Neanderthal-derived variants shape craniofacial enhancer activity at a human disease locus

  1. Kirsty Uttley
  2. Hannah J Jüllig
  3. Carlo De Angelis
  4. Julia MT Auer
  5. Ewa Ozga
  6. Hemant Bengani
  7. Hannah K Long

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Abstract

Facial appearance is one of the most variable morphological traits in humans, influenced by both rare and common genetic variants that can impact facial form between individuals and in disease. Deletion of an enhancer cluster 1.45 megabases upstream of the SOX9 gene (EC1.45) results in Pierre Robin sequence, a human craniofacial disorder characterised by underdevelopment of the lower jaw and frequently associated with cleft palate. We reasoned that single nucleotide variants in EC1.45 may cause more subtle alterations to facial morphology. Here, we took advantage of recent human evolution, and the distinct morphology of the Neanderthal lower jaw, to investigate the impact of three Neanderthal-derived single nucleotide variants on EC1.45 function and jaw development. Utilising a dual enhancer-reporter system in zebrafish, we observed enhanced Neanderthal regulatory activity relative to the human orthologue during a specific developmental window. At this same stage, we show that EC1.45 appears to be selectively active in neural crest- derived progenitor cells which lie in close apposition with and are transcriptionally related to precartilaginous condensations that contribute to craniofacial skeletal development. To examine the potential consequences of increased SOX9 expression in this specific cellular population during jaw development, we overexpressed human SOX9 specifically in EC1.45-active cells and observed an increase in the volume of developing cartilaginous precursors. Taken together, our work implicates Neanderthal-derived variants in increased regulatory activity for a disease- associated enhancer with the potential to impact craniofacial skeletal development and jaw morphology across recent hominin evolution.

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript by Uttley et al., describes the identification of a candidate sequence for enhancing craniofacial sox9 expression in Neanderthals and offers functional genomics evidence towards identification of candidate sequence variants in a cis regulatory element (CRE) responsible for jaw morphology variation in hominin evolution. They generated a transgenic zebrafish model for testing the activity of a previously characterised regulatory element in human, which when mutated causes Pierre Robin developmental disorder and its neanderthal counterpart which has been identified as a candidate enhancer by sequence similarity and by being a DMR in the Neanderthal genome. They show that the Neanderthal CRE is active similarly in distribution to its human counterpart but with elevated activity in anatomically loosely or unspecified cell types in zebrafish cartilaginous neural crest candidates, which they argue are matching the cells where the same enhancer is active in mammalian development. They then show by single cell transcriptomics the cell distribution for the enhancer activity in relation to neural crest subpopulations and trasncription factors involved in craniofacial development. Finally they carry out overexpression of SOX9 with the human enhancer variant in zebrafish and demonstrate morphology changes which they interpret as evidence towards the capacity of the enhancer to broaden mesenchymal condensations leading to change in jaw morphology.
    Taken together, the paper provides evidence for a predicted neanderthal regulatory element candidate to function as enhancer in a zebrafish model and evidence for this enhancer to carry sequence variation which can lead to overactivation in craniofacial cell types relevant to jaw morphology, which the authors interpret as the source of the cis regulatory mechanism for jaw morphology evolution in hominin evolution.

    Main comments:

    I found the conclusion on the functional divergence of sequence variants of Neanderthal v human enhancer convincing as they were provided by an elegant double reporter approach which offers internal control for variant comparison. However, i found the argument about the role of the sequence variant in craniofacial development less convincing

    1. Setting the aims I found the introduction to the topic and the setting of aims somewhat sketchy. It is not clear from the introduction, why the Neanderthal element was chosen for further study and why the SNVs in this one element were worth pursuing in the lack of broader understanding of the potentially complex regulatory element complexity at the Neanderthal Sox9 locus. While it is a very reasonable assumption, that a key CRE found and well characterised in human (by the authors in their seminal paper) is a worthy candidate for functional assessment, without better understanding of the overall locus conservation between human and Neanderthal this element may be one of many functionally redundant elements.
    2. Justification of the fish model in hominin gene regulation

    2.1. For the neanderthal element function to be compared to human in a valuable and informative fashion, one would expect that the host system i.e. the zebrafish is sufficiently conserved by offering a similar developmental context both in terms of gene regulation and in terms of anatomy. From the gene regulation perspective, i would expect that the analysis of the EC1.45 is based on expectation of similar regulatory information content to that in the fish homolog thus one can expect similar TF network activities on them and as a result one an test sequence variation effects relevant to endogenous regulatory interactions both in fish and hominins. However, there is no data shown for the relevance of fish regulatory background as a test system. No information is provided on the fish sox9 locus and its activity, or whether the fish homolog enhancer (or any sox9 enhancer that is expressed in the expected domains of craniofacial lineages and structures) has been identified and how it compares to the hominins. One expects that the hominin enhancers are active in domains of the zebrafish sox9 for the anatomical structures to give relevant readout. I would expect a comparison and match of the EC1.45 activity to ether endogenous sox9 by WISH or (although less accurate) a cross to one of the several sox9 reporter transgenic lines available on ZFIN.

    2.2. There is an argument about the regulatory networks being conserved (without references), this would need more arguments particularly in the context of Sox9/SOX9 regulation.

    1. Further to the justification of the fish model, from the anatomical perspective, the assessment of the parallels of zebrafish and mammalian craniofacial development need strengthening.

    3.1. While indeed transparency and external development helps the reporter transgenesis and argues for the fish model, but the generation time is actually comparable to mouse (in contrast to the statement in the introduction), however the understanding of zebrafish craniofacial development and its similarity to human is not well argued, and indeed very superficially compared in the manuscript. I found the anatomical analyses to be rather imprecise and difficult to compare. In the lack of direct comparisons and diagrams comparing mammalian and fish developmental structures and their origins, the statement of 'EC1.45 activity matches expression domains from mammalian development' or 'broadly recapitulate' to be an oversimplification and overstatement. The lineage tracing is an important evidence but again the anatomical homologies need to be more clearly visualized and the lineage history better explained.

    3.2. In a similar vein, direct comparison of human and Neanderthal adult morphologies (Figure 1B) would be very helpful.

    3.3. I was also confused why the sox10 reporter is used as reference (with no direct overlap of activity to the SOX9 associated EC1.45 reporter) rather than or alongside a sox9a reporter line or even comparison to endogenous sox9a activity by WISH (Figure 2). The anatomical details in Figure 2 would need to be extended with more precisely describing the cell types, where the transgene is active and how the homology to mammalian anatomies are established.

    3.4. Overall, the use of the fluorescence reporter is helpful for initial assessments but accurate enhancer activity profiling and comparison should be done by WISH, as mRNA is far more likely to follow the temporal activation dynamics and may explain fluorescence signal intensity differences, the latter important for correct interpretation of sequence variant effects (e.g. is the perceived higher expression by the Ne element is perhaps due to longer expression or earlier activation).

    1. Single cell transcriptomics This experiment was not only used to characterise transgenic reporter active cell types, but to establish transcription factor candidates relevant to neural crest differentiation regulated by EC1.45. What is somewhat confusing, is that the EC1.45 element activity domain is only partially and not predominantly overlapping with the twist1a expressing cells. The authors previously established Twist1 as key regulator of EC1.45 in craniofacial development. How do the authors explain the apparent little relevance of twist1a in regulating the enhancer in fish? Overall the lack of any attempt to link the SNVs to TFBS (including, if available that of the fish homolog sequences) is making the interpretation of the sequence variation harder. BTW, even of the fish elements are not directly identifiable by direct sequence alignment it may be possible to identify the fish homolog through phylogenetic footprinting with stepping stone species such as the non-duplicated paddlefish.
    2. Sox9 overexpression This experiment seems not to add too much to the main claim of the paper. While not essential, for this data to add more value, a comparison to that using the Neanderthal element would be more interesting and not a difficult experiment to carry out.
    3. Throughout the paper there is a lack of data on reproducibility of reporter activities. As random integration often leads to position effects, it is expected that more than one lines showing the same patterns is used to identify cell type and tissue specificities. This is lacking in the paper and is a concern, as for example, the human element activity in Fig. 1 appears to be different from that by in the dual reporter shown in Fig. 3.

    Minor points

    A request to the editor as much as the authors: please make sure that legends are on the same page with figures, it is very hard to follow manuscripts when one needs to scroll between 3 pages at the same time (text, figure, legend). This archaic separation inherited from decades ago when physical prints used to be submitted has no justification in the digital era but continues to make reviewer's life difficult. Similarly, there should be no limit, and it should be encouraged to label anatomical structures directly on panels to point out expression domains, highlight expression variation, or to make a panel more self-explanatory, while making sure that clarity is not lost.

    Figure 1A does not support the statement it is referenced to

    Figure 1B should include human anatomy in comparison and perhaps a schematic diagram of the hypothesized developmental morphogenesis divergence modelled in this paper

    Figure 1D should show why the authors argue the neanderthal is not the ancestral state (BTW, what does the fish homolog look like?)

    Figure 4A,B are better suited in Supplemental

    Significance

    Conceptual: identifying sequence variants in Neanderthal cis-regulatory element as potential source of evolutionary change in morphology.

    Technologically mostly following prior art, use of single cell in reporter analysis is technologically improvement on current standards, albeit somewhat rudimentary.

    The use of a tractable embryo model to explore a regulatory sequence change leading to morphology change has often been applied for carious aspects of evolutionary changes during development pioneering examples include the shh ZRA enhancer in fin/limb morphogenesis, or balean fin evolution (PMID: 9860988) or human versus ape hand evolution (PMID: 18772437), but this is the first for applying it to hominin evolution. This will be of interest to human geneticists, evolutionary geneticists and developmental geneticists.

    My expertise is in developmental gene regulation with the zebrafish model.

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    Referee #2

    Evidence, reproducibility and clarity

    The authors provide evidence that nucleotide sequence variants in a remote enhancer, E1.45, which is located 1.45 Mb upstream of the Sox9 promoter, probably contributed to subtle morphological differences in the lower jaws of Neanderthals and modern humans. The study employs the use of a cleverly-designed dual reporter gene for directly comparing the activities of the Neanderthal and modern human enhancers in transgenic zebrafish. The results are clear and convincing: the Neanderthal enhancer is significantly more active than the modern human enhancer.

    Here are a few minor recommendations that might help clarify aspects of the study:

    1. Is it possible to quantify the different enhancer activities in the zebrafish assays? Is it strictly a question of levels or are there also subtle differences in the timing and/or sites of expression during development?
    2. Is the Neanderthal form of the E1.45 enhancer ancestral for the hominids? If so, then reduced expression in modern humans is a derived trait. This could be stated more clearly.
    3. Are there potential transcription factor binding motifs associated with the SNVs?

    Significance

    The authors address one of the most compelling problems in biology: the evolutionary origins of modern humans. This study addresses the role of regulatory DNAs in the divergence of Neanderthals and modern humans. Sox9 is a good focus of study since it has been implicated in the development of craniofacial features in humans. The authors identified three SNVs (single nucleotide variants) in Neanderthal vs. modern human E1.45 enhancer sequences. Direct comparison of these enhancers provide compelling evidence that these SNVs cause upregulation of the Sox9 in Neanderthals. I think this is a very interesting finding and strongly endorse publication.

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    Referee #1

    Evidence, reproducibility and clarity

    This is an interesting paper that is logical continuation of authors previous work characterizing a human enhancer mutation implicated in Pierre Robin malformations that alters Sox9 expression. Here using zebrafish as a convenient model organism, the authors test the activity of the human enhancer compared to its Neanderthal ortholog. The results show that both enhancers drive reporter expression in the vicinity of forming cartilage condensations of the jaw. While both enhancers mediate reporter expression in neural crest derived cells, the Neanderthal sequence drives quantitatively higher expression than the orthologous human enhancer. Consistent with this, overexpression of Sox9 using the human enhancer caused an increase in cartilage volume. Altogether, this is a nicely done study that would be appropriate for publication after some revisions as detailed below.

    Major Revisions:

    1. The introduction seems overly long and a bit rambling so diminishes from the excitement of the work. It should be half the length and focus on the novelty of this question and findings.
    2. The authors should demonstrate that that human EC1.45 activity overlaps with Sox9 expression. This should be included in Figure 2.
    3. There are differences in level of enhancer activity signal between figures (e.g. seems lower in Fig. 3 than Fig. 2). Does enhancer activity vary between embryos or was the imaging protocol different?
    4. Some co-staining should be performed to show whether or not the enhancers are active in the same cells but at different levels or if they are actually in different cells.
    5. There is an important issue with the single cell RNA seq. Given that the cells were FACS sorted for +GFP and +Cherry, there seem to be many negative cells in their scRNAseq data. Perhaps the FACS gates (figure 4B) were not conservative enough? Did negative cells get included? Authors should verify that their clusters express both GFP and Cherry transcripts.
    6. From their scRNAseq data, they talk about enhancer activity in PA1, but this isn't discussed/shown in the enhancer reporter embryos. It would be appropriate to annotate PA1 in figures 2 and 3.
    7. Authors should quantify how many Sox9+ cells also have enhancer activity. Looking at the UMAPs in figure 4E and 4F, it actually looks like there is less enhancer activity in the Sox9 dense regions of the clusters.
    8. For the over-expression of Sox9 driven by EC1.45, it is important to first establish that EC1.45 activity does indeed overlap with Sox9 gene expression. Does Sox9 itself drive EC1.45?
    9. Importantly the authors do not discuss if the Neanderthal SNVs lie in TF binding sites? Which TF motifs? Are they conserved? Are those TF's expressed in the same cells as both enhancers?
    10. If you introduce the Neanderthal SNVs into the human sequence, do you gain enhancer activity?
    11. The over-expression experiments are tricky as they cause major developmental defects. Would it be possible to drive Sox9 expression at levels that better reflect those driven endogenously by the human versus Neanderthal enhancer?

    Minor Revisions:

    1. Figure 1 - authors should highlight that panel C is a zoom in of panel A.
    2. Figure 3 - Why does Human EC1.45 activity looks weaker here than it does in Figure 2.
    3. The first sentence of the last paragraph in the Introduction is unclear: "spatiotemporal developmental expression patterns for the human EC1.45 cluster during zebrafish development". Instead should read "reporter expression driven by the human EC1.45 enhancer over developmental time"

    Significance

    This is a nice paper that advances understanding of jaw development and has disease relevance as well as some evolutionary implications. Thus it is novel and would appeal to developmental biologist, the craniofacial community, and to some extent to evolutionary biologists.

  5. Version published to 10.1101/2024.09.24.614243 on bioRxiv