Integration of ATAC- and RNA-sequencing identifies chromatin and transcriptomic signatures in classical and non-classical zebrafish osteoblasts and indicates mechanisms of entpd5a regulation

Osteogenesis is one of the vital processes taking place in vertebrate embryos. In amniotes, including all mammals, osteoblasts are known to give rise to all cranial and axial bones through endochondral and intramembranous ossification (Mackie et al., 2008; Breeland et al., 2023). While most cranial bones form via intramembranous ossification, mammalian axial bones are formed via a cartilaginous template by osteoblasts derived from the somitic sclerotome. Despite the differences in modes of ossification, all osteoblasts are considered as a uniform cell type, experiencing similar ontogenetic stages which are controlled by similar genetic mechanisms (Amarasekara et al., 2021; Hartmann, 2009; Jensen et al., 2010).

Teleost and amphibian processes of endochondral and intramembranous ossification are conserved with amniotes (Weigele and Franz‐Odendaal, 2016). Although the osteoblasts, which generate bone in amniotes and anamniotes, are considered homologous, relatively recent work in teleost fish identified a bone producing cell type, which has not been described in anamniotes: the notochord sheath cell (NSC) (Grotmol et al., 2005; Lleras Forero et al., 2018). NSCs, also referred to as chordoblasts, form an epithelial layer around the vacuolated cells of the embryonic notochord (Figure 1A) and secrete the molecules making up the acellular, thick notochord sheath layer. NSCs are directly responsible for forming ossified chordacentra, ossified rings that are positioned in a segmental array within the sheath of the notochord. Chordacentra formation precedes the accumulation of sclerotomal osteoblasts around the notochord, which complete formation of the vertebral centra and in a later step also give rise to the dorsal and ventral arches (Fleming et al., 2015; Grotmol et al., 2005; Lleras Forero et al., 2018). Thus, formation of chordacentra is now considered the first stage of spine formation in teleost fish.

Figure 1

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Here, we coin the terms ‘classical’ and ‘non-classical’ osteoblast to describe zebrafish bone formation. Classical osteoblasts, highly homologous to the mammalian osteoblast, express osterix and generate the cranial bones and the late perichordal centra of the spine, while non-classical osteoblasts, with to date no known homologous cell type in amniotes, refer to the osterix- subset of NSCs capable of early chordacentra mineralisation. Zebrafish cranial classical osteoblasts originate from the cranial neural crest, share a common progenitor with cranial chondrocytes, and can be detected as early as 2 dpf using markers such as runx2b, entpd5a, and osterix (Huitema et al., 2012; Li et al., 2009). Axial classical osteoblasts are derived from the somitic sclerotome and contribute to the mineralisation of the finite vertebrae in a process very similar to the one observed in mammals. They are rather late in appearance, and osterix+ cells can only be found along the growing vertebral centra and arches from approximately 17 dpf onwards (Spoorendonk et al., 2008; Figure 1B and C).

Non-classical osteoblasts are derived from notochord progenitor cells (NPCs). Unlike classical osteoblasts, NSCs are distinct in their lack of expression of the transcription factor Osterix, commonly considered as an osteoblast master regulator in vertebrates (Nakashima et al., 2002; Wopat et al., 2018). Rather than osterix, expression of the early zebrafish bone marker ectonucleoside triphosphate diphosphohydrolase 5a (entpd5a) was demonstrated in bone-forming NSCs using BAC transgenic lines (Figure 1D). As early as 4 dpf entpd5a was shown to be expressed in NSCs in a segmental pattern, gradually progressing from anterior to posterior, predicting the precise location of the mineralised chordacentra (Lleras Forero et al., 2018; Wopat et al., 2018). entpd5a+ segments along the notochord are separated by intersegmental regions expressing cartilage markers, such as col9a2 (Figure 1E). entpd5a codes for a secreted hydrolase binding nucleoside di- and triphosphates, generating NMP and inorganic phosphate, a key mineral to initiate the mineralisation process of bone. Indeed, entpd5a mutant zebrafish embryos present with a complete lack of ossification (Huitema et al., 2012). In mouse mutants this phenotype has not been reported, although it was observed that homozygous mutants are smaller than their littermates (Read et al., 2009). Thus, NSCs, despite providing a morphologically uniform population of cells, are critical for initial chordacentrum ossification by expressing entpd5a in a segmental manner along the anterio-posterior axis. How this segmental expression pattern of entpd5a is established is not understood, but it is distinct from the one involved in somitogenesis (Lleras Forero et al., 2018). The role of the BMP and Notch signalling pathways has been highlighted in the segmenting notochord (Peskin et al., 2023; Wopat et al., 2018), but the transcriptional events governing entpd5a expression in classical osteoblasts and in the NSCs remain unknown.

The transcriptomics of mammalian classical osteoblasts have been investigated in some detail in the past decades. Genes such as Runx2, Dlx family factors, and Osterix play significant roles in different stages of osteoblast specification and maturation (Hassan et al., 2004; Hojo et al., 2022; Jensen et al., 2010; Li et al., 2008; Shirakabe et al., 2001). More recently, the assay for transposase-accessible chromatin (ATAC)-seq has been widely used in the field. In addition, an increasing number of studies integrate RNA-seq and ATAC-seq to provide deeper insight into gene regulation in populations of interest (Ackermann et al., 2016; Hao et al., 2022; Hojo et al., 2022; Yang et al., 2021; Yu et al., 2021). Such studies, however, are to date available neither for classical nor for non-classical zebrafish osteoblasts, rendering the molecular fingerprints and chromatin dynamics of zebrafish osteogenic cells to date elusive. Molecularly characterising zebrafish classical osteoblasts (Raman et al., 2024) is of particular interest for the field, as it allows for direct comparison between mammalian and teleost transcriptional regulation of osteogenesis. Specifically, it is crucial to acquire a deeper understanding of teleost osteoblasts in order to answer evolutionary questions regarding the cell type’s origin (Nguyen and Eames, 2020). Moreover, better understanding the mechanisms of regulation in non-classical osteoblasts is likely to highlight either novel ossification mechanisms that have not been appreciated to date or that are unique in teleosts.

In this study, we provide the first comprehensive resource of ATAC-seq and RNA-seq performed on classical and non-classical zebrafish osteoblasts, enabling studies of embryonic osteoblast development and differentiation. Comparisons across those populations and their closely related chondrocyte and intersegmental NSC populations allow a detailed assessment of gene expression profiles and chromatin regulation in different osteogenic cells, based on FAC-sorted cells from live embryos. We furthermore focus on the promoter structure of the early bone marker entpd5a, a gene critical for osteogenesis in zebrafish. We thus not only validate the usefulness and quality of the ATAC-seq data, but also demonstrate the complex mode of entpd5a regulation through identification of enhancers located both upstream of the ATG and within intronic regions, and suggest candidate transcription factors likely to be acting on the identified enhancer elements.

In recent years, research on teleost fish identified the ability of the embryonic notochord to produce bone in a manner independent of classical sclerotome-derived osteoblasts and to initiate the formation of mineralised chordacentra (Fleming et al., 2004; Lleras Forero et al., 2018). The cells responsible for this are non-classical osteoblasts within the notochord sheath, expressing the early bone marker entpd5a but not the classical osteoblast regulator osterix (Lleras Forero et al., 2018; Spoorendonk et al., 2008). Here, we aimed to better understand the genetic regulation and molecular signatures of classical and non-classical osteoblasts, and how those two populations differ from each other and from the closely related cranial chondrocytes and intersegmental NSCs. To this end, we characterised the newly generated transgenic line entpd5a:Gal4FF; UAS:GFP in combination with the previously described R2col2a1a:mCherry to isolate classical and non-classical osteoblasts, as well as cartilage and intersegmental cells from the head and trunk of zebrafish larvae. We employed ATAC-seq to reveal the differentially accessible chromatin domains in entpd5a+ cells of head and trunk populations and performed RNA-seq in equivalent cell populations to understand the gene expression signatures accompanying observed chromatin changes. This first compilation of combined ATAC-seq and RNA-seq data from teleost osteoblasts enables the genome-wide analysis of osteoblasts, chondrocytes, NSCs, and intervertebral disk precursors at both the chromatin and transcriptome level. This comprehensive dataset will be of use to a wide variety of researchers interested in osteogenesis, both within and outside the field of teleost developmental biology. For our immediate purposes, and as a proof of principle study, the data has allowed us to dissect the promoter organisation and to identify likely regulators of entpd5a, a critical gene in bone formation in zebrafish.

The ATAC-seq datasets of both entpd5a+ and entpd5a- cells in the head and trunk reveal the chromatin landscape in these skeletogenic cell populations. Although highly similar due to their respective origin from either cranial neural crest cells or NPCs, differential chromatin accessibility analysis on head and trunk populations revealed putative enhancer regions of interest for bone vs. cartilage generation. Here, we validate our chromatin accessibility datasets and prove the significance of performing such analyses by demonstrating the regulatory importance of open regions detected both upstream of entpd5a and within the gene’s intronic regions, thus highlighting a highly complex promoter organisation. Specifically, we demonstrate that a series of active enhancers responsible for driving expression in classical osteoblasts are located in a short stretch of non-coding sequence within 2.2 kb of the entpd5a ATG. Moreover, introns 1–3 contain regulatory regions required for entpd5a segmental activation in non-classical osteoblasts. Although we attempted to test several of the additional peaks found in non-coding areas surrounding the gene, more work is required to pinpoint the enhancers driving expression in cranial chondrocytes. In conclusion, the entpd5a:Gal4FF line has allowed us to refine the expression pattern of zebrafish entpd5a and to use ATAC-seq to elucidate the structure of its promoter.

We additionally performed RNA-seq in order to better understand the molecular signatures of the different cell populations and the functional significance of the observed chromatin configurations. As expected, our analyses reveal enrichment of bone-specific genes in entpd5a+ populations and, conversely, of cartilage-specific genes in entpd5a- populations. We went on to integrate the ATAC-seq and RNA-seq datasets to increase the predictive power of our analyses, which led to the determination of candidate transcriptional regulators for each cell type of interest. Our results suggest functional roles of Dlx family genes in classical osteoblasts, of Hox family regulators in non-classical osteoblasts, and of Foxa and Sox family members in entpd5a- cells of both the head and the trunk. It thus appears that gene expression is not regulated in the same manner in classical and non-classical osteoblasts, since the identified candidate regulators showed no overlap. In contrast, chondrocytes of the head and intersegmental NSCs demonstrated a surprising amount of overlap in putative regulators governing gene expression specific to their skeletogenic roles.

Identification of putative functions of Dlx family transcription factors in classical osteoblasts is compatible with the long-recognised roles of these factors in upregulating key osteoblast genes during all stages of osteoblast specification and maturation (Hassan et al., 2004; Li et al., 2008; Shirakabe et al., 2001). Significantly, Dlx factors have been implicated in recruiting Osterix, the master regulator of osteoblasts, to osteoblast enhancers in mice (Hojo et al., 2016). Interestingly, it has been shown that distinct Dlx family members become active in different stages of osteoblast development, with chick Dlx3 being able to activate mature osteoblast expression programmes (Li et al., 2008). Consistent with this, our analyses reveal enrichment of dlx3b expression in zebrafish classical osteoblasts. In non-classical osteoblasts, we identified several Hox factors amongst predicted regulators of skeletal fate genes. Hox family genes have long been known for their crucial roles in skeletal patterning (Fromental-Ramain et al., 1996a; Fromental-Ramain et al., 1996b; Wellik and Capecchi, 2003) and in the adult skeleton (Song et al., 2020). In future work, the binding affinity of zebrafish Dlx and Hox factors on the promoters of osteoblast genes such as entpd5a, as well as generating mutants in specific dlx and hox genes, would be appropriate means to obtain further insight into the gene regulatory network governing osteogenesis and axial patterning.

In regards to candidate regulators with putative roles in entpd5a- cells, Foxa2 and Foxa3 are transcription factors with confirmed roles in chondrogenesis (Ionescu et al., 2012). Furthermore, SOX9 is known as the master regulator of chondrocyte differentiation, and competition between SOX9 and FOXA have been shown to aid this process (Tan et al., 2018). Both entpd5a- cell populations investigated here differentially express the zebrafish genes foxa2, foxa3, and sox9a, while the second SOX9 orthologue, sox9b, is enriched in cranial entpd5a- cells. We thus postulate that the mechanism of chondrocyte differentiation is likely conserved between amniotes and zebrafish.

In summary, integration of ATAC-seq and RNA-seq has been valuable in predicting candidate regulators for skeletogenic cells found in teleost fish in a non-biased manner. Although this should not be viewed as a comprehensive list of regulators, it provides guidance in the first steps of elucidating genetic interactions taking place in these cell types, and particularly in the non-classical osteoblasts of the notochord, which have only been recently started to be analysed.

In addition to its use in better understanding the gene regulatory programmes acting in skeletogenic cells, our approach of performing both RNA-seq and ATAC-seq and analysing the data not only individually but also in an integrated manner provides a comprehensive resource that has hitherto not been available. Although osteoblasts of mammals and birds have been the subject of transcriptomic analyses for decades, the vast majority of RNA-seq and ATAC-seq studies have been performed using in vitro-differentiated cells (Hao et al., 2022; Liu et al., 2022; Yu et al., 2021). To our knowledge, only one study thus far used in vivo-derived mouse neonatal skeletal cells to perform both assays (Hojo et al., 2022). Furthermore, RNA-seq and ATAC-seq studies on osteoblasts of teleost fish remain extremely limited. With the exception of three studies: one in which scRNA-seq was performed on the zebrafish adult regenerating fin (Tang et al., 2022), another study where RNA-seq was performed on medaka osteoblasts (Phan et al., 2020), and a recent work on the transcriptomic signatures of classical osteoblasts isolated from zebrafish embryos at 4 dpf (Raman et al., 2024), we are not aware of RNA-seq and ATAC-seq analyses that have been performed on embryonic classical osteoblasts of teleost fish. Thus, our study fills a void and moves teleost osteoblasts on par with the single mouse study (Hojo et al., 2022) that provided both ATAC-seq and RNA-seq data.

Of note, the significance of studying the molecular signatures of osteoblasts in teleost fish has become more pressing in recent years. It has been observed that ‘aquatic’ and ‘amphibian’ osteoblasts and cartilage behave differently to ‘terrestrial’ osteoblasts in terms of gene expression (Nguyen and Eames, 2020). Specifically, the osteoblasts of teleost fish and amphibians, whose characteristics are putatively closer to a more ancestral state of skeletal differentiation compared to amniotes, appear to share gene expression with chondrocytes, a pattern which we also observed in our datasets, while amniote osteoblasts have suppressed chondrocyte markers in the course of evolution. Despite this, the vast majority of NGS studies have been carried out on osteoblasts of amniotes, leading to the establishment of an incomplete picture of the osteoblast (Nguyen and Eames, 2020). In this work, we show that the bone marker entpd5a is expressed in chondrocytes of the developing jaw, in a manner similar to osterix (Hammond and Schulte-Merker, 2009). Furthermore, we show that several other genes conventionally thought of as cartilage-specific, such as ccn6, sox9a, acana, and col9a2, are expressed at low levels in osteoblasts. These discoveries reinforce the arguments regarding the chondrocyte origin of osteoblasts, while also pointing towards cells that might be prone to transdifferentiate into osteoblasts (Hammond and Schulte-Merker, 2009; Thesingh et al., 1991; Zhou et al., 2014).

Moving beyond the classical osteoblasts, we also here provide means to investigate non-classical osteoblasts, a cell type confined to teleosts that has recently been shown to play crucial roles in the initiation of spine development (Grotmol et al., 2003; Lleras Forero et al., 2018; Renn et al., 2013). Very little is known to date about the mechanisms of gene regulation in these non-classical osteoblasts, limited to RNA-seq data that indicate a role for BMP and Notch signalling in patterning the cells of the notochord sheath (Wopat et al., 2018; Peskin et al., 2023). However, the gene regulatory interactions that are required to bring about the segmental expression of entpd5a remain unknown. We here demonstrate and experimentally validate a short piece of promoter sequence to be sufficient in governing entpd5a expression in NSCs, setting the stage for identifying the gene regulatory network that determines transcriptional control of this key factor in axial skeletogenesis.

Finally, this work provides insight into the mechanism of regulation of the early bone marker entpd5a, which we show is regulated in an unexpectedly dynamic manner throughout zebrafish embryonic development. entpd5a is not only of great interest because it acts as a specific osteoblast marker, but also because it is non-dispensable for bone development in zebrafish (Huitema et al., 2012). Importantly, entpd5a expression presents in a segmented manner along the zebrafish embryonic notochord, predetermining the locations of chordacentra mineralisation and future vertebrae position. This segmentation was shown to be independent of the somitic segmentation clock (Lleras Forero et al., 2018), arguing for an independent manner of generating the periodic appearance of entpd5a expression within NSCs. The molecular mechanism guiding this stereotypic pattern remains unclear (Fernández Arancibia et al., 2022).

Here, we show that entpd5a introns 1–3, together with enhancers located upstream of the ATG, drive segmental notochord expression of entpd5a. Furthermore, we used our NGS analyses to predict candidate regulators, including dlx, hox, and runx family genes, putatively binding on the identified enhancer sites to drive entpd5a expression in both classical and non-classical osteoblasts. Importantly, we identified a 37 bp region within 2.2 kb of the ATG, predicted to be able to bind Runx2a or Runx2b, as well as various members of the dlx gene family. runx2 family members are of particular interest to the process of osteogenesis, as mammalian Runx2 is known to play important roles in the regulation of osteoblast genes, both through its action as a pioneer factor, playing a crucial role in the plasticity between chondrocyte and osteoblast fates (Hojo et al., 2022), and through its interaction with co-factors to directly upregulate osteogenic genes (Lee et al., 2010; Nishio et al., 2006; Rashid et al., 2014). Although runx2a and runx2b were not thought to be expressed in mature osteoblasts in teleost fish (Huycke et al., 2012), via RNA-seq data we detect low-level expression of at least one of the two factors in entpd5a+ cells of the head and of the trunk, rendering them plausible entpd5a regulators in classical and non-classical osteoblasts. Our in silico predictions and presented hypotheses are to be validated by functional analyses (such as generating mutants), and this information can be built on to reveal gene regulatory network upstream of entpd5a in both types of zebrafish osteoblasts. Moreover, the role of Entpd5 in bone formation should be determined in other species and the evolutionary conservation of the mechanisms which we begin to identify here should be confirmed.

Overall, we here characterise the organisation of the complex promoter of entpd5a and identify critical genomic elements that govern transcriptional control of this early and highly specific marker of classical and non-classical osteoblasts. En route, we suggest candidate transcriptional regulators that can now be tested. We furthermore provide a resource with a wide range of possible uses in the field, making both ATAC-seq and RNA-seq datasets available that are based on live vertebrate chondrocytes and osteoblasts.

Processed as well as unprocessed ATAC-seq and RNA-seq data generated as part of this work can be accessed via Gene Expression Omnibus (accession numbers GSE267332 and GSE267333, respectively). Furthermore, code used in data processing has been deposited, and is freely available on Github (https://github.com/KleioP/ATAC_RNAseq_analysis/tree/Petratou-et-al.%2C-2024; copy archived at Petratou, 2026).

1. 1. Petratou K
   2. Schulte-Merker S

   (2024) NCBI Gene Expression Omnibus

   ID GSE267332. Integration of ATAC and RNA-Sequencing identifies chromatin and transcriptomic signatures in classical and non-classical zebrafish osteoblasts and indicates mechanisms of entpd5a regulation [ATAC].

   https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE267332
2. 1. Petratou K
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   (2024) NCBI Gene Expression Omnibus

   ID GSE267333. Integration of ATAC and RNA-Sequencing identifies chromatin and transcriptomic signatures in classical and non-classical zebrafish osteoblasts and indicates mechanisms of entpd5a regulation [RNA].

   https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE267333

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Author details

1. Kleio Petratou

   Institute of Cardiovascular Organogenesis and Regeneration, Faculty of Medicine, Universität Münster, Münster, Germany

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4687-1683

2. Martin Stehling

   Flow Cytometry Unit, Max Planck Institute for Molecular Biomedicine, Münster, Germany

   Contribution

   Resources, Investigation, Visualization

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0827-6357

3. Ferenc Müller

   Department of Cancer and Genomic Sciences, School of Clinical Sciences, College of Medicine and Health, University of Birmingham, Birmingham, United Kingdom

   Contribution

   Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0996-774X

4. Stefan Schulte-Merker

   Institute of Cardiovascular Organogenesis and Regeneration, Faculty of Medicine, Universität Münster, Münster, Germany

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Project administration, Writing – review and editing

   For correspondence

   schultes@ukmuenster.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3617-8807

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