Species-specific developmental timing is associated with global differences in protein stability in mouse and human

What determines the pace of embryonic development? Although many molecular mechanisms controlling developmental processes are evolutionarily conserved, the speed at which these operate can vary substantially between species. For example, the same genetic programme, comprising sequential changes in transcriptional states, governs the differentiation of motor neurons in mouse and human, but the tempo at which it operates differs between species. Using in vitro directed differentiation of embryonic stem cells to motor neurons, we show that the programme runs twice as fast in mouse as in human. We provide evidence that this is neither due to differences in signalling, nor the genomic sequence of genes or their regulatory elements. Instead, we find an approximately two-fold increase in protein stability and cell cycle duration in human cells compared to mouse. This can account for the slower pace of human development, indicating that global differences in key kinetic parameters play a major role in interspecies differences in developmental tempo.


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The events of embryonic development take place in a stereotypic sequence and at a characteristic tempo (1,2).
3 peaked at day 2 in mouse but not until day 6-8 in human (Fig. 2G, S1B). Differences in tempo have also been observed 93 between the differentiation of mouse and human pluripotent stem cells (28). To test whether the difference in 94 tempo of mouse and human MN differentiation represented a global change in the rate of developmental 95 progression we performed bulk transcriptomics. This revealed a similar pattern of gene expression changes in mouse 96 and human but the changes in mouse cells preceded their human orthologs (Fig. 2H). Cross-species comparison 97 showed a high degree of correlation although altered in time between mouse and human (Fig. 2I, S1D). Moreover, 98 the relative difference in developmental tempo appears constant throughout the differentiation process suggesting 99 a global temporal scaling -developmental allochrony -between mouse and human. 4 region and/or cis-regulatory elements might determine the tempo of development. To study sequence differences 142 between species, we focused our attention on Olig2; it is the major regulator of pMN and regulatory elements for 143 Olig2 have been characterized (34,35). We reasoned that if sequence differences were responsible for the different 144 temporal dynamics in mouse and human cells, we would be able to detect species-specific changes in the timing of 145 Olig2 expression if we introduced the human Olig2 locus into mouse cells. The human Olig2 gene is located on 146 chromosome 21 and we took advantage of the 47-1 mouse ESC line that contains the Hsa21q arm of human 147 chromosome 21 (36). We differentiated 47-1 (hereafter referred to as hChr21) alongside its parental line, which 148 lacked Hsa21q, from which it was generated (hereafter referred to as wt). The proportions of neural progenitors and 149 the dynamics of gene expression, measured by RNA expression, immunofluorescence and flow cytometry, were 150 similar between hChr21 and wt lines (Fig. 4A,B,S3A,B). We then assessed the timing of expression of the hOLIG2 151 allele. We detected induction of hOLIG2 at day 1 of differentiation ( Fig. 4C), 24h after addition of RA SAG. By contrast 152 in human cells, hOLIG2 induction is not induced until day 2-3 ( Fig. 2G). Thus, in mouse cells, hOLIG2 follows the same 153 dynamics of gene expression as mouse Olig2 (mOlig2), indicating that the temporal control of gene expression 154 depends on the cellular environment and not the species origin of the genomic sequence.

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To compare Olig2 expression levels between the mouse and human alleles, we performed single-molecule 156 Fluorescent In Situ Hybridization (smFISH) (Fig. 4D, S3C). We first assayed transcripts of Sox2 (mSox2), a transcription 157 factor expressed in all neural progenitors. The mean and variance in mSox2 transcripts were similar in both hChr21 158 and wt neural progenitors, supporting the comparability of the two cell lines (Fig. 4E). We then measured Olig2 159 transcripts using species specific probes. The number of mouse Olig2 (mOlig2) transcripts in hChr21 cells was lower 160 than in wt cells, but the mean total number of Olig2 transcripts in hChr21 cells, combining mouse and human alleles, 161 was higher than the mean number of transcripts in wt cells (Fig. 4E). This suggests that the number of transcripts 162 that cells express depends on the number of the alleles. 163 We next asked whether the levels of specific mRNAs were similar in human cells to those in mouse. To this end, we 164 performed smFISH in human neural progenitors for SOX2 (hSOX2) and OLIG2 (hOLIG2) (Fig. S3D, 4F). The median 165 number of hOLIG2 molecules in human cells at days 4, 6 and 8 was similar, indicating that the number of transcripts 166 is constant in cells (Fig. S3F). The number of Sox2 and Olig2 transcripts in human neural progenitors were higher 167 than in mouse (Fig. S3E). However, human neural progenitors were larger than mouse progenitors (data not shown) 168 and taking this into account allowed calculation of the concentration of mRNAs (RNAs/µm 2 ) in human and mouse 169 cells (Fig. 4G). Strikingly, the median concentration of hOLIG2 in mouse hChr21 cells was more similar to the

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Overall, we conclude that gene regulation in mouse cells follow mouse-specific characteristics, irrespective of the 173 species origin of the allele, suggesting that the species differences in gene expression dynamics are not encoded 174 within the regulatory genome of individual genes. nascent proteins replacing methionine in the medium with the methionine analog L-azidohomoalanine (AHA), and 191 used FACS to measure the stability of newly synthesized proteins upon removal of the amino acid analog over the 192 course of 48h (Fig S4C,D). We found that the half-life of the proteome in mouse neural progenitors was shorter than 193 in human progenitors (t1/2 = 8 ± 1.6 h in mouse versus t1/2=20.5 ± 5.2h in human day 4 or t1/2=18.5 ± 2.4 in human 194 day 8), an approximate 2.5 fold difference (Fig. 5C,D). This identifies a global difference in the protein lifetime 195 between mouse and human that corresponds to the difference in tempo.

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To test whether changes in protein stability could account for differences in developmental tempo, we took 197 advantage of a mathematical model of the GRN, which we had previously developed (38) (Fig. 5E). Doubling the 198 stability of the TFs to mimic human kinetics, resulted in a slower dynamic of the network with the same sequence of 199 gene expression, comparable to that observed experimentally (Fig. 5F). To explore further the connection between 200 changes in protein stability and GRN dynamics we measured the change in time of the onset of Olig2 as a function 201 of degradation rate. This revealed a superlinear relationship in which an increase in protein stability slows GRN 202 dynamics by slightly more than the fold increase in degradation rate (Fig. 5G, S4F). This confirms that an increase in 203 protein stability can explain the tempo changes in MN differentiation between mouse and human. In addition, the 204 analysis revealed that large changes in protein stability can lead to different sequences of gene expression, predicting 205 limits to the allochronies compatible with the GRN (Fig. S4F).

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A prediction that arises from this analysis is that the TFs comprising the GRN that regulate MN differentiation should 207 be more stable in human than mouse neural progenitors, and that a fold increase of protein stability close to 2 would 208 give a scaling factor of ~2.5. To this end, we performed pulse-chase experiments labeling nascent proteins with AHA, 209 conjugated labelled proteins to biotin and then streptavidin agarose beads to purify them This revealed that pan-210 neural proteins SOX1 and SOX2 had longer lifetimes than OLIG2 and NKX6.1 proteins in both species (Fig. 6A,S4G,H).

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The long half-life of mKATE2 raised the possibility that dilution, following cell division, contributed to the measured 226 decay rate (40). Differences in the cell cycle time between mouse and human neural progenitors could therefore 227 contribute to the difference in mKATE2 lifetime. To test this, we assayed total cell cycle length using cumulative EdU 228 labelling of mouse and human neural progenitors (Fig. 6D,E, S5C,D) (41). Cell cycle duration in equivalent staged 229 neural progenitors from mouse and human was 10.8h ± 8.3h compared to 28.4h ± 13.9h, respectively. Thus, similar 230 to the proteome, the cell cycle operates twice as fast in mouse compared to human. Since progress through the cell 231 cycle is controlled by protein degradation (42, 43), the difference in cell cycle rate between mouse and human cells 232 may also be a consequence of a global change in protein stability.

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Taken together, these data indicate that the dynamics of the gene regulatory network associated with the embryonic 234 generation of MNs progresses 2-3 times faster in mouse than in human cells. A similar difference in the tempo of 235 the segmentation clock between mouse and human has also been observed (9, 11). These differences do not appear 236 to arise from a bottleneck caused by a specific rate limiting event in MN generation. Moreover, neither changes in 237 the dynamics of signalling nor variations in genomic regulatory sequences appear to account for the species-specific 238 tempos. Instead, the correlated ~2.5 fold differences in cell cycle length and general protein stability suggest that 6 the temporal scaling in developmental processes results from global differences in key kinetic parameters that 240 broadly affect the tempo of molecular processes. What sets this global tempo remains to be determined but could 241 involve the differences in the rates of pivotal molecular processes such as global changes in proteostasis or 242 differences in the overall metabolic rate of cells. How these affect the pace at which GRNs elaborate and how such 243 variations are assimilated to ensure the development of robust and appropriately proportioned tissues will need to