝑑𝑑 𝑑𝑑𝑑𝑑 − δ𝑚𝑚 9 = ( ) p (7) 𝑑𝑑𝑑𝑑 𝑇𝑇𝑇𝑇 𝑝𝑝 10 where𝑑𝑑𝑑𝑑 βrT𝛼𝛼𝛼𝛼 is the𝑡𝑡 −transcriptionτ − δrate of the rTetOne promoter. 11 The solution of this is

12 p(t) = (t < τTxTl) 𝑏𝑏 δ𝑝𝑝 13 p(t) = ( ) ( ) (t ≥ τTxTl) (8) 𝑏𝑏 δ𝑚𝑚 −δ𝑝𝑝 𝑡𝑡 − τ𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 −δ𝑚𝑚 𝑡𝑡 − τ𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 14 where δ =𝑚𝑚 − δ𝑝𝑝 �/δ𝑝𝑝 𝑒𝑒. − 𝑒𝑒 �

15 As for δp, the value estimated in the degradation assay was used. τTxTl and δm, together 𝑏𝑏 𝛼𝛼β𝑟𝑟𝑟𝑟 δ𝑚𝑚 16 with a and b, were estimated in Python by simultaneously fitting the data of expression 17 delay assay and degradation assay to the equations (5) and (8), respectively, with SciPy’s 18 basin-hopping algorithm. 19 20 4. Degradation and expression delay assays of other genes 21 For the degradation assay, the overexpression of a fusion construct of a target gene and 22 NLuc was regulated by the rTetOne system. The construct was introduced into mouse 23 ESCs or human iPSCs. After PSM cells or NPCs were induced in the presence of Dox 24 (100 ng/m), the expression of the fusion protein was initiated by washing out Dox and 25 changing the medium into CDMi containing protected furimazine (5 µM in human NPCs, 26 1 µM in the other cell types). After the NLuc signal was confirmed 5-8 hours later, the 27 expression of the fusion protein was halted by Dox (300 ng/ml), and the decay of NLuc

28 signal was monitored with Kronos Dio luminometer. To estimate the degradation rate (δp) 29 of the fusion protein, the slope of log-transformed data was calculated with the least 30 square method of R.

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1 For the expression delay assay, the overexpression of a fusion construct of FLuc (w/ stop 2 codon) and a target gene (w/ intron) was regulated by the TetOne system. The construct 3 was introduced into mouse ESCs or human iPSCs. After PSM cells or NPCs were induced 4 in the absence of Dox, the medium was changed into CDMi containing D-luciferin (200 5 µM). Six hours after the medium change, the expression of the fusion construct was 6 initiated by Dox (300 ng/ml), and the onset of FLuc signal was monitored with Kronos 7 Dio luminometer. To estimate the sum of the transcription delay, intron delay, and

8 translation delay (τTxInTl) of the fusion construct, a model for expression delay assay was 9 constructed. 10 Expression delay model:

11 = m (t < τTx + τIn) 𝑑𝑑𝑚𝑚 𝑑𝑑𝑑𝑑 − δ𝑚𝑚 12 = m (t ≥ τTx + τIn) (9) 𝑑𝑑𝑚𝑚 𝑑𝑑𝑑𝑑 β𝑇𝑇 − δ𝑚𝑚 13 = ( ) p (10) 𝑑𝑑𝑑𝑑 𝑇𝑇𝑇𝑇 𝑝𝑝 14 The𝑑𝑑𝑑𝑑 solution𝛼𝛼𝛼𝛼 𝑡𝑡of −thisτ is − δ

15 p(t) = 0 (t < τTxInTl)

16 p(t) = ( ) ( ) + (t ≥ τTxInTl) (11) 𝑎𝑎 −δ𝑚𝑚 𝑡𝑡 − τ𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 δ𝑚𝑚 −δ𝑝𝑝 𝑡𝑡 − τ𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 𝑎𝑎 17 where δ𝑚𝑚− δ=𝑝𝑝 �𝑒𝑒 + + . − δ𝑝𝑝 𝑒𝑒 � δ𝑝𝑝

18 τTxInTl for each target gene, together with δm, δp, and a, were estimated in Python by fitting τ𝑇𝑇𝑇𝑇 𝑇𝑇𝐼𝐼𝑇𝑇𝑇𝑇 τ𝑇𝑇𝑇𝑇 τ𝐼𝐼𝐼𝐼 τ𝑇𝑇𝑇𝑇 19 the data of expression delay assay to the equation (11) with SciPy’s basin-hopping 20 algorithm. 21 22 5. Intron delay assay 23 The HES7 promoter-NLuc-stop-HES7 (w/o intron) and HES7 promoter-FLuc-stop-HES7 24 (w/ intron) reporter constructs were introduced into mouse ESCs or human iPSCs. After 25 PSM cells were induced, the medium was changed into CDMi containing protected 26 furimazine (1 µM) and D-luciferin (1 mM), and the oscillations of the NLuc and FLuc 27 signals were simultaneously monitored with Kronos Dio luminometer. To estimate the

28 intron delay (τIn) of HES7, the oscillation phase difference between the ‘w/o intron’ and 29 ‘w/ intron’ reporters was estimated by calculating their cross correlation with R. To 30 normalize the difference in the maturation/degradation time between NLuc and FLuc, 31 cells containing the HES7 promoter-NLuc-stop-HES7 (w/o intron) and HES7 promoter- 32 FLuc-stop-HES7 (w/o intron) constructs were also created, and the phase difference

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1 between the NLuc and FLuc reporters was subtracted from that between the w/o intron 2 and w/ intron reporters. 3 4 6. Repression delay assay 5 First, the expression delay assay of FLuc reporter was performed to estimate the

6 degradation rates of the mRNA (δf) and protein (δF) of FLuc as well as the

7 transcription/translation delay (τTxTlF) of FLuc as described in the section of Expression 8 delay assay of other genes. Next, the overexpression of a fusion construct of HES7 (w/o 9 intron) and NLuc was regulated by the TetOne system. The expression of HES7 promoter- 10 FLuc reporter was repressed by the HES7-NLuc protein. The constructs were introduced 11 into mouse ESCs or human iPSCs where the endogenous HES7 was knocked out. After 12 PSM cells were induced in the absence of Dox, the medium was changed into CDMi 13 containing D-luciferin (200 μM). Six hours after the medium change, the expression of 14 HES7-NLuc protein was initiated by Dox (300 ng/ml), and the onset of decline in the 15 FLuc reporter signal was monitored with Kronos Dio luminometer. To estimate the

16 repression delay (τRp) of HES7, a model for the repression delay assay was constructed. 17 Repression delay model:

18 p(t) = 0 (t < τTxTl)

19 p(t) = ( ) ( ) + (t ≥ τTxTl) (5) 𝑎𝑎 −δ𝑚𝑚 𝑡𝑡 − τ𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 δ𝑚𝑚 −δ𝑝𝑝 𝑡𝑡 − τ𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 𝑎𝑎 δ𝑚𝑚− δ𝑝𝑝 �𝑒𝑒 − δ𝑝𝑝 𝑒𝑒 � δ𝑝𝑝 20 = f (12) ( ) 𝑑𝑑𝑑𝑑 𝛽𝛽 𝑝𝑝 𝑡𝑡 − τ − τ 𝑛𝑛 𝑑𝑑𝑑𝑑 𝑅𝑅𝑅𝑅 𝑇𝑇𝑇𝑇𝑇𝑇 𝑓𝑓 1 + � 𝐾𝐾 � − δ 21 = ( ) F (13) 𝑑𝑑𝑑𝑑 𝐹𝐹 𝑇𝑇𝑇𝑇𝑇𝑇 𝐹𝐹 22 where𝑑𝑑𝑑𝑑 f αand𝑓𝑓 F𝑡𝑡 are− theτ mRNA− andδ protein concentrations of FLuc, respectively. τTxf and

23 τTlF are the transcription and translation delays of FLuc (τTxTlF = τTxf + τTlF), and αF is the 24 translation rate of FLuc. The numerical calculation was performed with Python, and the 25 resulting F(t) was multiplied by C(t) to incorporate the effect of cell population growth.

26 C(t) = (14) ( ) f𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛 1 −𝛾𝛾𝛾𝛾 1 + C − 1 𝑒𝑒 27 where C0 is the 0initial cell density, γ is the growth rate, and fnorm is the scaling factor for

28 luminescence. As for δp, δm, δF, δf, τTxTl, and τTxTlF, measured values were used. The data of

29 repression delay assay were fitted to F(t)×C(t) manually. The fitting was good when τRp 30 = 0 with both murine and human parameters. 31 32 Acknowledgments

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1 We thank to J. Sharpe, V. Trivedi, X. Diego, and C. Villava for their comments. We also 2 thank to M. Ogawa and T. Tsuji for helping μCT scan of knock-in mice, to the Laboratory 3 for Animal Resources and Genetic Engineering (LARGE), RIKEN BDR, for generating 4 transgenic mice and animal housing, and to M. Matsumiya for helping oscillation 5 analyses. This work was supported by internal grants from RIKEN and EMBL, Takeda 6 Science Foundation (to M.E.), Grant-in-Aid for Scientific research (KAKENHI) 7 programs from Ministry of Education Culture, Sports, Science, and Technology (MEXT) 8 (16KT0178, 17H05777 to M.M.), and RIKEN Special Postdoctoral Researchers (SPDR) 9 fellowship (to M.M.). 10 11 Author contributions 12 M.M., M.E., and C.A. conceived the usage of murine and human PSM. M.M. and M.E. 13 conceived the swapping of HES7 loci as well as comparison of biochemical parameters 14 between mouse and human. M.M. and M.E. designed the work and wrote the manuscript. 15 M.M. performed most of the experiments and analyzed the data. H.H. and C.A. developed 16 the induction protocol of mouse PSM. Y.Y., M.I., J.T., and C.A. developed the induction 17 protocol of human PSM. J.G.-O. constructed mathematical models and fitted them to the 18 experimental data. K.Y. and R.K. measured the oscillation period in mouse embryos. 19 20 Competing interests 21 The authors declare no competing interests.

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1 Figure legends 2 3 Figure 1 Cell autonomous period difference between murine and human 4 segmentation clocks 5 a, Induction of PSM-like cells. Mouse ESCs were pretreated with ACTIVIN A and bFGF 6 to induce mouse EpiLCs. Mouse EpiLCs and human iPSCs were treated with WNT 7 agonist, bFGF, TGFβ inhibitor, and BMP inhibitor to induce the PSM fate. Scale bars: 8 200 µm. b, HES7 reporter activities in murine and human PSM in time-lapse imaging. 9 Kymographs indicate the spatio-temporal signals along the line between the point A and 10 B. Scale bars: 400 µm. See also Supplementary Video 1. c, Oscillatory HES7 reporter 11 activity measured with a luminometer. Original (top) and detrended (bottom) signals of 12 three independent experiments. d, Overlay of mean murine and human signals shown in 13 c. e. Oscillation periods estimated from c. f, Effects of NOTCH signaling on the 14 oscillation periods. Murine or human PSM were treated with NOTCH inhibitor DAPT 15 (10 µM). Representative of three independent experiments. g, Periods estimated from f 16 and Supplementary Fig. 1b. h, Time-lapse imaging of single cells. Mouse PSM cells were 17 sparsely split, and the oscillatory HES7 reporter activity in a single cell was monitored. 18 See also Supplementary Video 2. Ph: Phase image; Luc; Luciferase image. Scale bar: 20 19 µm. i, Periods of single murine and human PSM cells estimated from Supplementary Fig. 20 2b, c. All p-values are from two-sided student’s t-test. 21 22 Figure 2 Effects of sequence differences between murine and human HES7 loci on 23 the oscillation period 24 a, Swapping of the human and murine HES7 loci with CRISPR/Cas9-mediated 25 homologous recombination. The HES7 locus was defined as the region ranging from the 26 end of 3’UTR of the adjacent gene, PER1, to the end of 3’UTR of HES7. b, Southern 27 blotting of mouse ESCs containing the human HES7 locus with probes against murine 28 (left) and human (right) HES7 sequences. Wt: mouse HES7/mouse HES7; Hetero swap: 29 human HES7/mouse HES7; Homo swap: human HES7/human HES7. c, Oscillatory 30 HES7 reporter activity in mouse PSM containing the human HES7 locus. Mean of three 31 independent experiments. d, Periods estimated from Supplementary Fig. 4a. The period 32 of wild-type human PSM shown in Fig. 1e is displayed as a control. P-values are from 33 two-sided Dunnett’s test. e, Phenotypes of the knock-in mice containing the human HES7 34 locus. Four-week old mice were scanned with µCT. f, g, Ex vivo tail bud cultures of the 35 mouse embryos containing the human HES7 locus. The tail buds of E10.5 mouse embryos 36 were cultured (f), and the oscillatory HES7 reporter activity was monitored (g). Signals

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1 were averaged within the yellow circle, and a representative of three independent 2 experiments is shown. h, Periods estimated from g and Supplementary Fig. 4b. P-value 3 is from two-sided paired t-test. i-o, Knock-out (KO) and rescue assay. j, m, Endogenous 4 HES7 genes were knocked out in murine (j) and human (m) cells to disrupt HES7 5 oscillation. k, n, The disrupted oscillations were rescued with either an exogenous 6 construct containing a promoter, exons, introns, and UTRs of mouse HES7 (mHES7) or 7 human HES7 (hHES7). Mean of three independent experiments. The mean data of KO 8 shown in j, m are displayed as a control. l, o, Periods estimated from Supplementary Fig. 9 4c. P-values are from two-sided student’s t-test. 10 11 Figure 3 Measuring biochemical parameters of HES7 that determine the oscillation 12 period 13 a, Schematic representation of the negative feedback loop of HES7. The biochemical 14 parameters that determine the oscillation period, i.e., delays and degradation rates, were

15 measured in the indicated panels. τTx: Transcription delay; τIn: Intron delay; τTl:

16 Translation delay; τRp: Repression delay; δm: Degradation rate of mRNA; δp: Degradation 17 rate of protein. b, Degradation assay of HES7. The transcription of the fusion construct 18 of HES7 and NanoLuc (NLuc) was halted upon Doxycycline (Dox) addition at t = 0 (top), 19 and the decay of NLuc signal was monitored (bottom). Either mouse HES7 or human 20 HES7 sequence was used in murine or human PSM. Mean of three independent 21 experiments. c, Inverse degradation rates of HES7 protein estimated from Supplementary 22 Fig. 6a. d, Expression delay assay of HES7. The transcription of HES7-NLuc construct 23 was induced upon Dox addition at t = 0 (top), and the onset of NLuc signal was monitored 24 in either murine or human PSM (bottom). e, Fitting of the HES7 degradation data shown 25 in b (Mouse HES7 in mouse PSM and Human HES7 in human PSM) (top) and fitting of 26 HES7 expression delay shown in d (Ex1) (bottom). f, Transcription/translation delays of 27 HES7 (top) and inverse degradation rates of Hes7 mRNA (bottom) estimated from e and 28 Supplementary Fig. 6b. g, h, Intron delay assay of HES7. Three reporter constructs were 29 used (h). Stop: stop codon. Dual reporter assays with NLuc and Firefly luciferase (FLuc) 30 were performed (g, top), and the cross correlation functions of NLuc and FLuc signals 31 were calculated (g, bottom) in either murine or human PSM. Mean of three independent 32 experiments. i, Intron delays of HES7 estimated from Supplementary Fig. 7. j, Repression 33 delay assay of HES7. The transcription of HES7-NLuc was induced upon Dox addition 34 at t = 0, and the induced HES7 protein repressed the transcription of the FLuc reporter in 35 either murine or human PSM (top). Fitting of the repression data with the parameter 36 repression delay = 0 (bottom). Mean of three independent experiments. k, Simulating

21 bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1 HES7 oscillations with measured biochemical parameters. Hill coefficient n = 3. The 2 other parameters are summarized in Supplementary Table 1. l, Periods estimated by 3 computing the power spectra of simulated oscillations with different values of repression 4 Hill coefficient. All p-values are from two-sided student’s t-test. 5 6 Figure 4 Generality of slower biochemical reactions in human cells 7 a, b, Degradation assay of other genes expressed at the PSM stage. The transcription of a 8 gene of interest (GOI) fused with NLuc was halted upon Dox addition at t = 0 (b), and 9 the decay of NLuc signal was monitored in either murine or human PSM (a). c, Inverse 10 protein degradation rates of other PSM marker genes estimated from Supplementary Fig. 11 9. The HES7 degradation rate shown in Fig. 3c is displayed as a control. d, e, Expression 12 delay assay of other genes expressed at the PSM stage. The transcription of FLuc-GOI 13 fusion construct flanked by a stop codon was induced upon Dox addition at t = 0 (e), and 14 the onset of FLuc signal was monitored in either murine or human PSM (d). Note that the 15 delay measured here is the sum of the transcription delay of the fusion construct, intron 16 delay of GOI, and translation delay of FLuc. Brachyury T and CDX2 were not used due 17 to their long introns. f, Delays of other PSM genes estimated from Supplementary Fig. 18 10. g, In vitro differentiation of NPCs from mouse ESCs and human iPSCs. PAX6 is a 19 neural marker gene. Scale bars: 200 µm. h, Degradation assay in NPCs. The transcription 20 of GOI-NLuc was halted upon Dox addition at t = 0, and the decay of NLuc signal was 21 monitored in either murine or human NPCs. i, Inverse protein degradation rates in NPCs 22 estimated from Supplementary Fig. 11. j, Expression delay assay in NPCs. The 23 transcription of FLuc-GOI fusion construct flanked by a stop codon was induced upon 24 Dox addition at t = 0, and the onset of FLuc signal was monitored in either murine or 25 human NPCs. PAX6 was not used due to its long introns, and HES7 in NPCs was used as 26 a control. k, Delays in NPCs estimated from Supplementary Fig. 12. l, Proposed scheme. 27 Murine and human cells have different cellular environments that affect the speeds of 28 several biochemical reactions. All p-values are from two-sided student’s t-test. 29 30 Supplementary Figure 1 Oscillations and synchronization of the segmentation clock 31 a, Scheme of the segmentation clock. HES7 is a transcription repressor that inhibits its 32 own promoter, giving rise to an oscillatory expression. Oscillations in individual cells are 33 synchronized through intercellular communications driven by NOTCH signaling. WNT 34 and FGF signaling pathways also modulate the segmentation clock. b, Effects of 35 inhibiting NOTCH signaling on the HES7 oscillation. The data of Ex2 is also shown in 36 Fig. 1f.

22 bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1 2 Supplementary Figure 2 Oscillation in a single cell 3 a, Time-lapse imaging of single cells. Human PSM cells were sparsely split, and the 4 oscillatory HES7 reporter activity in a single cell was monitored. See also Supplementary 5 Video 2. Scale bar: 20 µm. b, Oscillations in eight single mouse cells. c, Oscillations in 6 eight single human cells. 7 8 Supplementary Figure 3 Southern blotting verifying the interspecies genome 9 swapping of the HES7 loci 10 a, Mouse ESCs (EB5) containing the human HES7 locus. The cropped version is shown 11 in Fig. 2b. b, Mouse ESCs (TT2) containing the human HES7 locus. Two different lines 12 of hetero swap (clones 1 and 7) were used for knock-in mouse generation. 13 14 Supplementary Figure 4 HES7 swapping and KO-and-rescue assay 15 a, Oscillatory HES7 reporter activity in mouse PSM containing the human HES7 locus. 16 Means are shown in Fig. 2c. b, Ex vivo tail bud cultures of the mouse embryos containing 17 the human HES7 locus. Repeat experiments of Fig. 2f, g. c, Rescue of the oscillation by 18 either mouse HES7 or human HES7 construct in HES7-knock-out cells. Means are shown 19 in Fig. 2k, n. 20 21 Supplementary Figure 5 Knock-in mice containing the human HES7 locus 22 a, Phenotypes of swap mice derived from ESC clone 1 shown in Supplementary Fig. 3b. 23 b, Phenotypes of swap mice derived from ESC clone 7 shown in Supplementary Fig. 3b. 24 The pictures of clone 7 Wt and Homo swap are also shown in Fig. 2e. 25 26 Supplementary Figure 6 Fitting of the degradation rates and delays of HES7 27 a, Fitting of the degradation rate of HES7 protein. Mouse data at 60-90 min and human 28 data at 100-300 min were used for the fitting. b, Fitting of the transcription/translation 29 delay of HES7 shown in Fig. 3d (Ex2, Ex3). Fitting of Ex1 is shown in Fig. 3e. The same 30 data of degradation assay as Fig. 3e was used for fitting. 31 32 Supplementary Figure 7 Measurements of the intron delays of HES7 33 Dual reporter assays and cross correlation functions of NLuc and FLuc signals. Means 34 are shown in Fig. 3g. 35 36 Supplementary Figure 8 Measurements of the repression delays of HES7

23 bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1 a, Measurements of the repression delays of HES7. Means are shown in Fig. 3j. b,

2 Measurements and fitting of the degradation rates of mRNA and protein of FLuc (δf, δF;

3 top) and the sum of transcription (τTxf) and translation (τTlF) delays of FLuc (bottom).

4 Mean of three independent experiments. Estimated mouse parameters: δf = 0.021, δF =

5 0.021, τTxTlF (i.e., τTxf + τTlF) = 29.3; Estimated human parameters: δf = 0.014, δF = 0.014,

6 τTxTlF = 32.1. 7 8 Supplementary Figure 9 Measurements of the protein degradation rates of other 9 PSM marker genes 10 Fitting of the protein degradation rates of genes expressed at the PSM stage. Mouse data 11 at 100-300 min (100-200 min for GBX2) and human data at 200-400 min shown in Fig. 12 4a were used for the fitting. 13 14 Supplementary Figure 10 Measurements of the delays of other PSM marker genes 15 Fitting of the delays of gene expressed at the PSM stage. The original data are shown in 16 Fig. 4d. 17 18 Supplementary Figure 11 Measurements of the protein degradation rates in NPCs 19 Fitting of the protein degradation rates in NPCs. Mouse data at 50-150 min and human 20 data at 100-200 min shown in Fig. 4 h were used for the fitting. 21 22 Supplementary Figure 12 Measurements of the delays in NPCs 23 Fitting of the delays in NPCs. The original data are shown in Fig. 4j. 24 25 Supplementary Text 1 Parameter dependency of simulated oscillation periods 26 27 Supplementary Table 1 Biochemical parameters of HES7 28 29 Supplementary Table 2 Genetic constructs 30 31 Supplementary Video 1 32 Time-lapse imaging of HES7 reporter activity in murine (left) and human (right) PSM. 33 34 Supplementary Video 2 35 Time-lapse imaging of HES7 reporter activity of a single PSM cell in a sparse culture. 36

24

bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was Mousenot certified by peer review) is the author/funder,WNT whoMouse has granted(presomitic bioRxiv a license to display theHuman preprint in perpetuity.WNT It is madeHuman a available under aCC-BY 4.0 International license. ESCs FGF PSM mesoderm) iPSCs FGF PSM ACTIVIN TGFbi TGFbi FGF BMPi BMPi EpiLCs

b Mouse PSM A Space B Human PSM A Space B 0 0

A B B A Time (min) Time (min) HES7 reporter activity HES7 reporter activity 1420 1420

d Mouse Mean Human Mean c Mouse PSM Human PSM Original Original -1.0 0.0 1.0

HES7 reporter activity 0 100 200 300 400 500 600 Ex1 Ex2 Ex3 Mean Ex1 Ex2 Ex3 Mean Time (min) 0.0 0.6 1.2 0.0 0.6 1.2 0 100 200 300 400 500 600 0 500 1000 1500 e p = 5.6E-07 Detrend Detrend HES7 reporter activity Period (min) -1.0 0.0 1.0 -1.0 0.0 1.0 0 100 200 300 400 500 600 0 500 1000 1500 Time (min) Time (min) 0 100 200 300 400 Mouse PSM Human PSM f Original Original g

Mouse Cont Mouse DAPT Human Cont Human DAPT

0.0 0.6 1.2 0.0 0.6 1.2 p = 0.44 0 100 200 300 400 500 600 0 500 1000 1500 p = 0.999

Detrend Detrend Period (min) 0 100 200 300 400 0 100 200 300 400

HES7 reporter activity Cont DAPT Cont DAPT -1.0 0.0 1.0 -1.0 0.0 1.0 Mouse PSM Human PSM 0 100 200 300 400 500 600 0 500 1000 1500 Time (min) Time (min)

p = 1.2E-08 h Mouse single PSM cell HES7 reporter activity 0 min 280 min Luc i

Ph

300 min 580 min

Luc Period (min)

Ph 0 100 200 300 400 Mouse Human single cell single cell

Fig. 1 bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Mouse ESCs Mouse ESCs hPER1 hHES7 hHES7 hHES7 a Hetero Homo Hetero Homo Human 3’ UTR Promoter + 5’ UTR CDS 3’ UTR Wt swap swap Wt swap swap genome (BAC) b 20 kb 23.1 kb CRISPR/Cas9 mediated homologous recombination Mouse ESC genome mPER1 mHES7 mHES7 mHES7 9.4 kb 14 kb 3’ UTR Promoter + 5’ UTR CDS 3’ UTR Mouse HES7 probe Human HES7 probe Mouse PSM

Wt Hetero swap Homo swap p = 0.14 c d p = 0.0034 Period (min) 0 100 200 300 400

-1.0 0.0 1.0 Hetero Homo HES7 reporter activity Wt (Wt) 0 100 200 300 400 500 600 swap swap Time (min) Mouse PSM Human PSM

f Mouse embryo h e Wt mouse Homo swap mouse Wt Homo swap p = 0.035 Period (min) 0 100 200 300 400 Wt Homo swap g Mouse embryo Wt embryo Homo swap embryo

Wt: Hetero swap: Homo swap: Normal (n=5) Normal (n=10) Kinked short tail and/or Kinked tail (n=1) curved spine (n=3) -1.0 0.0 1.0 HES7 reporter activity 0 100 200 300 400 500 600 Time (min)

Endogenous HES7 i Knock-out Exogenous mouse HES7 Endogenous HES7 Rescue Endogenous HES7 Exogenous human HES7

Normalized j HES7 KO in mouse PSM k l to max value p = 0.23 KO Rescue mHES7 Rescue hHES7 0.0 0.6 1.2 0 100 200 300 400 500 600

Detrend Period (min) Ex1 Ex2 Ex3 Mean 0.0 0.6 1.2 0 100 200 300 400 0 100 200 300 400 500 600 HES7 reporter activity Time (min) HES7 reporter activity Rescue Rescue

-1.0 0.0 1.0 mHES7 hHES7 0 100 200 300 400 500 600 Time (min) Mouse PSM o m HES7 KO in human PSM n

KO Rescue mHES7 Rescue hHES7 0.0 0.6 1.2 p = 0.50 0 500 1000 1500 Detrend

Ex1 Ex2 Ex3 Mean Period (min) 0.0 0.6 1.2 0 100 200 300 400

0 500 1000 1500 HES7 reporter activity HES7 reporter activity Time (min) Rescue Rescue

-1.0 0.0 1.0 mHES7 hHES7 0 500 1000 1500 Fig. 2 Time (min) Human PSM bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made δm δp τ Delay available under aCC-BYrTetOne 4.0 International license. , a : b Dox HES7-NLuc φ c δ Degradation : (w/o intron) HES7 p = 8.4E-04 HES7 Mouse HES7 in mouse PSM (Fig. 3f) Human HES7 in mouse PSM τTx Mouse HES7 in human PSM (min) Human HES7 in human PSM p τIn (3i) δm (Fig. 3f) δ mRNA φ 1/

Tl (3f)

τ 0 20 40 60 80 δp (3c) Protein HES7-NLuc activity Mouse HES7 Human HES7 τRp (3j) φ in mouse PSM in human PSM 0.0 0.2 0.4 0.6 0.8 1.0 0 50 100 150 200 250 300 Time (min) f p = 5.4E-04 (min) Mouse PSM Human PSM

d Tx + Tl Tl τ τ e Fitting Fitting

TetOne τ Dox HES7-NLuc Protein +

(w/o intron) Tx τ 0 10 20 30 40 0.0 0.6 1.2 0.0 0.6 1.2 0 50 100 150 0 100 200 300 Mouse PSM Ex1 p = 1.3E-05 Mouse PSM Ex2 Mouse PSM Ex3 Mouse PSM Mean HES7-NLuc activity

Human PSM Ex1 (min) m

Human PSM Ex2 δ HES7-NLuc activity Human PSM Ex3 Human PSM Mean 1/ 0.0 0.6 1.2 0.0 0.6 1.2 0.0 0.2 0.4 0.6 0.8 1.0 0 50 100 150 0 100 200 300 0 50 100 150 200 Time (min) 0 10 20 30 Time (min) Mouse PSM Human PSM

Mouse PSM Human PSM g Nluc, w/o intron Nluc, w/o intron Nluc, w/o intron Nluc, w/o intron h Fluc, w/ intron Fluc, w/o intron Fluc, w/ intron Fluc, w/o intron HES7 NLuc-stop-HES7 (w/o intron) HES7 FLuc-stop-HES7 (w/ intron) HES7 FLuc-stop-HES7 (w/o intron) -1.0 0.0 1.0 -1.0 0.0 1.0 -1.0 0.0 1.0 -1.0 0.0 1.0 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 i 3 - 4 2 4 1 3 (min)

In 1 - 2 τ Cross correlation Reporter activity -1.0 0.0 1.0 -1.0 0.0 1.0 -1.0 0.0 1.0 -1.0 0.0 1.0

0 10 20 30 40p = 50 5.8E-04 0 50 100 150 0 50 100 150 0 50 100 150 0 50 100 150 Time (min) Time (min) Mouse PSM Human PSM

TetOne j Dox HES7-NLuc k Human simulation l τRp HES7 Mouse simulation 400 HES7-NLuc FLuc 1 Human simulation 0.8 300

0.6 200 0.4 Period (min) HES7 protein 100 0.2 Mouse simulation Mouse PSM Human PSM Fluc activity Fitting (τRp = 0) Fitting (τRp = 0) 0 0 0 500 1000 1500 1.5 2 2.5 3 3.5 4 0.0 0.6 1.2 0.0 0.6 1.2 0 100 200 300 0 100 200 300 400 Time (min) Hill coefficient, n Time (min)

Fig. 3 bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. rTetOne δp a GBX2 MSGN1 TBX6 b Dox GOI-NLuc φ (w/o intron)

0.0090 Mouse PSM c 0.096 0.084 0.0 0.6 1.2 0.0 0.6 1.2 0.0 0.6 1.2 Human PSM 0.0053 0 200 400 0 200 400 0 200 400

NLuc activity CDX2 EVX1 T p = 4.4E-04 3.0E-04 (min) p δ 1/ 0.0 0.6 1.2 0.0 0.6 1.2 0.0 0.6 1.2 0 200 400 0 200 400 0 200 400 Time (min) 0 100 200 300 400 Mouse PSM Ex1 Human PSM Ex1 (HES7) GBX2 MSGN1 TBX6 CDX2 EVX1 T Mouse PSM Ex2 Human PSM Ex2 Mouse PSM Ex3 Human PSM Ex3 Mouse PSM Mean Human PSM Mean + + TetOne τTx τIn τTl eDox Protein HES7 w/ intron TBX6 GBX2 FLuc-stop-GOI (w/ intron) d p = 0.0022 0.0035 f Mouse PSM Human PSM 0.0 0.6 1.2 0.0 0.6 1.2 0.0 0.6 1.2 (min) 0 100 200 300 0 100 200 300 0 100 200 300 Tl 0.025 MSGN1 EVX1 τ FLuc activity + 0.065 In

τ 0.0068 + Tx τ 0.0 0.6 1.2 0.0 0.6 1.2

0 100 200 300 0 100 200 300 0 10 20 30 40 50 60 Time (min) HES7 TBX6GBX2 MSGN1 EVX1 w/ intron

0.034 OTX2 FOXG1 Mouse NPCs g Mouse ESCs Human iPSCs h i Human NPCs 0.018 Differentiation p = 0.0011 (min) p 0.0 0.6 1.2 0.0 0.6 1.2

δ 0.029 Neural progenitor cells (NPCs) 0 200 400 0 200 400 Mouse Human 1/ PAX6 SOX1 NLuc activity 0 100 200 300 400 500 Phase OTX2 FOXG1PAX6 SOX1 0.0 0.6 1.2 0.0 0.6 1.2 p = 0 200 400 0 200 400 k 1.3E-05 Time (min) Mouse NPCs Mouse NPCs Ex1 Human NPCs Ex1 Human NPCs (min)

Mouse NPCs Ex2 Human NPCs Ex2 Tl

Mouse NPCs Ex3 Human NPCs Ex3 τ

PAX6 Mouse NPCs Mean Human NPCs Mean + 0.012

In 0.0045

τ 0.0081 HES7 w/ intron OTX2 + Tx

j τ 0 10 20 30 40 50 HES7 OTX2 FOXG1 SOX1 Merge w/ intron 0.0 0.6 1.2 0.0 0.6 1.2 0 100 200 300 0 100 200 300 FOXG1 SOX1 FLuc activity l Species-specific cellular environments

Mouse Human cell cell

0.0 0.6 1.2 0.0 0.6 1.2 Fast reactions Slow reactions 0 100 200 300 0 100 200 300 Time (min)

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a Intercelllar communication through NOTCH / WNT / FGF signaling

HES7 HES7

HES7 HES7

Oscillation Oscillation

b Mouse PSM Human PSM Cont Ex1 Ex2 Ex3 Cont Ex1 Ex2 Ex3 DAPT Ex1 Ex2 Ex3 DAPT Ex1 Ex2 Ex3

Original Original 0.0 0.6 1.2 0.0 0.6 1.2 0 100 200 300 400 500 600 0 500 1000 1500

Detrend Detrend Hes7 reporter activity Hes7 reporter activity -1.0 0.0 1.0

0 100 200 300 400 500 600 -1.0 0.0 1.0 0 500 1000 1500 Time (min) Time (min)

Supplementary Fig. 1 bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made a Human single PSM cell availableHES7 under reporter aCC-BY 4.0activity International license. 0 min 280 min Luc

Ph

300 min 580 min Luc

Ph

600 min 880 min Luc

Ph

b Mouse single PSM cell

Cell #1 Cell #2 Cell #3 Cell #4 -1.0 0.0 1.0 -1.0 0.0 1.0 -1.0 0.0 1.0 -1.0 0.0 1.0 0 200 400 600 800 0 200 400 600 800 0 200 400 600 800 0 200 400 600 800 HES7 reporter activity

Cell #5 Cell #6 Cell #7 Cell #8 -1.0 0.0 1.0 -1.0 0.0 1.0 -1.0 0.0 1.0 -1.0 0.0 1.0 0 200 400 600 800 0 200 400 600 800 0 200 400 600 800 0 200 400 600 800 Time (min)

c Human single PSM cell -1.0 0.0 1.0 -1.0 0.0 1.0 -1.0 0.0 1.0 Cell #1 Cell #2 Cell #3 -1.0 0.0 1.0 Cell #4 0 500 1000 1500 0 500 1000 1500 0 500 1000 1500 0 500 1000 1500 HES7 reporter activity -1.0 0.0 1.0 -1.0 0.0 1.0 -1.0 0.0 1.0 -1.0 0.0 1.0 Cell #5 Cell #6 Cell #7 Cell #8 0 500 1000 1500 0 500 1000 1500 0 500 1000 1500 0 500 1000 1500 Time (min)

Supplementary Fig. 2 bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

a Mouse ESCs (EB5) for PSM induction b Mouse ESCs (TT2) for knock-in mice

WT Hetero Homo WT Hetero Homo WT Hetero Hetero WT Hetero Hetero EB5 swap swap EB5 swap swap TT2 swap swap TT2 swap swap 1 7 1 7

23.1 kb

23.1 kb

9.4 kb

9.4 kb 6.6 kb

6.6 kb

4.4 kb

4.4 kb

2.3 kb 2.0 kb 2.3 kb

Mouse HES7 probe Human HES7 probe Mouse HES7 probe Human HES7 probe (15.3 kb) (9.6 kb) (15.3 kb) (9.6 kb)

Supplementary Fig. 3 bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. a Wt Ex1 Ex2 Ex3 Hetero swap Ex1 Ex2 Ex3 Homo swap Ex1 Ex2 Ex3

-1.0 0.0 1.0 Mouse PSM HES7 reporter activity 0 100 200 300 400 500 600 Time (min)

Mouse embryo Wt embryo Homo swap embryo b Wt Homo swap

Ex2 -1.0 0.0 1.0

HES7 reporter activity 0 100 200 300 400 500 600

Wt Homo swap

Ex3 -1.0 0.0 1.0

HES7 reporter activity 0 100 200 300 400 500 600 Time (min)

c Rescued with mHES7 construct Ex1 Ex2 Ex3 Rescued with hHES7 construct Ex1 Ex2 Ex3 Mouse HES7-KO PSM Human HES7-KO PSM 0.0 0.5 1.0 1.5 0.0 0.6 1.2 0 100 200 300 400 500 600 0 500 1000 1500 HES7 reporter activity -1.0 0.0 1.0 -1.0 0.0 1.0 0 100 200 300 400 500 600 0 500 1000 1500 Time (min) Time (min)

Supplementary Fig. 4 bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

a Mice derived from ESC clone 1 b Mice derived from ESC clone 7

Wt Hetero swap Homo swap Wt Hetero swap Homo swap

Wt: Hetero swap: Homo swap: Wt: Hetero swap: Homo swap: Normal (n=3) Normal (n=5) Kinked short tail Normal (n=2) Normal (n=5) Kinked short tail Kinked tail (n=1) (n=1) and/or curved spine (n=2)

Supplementary Fig. 5 bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

a Ex1 Ex2 Ex3 Data Fitting Mouse HES7 in mouse PSM Slope = -0.032 -0.034 -0.034 -5 -4 -3 -2 -1 0 -5 -4 -3 -2 -1 0 -5 -4 -3 -2 -1 0 0 100 200 300 0 100 200 300 0 100 200 300

Data

Log(HES7-NLuc activity) Fitting Human HES7 in human PSM Slope = -0.019 -0.016 -0.017 -5 -4 -3 -2 -1 0 -5 -4 -3 -2 -1 0 -5 -4 -3 -2 -1 0 0 100 200 300 0 100 200 300 0 100 200 300 Time (min)

b Mouse PSM Human PSM Ex2 Ex3 Ex2 Ex3

Data Data Fitting Fitting 0.0 0.6 1.2 0.0 0.6 1.2 0.0 0.6 1.2 0.0 0.6 1.2 HES7-NLuc activity 0 50 100 150 0 50 100 150 0 100 200 300 0 100 200 300

Time (min) Time (min)

Supplementary Fig. 6 bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Mouse PSM Human PSM

Nluc, w/o intron Nluc, w/o intron Nluc, w/o intron Nluc, w/o intron Fluc, w/ intron Fluc, w/o intron Fluc, w/ intron Fluc, w/o intron Reporter acrivity -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400

xcor = 25 xcor = 10 xcor = 40 xcor = 5 Experiment_1 Cross correlation -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 0 50 100 150 0 50 100 150 0 50 100 150 0 50 100 150 Time (min) Time (min) Reporter acrivity -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400

xcor = 25 xcor = 10 xcor = 45 xcor = 10 Experiment_2 Cross correlation -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 0 50 100 150 0 50 100 150 0 50 100 150 0 50 100 150 Reporter acrivity -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400

xcor = 20 xcor = 10 xcor = 45 xcor = 5 Experiment_3 Cross correlation -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0 0 50 100 150 0 50 100 150 0 50 100 150 0 50 100 150

Supplementary Fig. 7 bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Mouse PSM Human PSM a Ex1 Ex2 Ex3 Ex1 Ex2 Ex3 1.2 1.2 0.6 0.6 Fluc activity 0.0 0.0

0 100 200 300 0 100 200 300 400 Time (min)

rTetOne δf, δF b Dox FLuc φ

1.2 Mouse PSM 1.2 Human PSM Fitting Fitting 0.6 0.6 Fluc activity 0.0 0.0

0 100 200 300 0 100 200 300 Time (min)

+ TetOne τTxf τTlF Dox FLuc Protein 1.2 1.2 0.6 0.6 Fluc activity 0.0 0.0

0 100 200 300 0 100 200 300 Time (min)

Supplementary Fig. 8 bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Mouse PSM available under aCC-BY 4.0 International licenseHuman. PSM Fitting Fitting

Mouse Ex1 Mouse Ex2 Mouse Ex3 Human Ex1 Human Ex2 Human Ex3

Slope = -0.0080 -0.0073 -0.0077 Slope = -0.0049 -0.0054 -0.0050 0.0 0.0 0.0 0.0 0.0 0.0

GBX2 Log(NLuc activity) Log(NLuc activity) -2.0 -2.0 -2.0 -2.0 -2.0 -2.0 0 400 0 400 0 400 0 400 0 400 0 400 Time (min) Time (min) -0.0066 -0.0066 -0.0071 -0.0048 -0.0049 -0.0050 0.0 0.0 0.0 0.0 0.0 0.0

MSGN1 -2.0 -2.0 -2.0 -2.0 -2.0 -2.0 0 400 0 400 0 400 0 400 0 400 0 400

-0.0043 -0.0043 -0.0040 -0.0033 -0.0034 -0.0036 0.0 0.0 0.0 0.0 0.0 0.0

TBX6 -2.0 -2.0 -2.0 -2.0 -2.0 -2.0 0 400 0 400 0 400 0 400 0 400 0 400

-0.0036 -0.0037 -0.0036 -0.0031 -0.0034 -0.0035 0.0 0.0 0.0 0.0 0.0 0.0

CDX2 -2.0 -2.0 -2.0 -2.0 -2.0 -2.0 0 400 0 400 0 400 0 400 0 400 0 400

-0.0033 -0.0032 -0.0035 -0.0031 -0.0031 -0.0032 0.0 0.0 0.0 0.0 0.0 0.0

EVX1 -2.0 -2.0 -2.0 -2.0 -2.0 -2.0 0 400 0 400 0 400 0 400 0 400 0 400

-0.0027 -0.0029 -0.0028 -0.0032 -0.0031 -0.0031 0.0 0.0 0.0 0.0 0.0 0.0

T -2.0 -2.0 -2.0 -2.0 -2.0 -2.0 0 400 0 400 0 400 0 400 0 400 0 400

Supplementary Fig. 9 bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Mouse PSM Human PSM Fitting Fitting

Mouse Ex1 Mouse Ex2 Mouse Ex3 Human Ex1 Human Ex2 Human Ex3 1.0 1.0 1.0 1.0 1.0 1.0

HES7 w/ intron FLuc activity FLuc activity 0.0 0.0 0.0 0.0 0.0 0.0 0 200 0 200 0 200 0 300 0 300 0 300 Time (min) Time (min) 1.0 1.0 1.0 1.0 1.0 1.0

TBX6 0.0 0.0 0.0 0.0 0.0 0.0 0 200 0 200 0 200 0 300 0 300 0 300 1.0 1.0 1.0 1.0 1.0 1.0

GBX2 0.0 0.0 0.0 0.0 0.0 0.0 0 200 0 200 0 200 0 300 0 300 0 300 1.0 1.0 1.0 1.0 1.0 1.0

MSGN1 0.0 0.0 0.0 0.0 0.0 0.0 0 200 0 200 0 200 0 300 0 300 0 300 1.0 1.0 1.0 1.0 1.0 1.0

EVX1 0.0 0.0 0.0 0.0 0.0 0.0 0 200 0 200 0 200 0 300 0 300 0 300

Supplementary Fig. 10 bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Mouse NPCs Human NPCs Fitting Fitting

Mouse Ex1 Mouse Ex2 Mouse Ex3 Human Ex1 Human Ex2 Human Ex3

Slope = -0.0088 -0.011 -0.010 Slope = -0.0049 -0.0043 -0.0040 0.0 0.0 0.0 0.0 0.0 0.0 0.0

OTX2 Log(NLuc activity) Log(NLuc activity) -2.0 -2.0 -2.0 -2.0 -2.0 -2.0 0 400 0 400 0 400 0 400 0 400 0 400 Time (min) Time (min)

-0.0066 -0.0090 -0.0081 -0.0060 -0.0058 -0.0052 0.0 0.0 0.0 0.0 0.0 0.0

FOXG1 -2.0 -2.0 -2.0 -2.0 -2.0 -2.0 0 400 0 400 0 400 0 400 0 400 0 400

-0.0050 -0.0067 -0.0059 -0.0034 -0.0031 -0.0042 0.0 0.0 0.0 0.0 0.0 0.0

PAX6 -2.0 -2.0 -2.0 -2.0 -2.0 -2.0 0 400 0 400 0 400 0 400 0 400 0 400

-0.0043 -0.0041 -0.0038 -0.0022 -0.0020 -0.0032 0.0 0.0 0.0 0.0 0.0 0.0

SOX1 -2.0 -2.0 -2.0 -2.0 -2.0 -2.0 0 400 0 400 0 400 0 400 0 400 0 400

Supplementary Fig. 11 bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Mouse NPCs Human NPCs Fitting Fitting

Mouse Ex1 Mouse Ex2 Mouse Ex3 Human Ex1 Human Ex2 Human Ex3 1.0 1.0 1.0 1.0 1.0 1.0

HES7 FLuc activity w/ intron FLuc activity 0.0 0.0 0.0 0.0 0.0 0.0 0 150 0 150 0 150 0 300 0 300 0 300 Time (min) Time (min) 1.0 1.0 1.0 1.0 1.0 1.0

OTX2 0.0 0.0 0.0 0.0 0.0 0.0 0 150 0 150 0 150 0 300 0 300 0 300 1.0 1.0 1.0 1.0 1.0 1.0

FOXG1 0.0 0.0 0.0 0.0 0.0 0.0 0 150 0 150 0 150 0 300 0 300 0 300 1.0 1.0 1.0 1.0 1.0 1.0

SOX1 0.0 0.0 0.0 0.0 0.0 0.0 0 150 0 150 0 150 0 300 0 300 0 300

Supplementary Fig. 12 bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Supplementary Text 1: Parameter dependency of simulated oscillation periods

The mathematical model of the HES7 system that we use to describe the behavior of the segmen- tation clock is [1]: dm β = n − δmm (1) dt  p(t−τm)  1 + K dp = αm(t − τ ) − δ p , (2) dt p p where m and p are the concentrations of HES7 mRNA and protein, respectively, α and β are the translation and transcription rates, K is the repression threshold, n is the repression Hill

coefficient, δm and δp are the degradation rates of the mRNA and protein, which we have measured

experimentally as explained in the main text. The mRNA delay τm is composed by the repression

delay τRp, the transcription delay τTx, and the intron delay τIn, all of which we have measured

experimentally as well. The protein delay τp, in turn, corresponds to the translation delay τTl, which was also quantified using our experimental observations.

This system has a fixed point (m∗, p∗) for which the two derivatives above are zero, which obeys:

β δ δ p∗ = m p (3) p∗
n α 1 + K

The stability of this fixed point can be analyzed by assuming the following temporal response to a small perturbation (a, b):

m(t) = m∗ + a exp(λt) (4) p(t) = p∗ + b exp(λt) (5)

Introducing expressions (4)-(5) into Eqs. (1)-(2), linearizing around a = b = 0, and imposing that a solution of the form (4)-(5) exists with nonzero a and b, leads to the following transcendental characteristic equation for the eigenvalues λ:

n(δ δ )2 p∗n+1 (λ + δ )(λ + δ ) + m p exp(−λ(τ + τ )) = 0 , (6) m p αβ Kn m p

In general the eigenvalues are complex numbers λ = σ + iω. The eigenvalue with highest real part determines the stability of the fixed point, with σ < 0 corresponding to an unstable fixed point, and σ > 0 to a stable one. The corresponding imaginary part establishes the frequency at which the system oscillates towards the fixed point (if σ < 0) or away from it (if σ > 0). In the case of an unstable fixed point with ω 6= 0, the system usually falls on a limit cycle whose period can be expected to be close to 2π/ω. Taking these considerations into account, Eq. (6) shows that the period of the HES7 oscillations does not depend on the mRNA and protein delays separately,

but only on the total delay τm + τp.

1 bioRxiv preprint doi: https://doi.org/10.1101/650648; this version posted May 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

For the parameters that we consider in this paper, p∗  K, as can be seen in Fig. 1, which represents graphically the solution of Eq. (3) as the crossing point between its left-hand side (blue line) and right-hand side (orange line). In the limit p∗  K, Eq. (3) has the following approximate solution: αβKn p∗n+1 = (7) δmδp

Inserting expression (7) into the characteristic equation (6) leads to:

(λ + δm)(λ + δp) + nδmδp exp(−λ(τm + τp)) = 0 , (8) Considering again that the imaginary part of the eigenvalue with the highest real part gives us an estimate of the oscillation period, we can observe from Eq. (8) that the period of the HES7 oscillations does not depend on the values of the translation and transcription rates α and β, nor on the repression threshold K.

mouse human K pst K pst 101 101 l.h.s. l.h.s. 100 100 r.h.s. r.h.s. 10 1 10 1 10 2 10 2 10 3

10 3 10 4 0 200 400 600 800 1000 0 200 400 600 800 1000 p p

Figure 1: Graphical determination of the fixed point of the HES7 model used in this paper, for the parameters corresponding to the mouse (left) and human (right) cells.

Finally, if we focus on the bifurcation point (σ = 0), we can obtain in closed form the period of the oscillations at that point by computing ω. To that end, we write the real and imaginary parts of Eq. (8) for λ = iω and divide one by the other, to reach the following transcendental equation:

ω(δm + δp) tan(ωτ) = 2 (9) ω − δmδp We can thus see that at the bifurcation point, the period of the oscillations does not depend on n, but only on the degradation rates of the mRNA and the protein, and on the total delay. These observations are reproduced by our numerical simulations.

References

[1] J. Lewis, “Autoinhibition with transcriptional delay: a simple mechanism for the zebrafish somitogenesis oscillator,” Current Biology, vol. 13, no. 16, pp. 1398–1408, 2003.

2