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

Although many molecular mechanisms controlling developmental processes are evolutionarily conserved, the speed at which the embryo develops can vary substantially between species. For example, the same genetic program, 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 program runs more than twice as fast in mouse as in human. This is not due to differences in signaling, nor the genomic sequence of genes or their regulatory elements. Instead, there is 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 and suggests that differences in protein turnover play a role in interspecies differences in developmental tempo. The events of embryonic development take place in a stereotypic sequence and at a characteristic tempo (1, 2). Although the order and underlying molecular mechanisms are often indistinguishable between different species, the timescale and pace at which they progress can differ substantially. For example, compared to their rodent counterparts, neural progenitors in the primate cortex progress more slowly through a temporal sequence of Correspondence to: james.briscoe@crick.ac.uk, teresa.rayon@crick.ac.uk. Author Contributions T.R. and J.B. conceived the project, interpreted the data, and wrote the manuscript with input from all authors. T.R. designed and performed experiments and data analysis. D.S. designed and performed experiments and data analysis. R.P.C. performed theoretical modelling and data analysis. L.G.P. designed experiments and performed data analysis for smFISH. C.B. performed bioinformatic analysis. M.M. performed embryo work, generated and characterized the Ptch1::T2A-mKate2 mouse ES cell line. K.E. performed embryo work. J.L. analysed embryo data. E.M. and V.T. provided reagents and feedback. Competing Interests The authors declare no competing or financial interests. Europe PMC Funders Group Author Manuscript Science. Author manuscript; available in PMC 2021 March 18. Published in final edited form as: Science. ; 369(6510): . doi:10.1126/science.aba7667. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts neuronal subtype production (3, 4). Moreover, the duration of cortical progenitor expansion differs between species of primates, at least partly accounting for differences in brain size (5, 6). Even in more evolutionary conserved regions of the central nervous system (CNS) there are differences in tempo. The specification of neuronal subtype identity in the vertebrate spinal cord involves a well-defined gene regulatory program comprising a series of changes in transcriptional state as cells acquire specific identities as neural progenitors differentiate to post-mitotic neurons (7). The pace of this process differs between species, despite the similarity in the regulatory program and the structural and functional correspondence of the resulting spinal cords. The differentiation of motor neurons (MNs), a prominent neuronal subtype of the spinal cord, takes less than a day in zebrafish, 3-4 days in mouse, but ~2 weeks in human (8, 9). Moreover, differences in developmental tempo are not confined to the CNS. The oscillatory gene expression that regulates the sequential formation of vertebrate body segments – the segmentation clock – has a period that ranges from ~30mins in zebrafish, to 2-3h in mouse, and 5-6h in human (10–12). It is unclear as to what causes the interspecies differences in developmental tempo, termed developmental allochrony. To address this question, we compared the generation of mouse and human MNs. Progenitors of the spinal cord initially express the transcription factors (TFs) Pax6 and Irx3 (13). Exposure to Sonic Hedgehog (Shh), emanating from the underlying notochord, results in ventrally located progenitors inducing Nkx6.1 and Olig2. This downregulates Pax6 and Irx3 (14). Progenitors expressing Olig2 and Nkx6.1 are termed pMNs and these either differentiate into post-mitotic MNs, which express a set of TFs including Hb9/Mnx1 and Isl1, or transition into p3 progenitors that express Nkx2.2 (15). This gene regulatory network (GRN), in which Olig2 represses Irx3 and Pax6 and promotes the differentiation of MNs, is conserved across vertebrates (16). We used in vitro differentiation of MNs from mouse and human embryonic stem cells (ESCs) to investigate the pace of differentiation. We find that MN differentiation in vitro recapitulates species-specific global timescales observed in the embryos, lasting ~3 days in mouse and more than a week in human. We show that increased levels of signalling are unable to speed up the rate of differentiation of human cells. Moreover, by assaying the expression of a human gene, with its regulatory landscape, in a mouse context, we rule out the possibility that species differences in genomic sequence plays a major role in temporal scaling. Finally, we show that differences in protein degradation can explain the differences in developmental tempo. Results and Discussion The characteristic spatial-temporal changes in gene expression and the regulatory interactions between the genes responsible for neural tube development are well described (17). Despite the conservation of the GRN across vertebrates, only limited analysis has been performed on the relevant stages of human development (18, 19). We performed immunostainings on mouse and human embryonic spinal cords at brachial levels at equivalent stages (20) to more accurately correlate the major developmental events of neural differentiation processes in vivo between mouse and human (Fig. 1A). The dorsoventral (DV) length of the neural tube increases at the same rate in mouse and human (Fig S1A), Rayon et al. Page 2 Science. Author manuscript; available in PMC 2021 March 18. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts and the shifts in gene expression are similar between mouse and human (Fig S1D). At their maximum extents, the OLIG2-expressing pMN domains comprise a large proportion of ventral progenitors, occupying approximately 30% of the DV length of the neural tube in mouse and a ~15% larger domain in human embryos (Fig. 1B, S1E). Consistent with this, there were more MN progenitors (pMN) in human but similar numbers of interneuron progenitors in mouse and human (Fig S1F). Over the following two days of mouse development, from E9.5 to E11.5, many post-mitotic MNs differentiate (Fig. 1C) resulting in a marked reduction in the size of the pMN domain (Fig. 1B), despite the continued proliferation of the progenitors (9). The proportion of neurons is higher in human compared to mouse (Fig S1B). By contrast, the pace of development is noticeably slower in human embryos. At Carnegie Stage (CS) 11 the pMN occupies a large proportion of the human neural tube, similar to the pMN in E9.0 mouse embryos. During the following 1-2 weeks of development (CS13-19, Fig. 1B), the size of the pMN decreases as MNs accumulate (Fig. 1C), but the rate of this change is slower than seen in mouse. MN production decreases at ~E11.5 in mouse whereas MN production continues to at least CS17 in human (Fig S1C), and glial progenitors, co-expressing SOX9 and NFIA, begin to arise in both species at these stages (Fig. 1D). Together, the data indicate an equivalent progression in neural tube development of mouse and human that lasts around 3 days in mouse and over a week in human (Fig. 1A). We examined whether interspecies tempo differences were preserved in vitro. Methods for the differentiation of MNs from ESCs, which mimic in vivo developmental mechanisms, have been established for both mouse and human (21–24). To ensure comparison of similar axial levels in both species, we initially exposed mouse ESCs to a 20h pulse of WNT signalling, and human ESCs to a 72h pulse (21, 25). This generated cells with a posterior epiblast identity – so called neuromesodermal progenitors – that express a suite of genes including T/TBXT, SOX2 and CDX2 (21, 26) (Fig. S2A). These were then exposed to 100nM of Retinoic Acid (RA), which acts as a neuralizing signal, and to 500nM Smoothened agonist (SAG) that ventralises neural progenitors (27) (Fig. 2A,B). For both mouse and human, this resulted in the efficient generation of pMN expressing OLIG2 (Fig. 2C,D, S2B,C), and MNs expressing ISLET1 (ISL1), HB9/MNX1 and neuronal class III beta-tubulin (TUBB3) (Fig. 2E,F). Progenitors that had not differentiated into neurons switched from OLIG2 expressing pMN to p3 progenitors expressing NKX2.2 (Fig. 2C,D). Mouse and human MNs expressed HOXC6, characteristic of forelimb level spinal cord MNs (28) (Fig. 2F), indicating pMN and MNs with similar axial levels were being produced in both cases. Comparison of the two species revealed the same sequence of gene expression changes: expression of Pax6 in newly induced neural progenitors, followed by the expression of the pMN marker Olig2, which precedes the induction of post-mitotic MN markers, including Isl1 (Fig. 2C-G, S2B). But the rate of progression differed. Immunofluorescence and RTqPCR assays for specific components of the GRN indicated that, after the addition of RA and SAG, the onset of ISL1 expression took 2-3 days in mouse, but ~6 days in human (Fig. 2E-G,K), consistent with the slower developmental progression in the developing human embryonic spinal cord. Moreover, Olig2 induction peaked after 2-3 days in mouse and 6-8 days in human (Fig. 2G, S2B). Differences in tempo have also been observed between the Rayon et al. Page 3 Science. Author manuscript; available in PMC 2021 March 18. E uope PM C Fuders A uhor M ancripts E uope PM C Fuders A uhor M ancripts differentiation of mouse and human pluripote