Some of the most common birth defects involve improper development of the head and face. One such birth defect is called craniosynostosis. Normally, an infant’s skull bones are not fully fused together. Instead, they are held together by soft tissue that allows the baby’s skull to more easily pass through the birth canal. This tissue also houses specialized cells called stem cells that allow the brain and skull to grow with the child. But in craniosynostosis these stem cells behave abnormally, which fuses the skull bones together and prevents the skull and brain from growing properly during childhood.

One form of craniosynostosis called Saethre-Chotzen syndrome is caused by mutations in one of two genes that ensure the proper separation of two bones in the roof of the skull. Mice with mutations in the mouse versions of these genes develop the same problem and are used to study this condition. Mouse studies have looked mostly at what happens after birth. Studies looking at what happens in embryos with these mutations could help scientists learn more. One way to do so would be to genetically engineer zebrafish with the equivalent mutations. This is because zebrafish embryos are transparent and grow outside their mother’s body, making it easier for scientists to watch them develop.

Now, Teng et al. have grown zebrafish with mutations in the zebrafish versions of the genes that cause Saethre-Chotzen syndrome. In the experiments, imaging tools were used to observe the live fish as they developed. This showed that the stem cells in their skulls become abnormal much earlier than previous studies had suggested. Teng et al. also showed that similar stem cells are responsible for growth of the skull in zebrafish and mice.

Babies with craniosynostosis often need multiple, risky surgeries to separate their skull bones and allow their brain and head to grow. Unfortunately, these bones often fuse again because they have abnormal stem cells. Teng et al. provide new information on what goes wrong in these stem cells. Hopefully, this new information will help scientists to one day correct the defective stem cells in babies with craniosynostosis, thus reducing the number of surgeries needed to correct the problem.

Craniofacial anomalies are among the most common congenital defects, encompassing cleft lip and palate, facial malformations, and abnormalities in the flat bones forming the top of the skull. Craniosynostosis involves the premature fusion of the skull bones at sutures, fibrous structures that join the bones. During normal development, cranial sutures hold bones of the skull in place while providing malleability required during childbirth. Cranial sutures also contain stem cells that allow for continued skull bone growth as the brain and head structure expands (Zhao et al., 2015). While human skull bones eventually fuse later in life, precocious bone fusion in craniosynostosis correlates with region-specific defects in bone growth that often negatively impact growth of the underlying brain. Genetic causes of craniosynostosis include mutations in genes that participate in developmental signaling pathways, including FGFRs 1, 2, and 3 (Hajihosseini, 2008), TGFBRs 1 and 2 (Loeys et al., 2005), and the Notch ligand JAGGED1 (Kamath et al., 2002). A central unanswered question is the extent to which defects in these pathways result in a failure to maintain postnatal stem cells once the sutures have formed, or whether these pathways have roles in the specification and maintenance of bone progenitors during earlier phases of bone growth preceding suture formation (Flaherty et al., 2016). Most studies have focused on the maintenance of postnatal sutural stem cells, as these stem cells have only been recently marked with a variety of reporters in mice, for example based on Gli1 (Zhao et al., 2015), Axin2 (Maruyama et al., 2016), and Prrx1 (Wilk et al., 2017). Less is known, however, about how potential defects in the embryonic progenitors that grow the skull bones may prefigure suture loss, although a few reports describe altered bone formation during embryonic stages in craniosynostosis mutants (Merrill et al., 2006). The ex utero development and transparency of zebrafish provide a unique opportunity to track the progenitors that form the skull bones, and to better understand how defects in the dynamics of bone growth relate to a later ability to form and maintain sutures once the bones come together (Quarto and Longaker, 2005; Laue et al., 2011; Kague et al., 2016; Topczewska et al., 2016).

A striking feature of many forms of craniosynostosis is that only particular sutures are affected. For example, in Saethre-Chotzen syndrome, the second most common form of craniosynostosis, the coronal suture is selectively lost. The majority of Saethre-Chotzen patients harbor heterozygous loss-of-function mutations in TWIST1 or TCF12, which encode basic helix-loop-helix transcription factors (el Ghouzzi et al., 1997; Howard et al., 1997; Sharma et al., 2013). Similarly, mice lacking one copy of Twist1, or compound heterozygous for Twist1 and Tcf12, display loss of just the coronal suture (Sharma et al., 2013). Analysis of mouse models has led to an appreciation of proper cell migration (Yoshida et al., 2008; Ting et al., 2009; Roybal et al., 2010; Deckelbaum et al., 2012), segregation of osteogenic and non-osteogenic cells at the sutural boundary (Merrill et al., 2006; Ting et al., 2009; Yen et al., 2010), and maintenance of postnatal stem cells (Zhao et al., 2015) in suture patency. However, it remains unknown the extent to which defects in early progenitors versus postnatal stem cells account for suture loss. As fish are amenable to repeated live imaging of calvarial bone growth and suture development outside the mother, this model provides an opportunity to correlate early defects in osteoprogenitors and bone growth with later suture loss in individual mutants, particularly in cases where the synostosis phenotype is variably penetrant. However, a potential complication is that the coronal suture of zebrafish (Kague et al., 2012; Mongera et al., 2013), as well as those of some amphibians (Piekarski et al., 2014) and birds (Matsuoka et al., 2005), forms at a mesoderm/mesoderm boundary, contrasting with the mammalian coronal suture that forms at a unique interface between the neural-crest-derived frontal bone and the mesoderm-derived parietal bone (Ishii et al., 2015) (Figure 1A). Mammalian sutures may therefore not be considered evolutionarily homologous to the sutures of these other vertebrates, at least from a strict embryological perspective. Should the neural-crest/mesoderm boundary be a factor in coronal sensitivity, non-mammalian coronal sutures might not be susceptible to the same genetic perturbations that cause coronal-specific synostosis in mammals.

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We report in this study the generation of a zebrafish model of Saethre-Chotzen syndrome that faithfully recapitulates the craniosynostosis phenotype seen in mice and humans with heterozygous mutations in TCF12 and TWIST1. The similarity in the genetic interaction between Twist1 and Tcf12 in mice, humans, and fish, despite differences in the cell lineages that give rise to the bones, suggests that the underlying processes of coronal suture development and craniosynostosis are deeply conserved. We demonstrate that in tcf12; twist1b mutant fish, the frontal and parietal bones grow abnormally. In mutants, skull bones initiate normally, yet early bone growth is accelerated across the skull. However, subsequent bone growth selectively stalls at the future coronal suture that is destined to fuse. Moreover, sequential live imaging of individual mutant fish shows that the degree of later bone stalling predicts which animals will lose the coronal suture. We observe a similar misregulation of bone growth in Tcf12^+/-; Twist1^+/- mutant mice, with tissue-specific removal of Twist1 resulting in selective overgrowth of the frontal or parietal bones. Further, Twist1 function must be perturbed within both neural crest- and mesoderm-derived bones, and not just the mesoderm-derived postnatal sutural mesenchyme, to prevent suture formation. At least in mice, we find that these altered bone growth dynamics may be due to changes in osteoblast proliferation and eventual depletion of Gli1⁺ and Gremlin1⁺ putative bone progenitors prior to the fusion of the bones. These findings demonstrate that Tcf12 and Twist1 have a conserved early function during skull bone growth to regulate the sustained production of osteoblasts, possibly through maintenance of osteoprogenitors, and that the coronal suture is particularly sensitive to defects in this process.

Discovery of a selective requirement for tcf12 and twist1b in coronal suture formation in zebrafish has allowed us to gain a better understanding of the developmental basis of suture loss in Saethre-Chotzen syndrome. The similarity of coronal suture defects from humans to mice to zebrafish is striking, although each species displays unique dosage sensitivities to loss of TWIST1 and TCF12. In humans, haploinsufficiency of TWIST1 or TCF12 can lead to suture loss. In mice, haploinsufficiency of Twist1 also results in coronal suture loss, yet haploinsufficiency of Tcf12 does not, despite enhancing the penetrance of suture defects in Twist1^+/- mice (Sharma et al., 2013). In zebrafish, only loss of one of two Twist1 homologs (twist1b) along with loss of tcf12 results in suture loss. It remains unclear why humans are more sensitive to Twist1 and Tcf12 dosage than mice and zebrafish, although a similar phenomenon has been observed with other synostosis genes (e.g. JAG1) (Teng et al., 2017).

Repeated live imaging of individual mutant fish revealed a strong correlation between the extent of altered bone growth and later suture loss. Whereas the initiation of the frontal and parietal bones was largely unaffected in mutants, we observed increased proliferation and osteoblast production at the mutant bone fronts in both fish and mice. There were some subtle differences between fish and mice, with larger increases in proliferative osteoblasts in mutant fish than mice, which might reflect species-specific or staging differences. Nonetheless, mutants in both species displayed accelerated growth of both the frontal and parietal bones, which often resulted in abnormal shapes likely due to growth variations along individual bone fronts. In particular, we found that increased diagonal growth in mutants brought the parietal and frontal bones together at the prospective medial regions of the coronal suture much earlier than in wild type animals, which correlates with the medial region of the coronal suture being most commonly fused in mutants. This altered directional growth might be one reason why the coronal suture is preferentially affected in both zebrafish and mouse Twist1/Tcf12 mutants, despite the different origins of the coronal suture in these species.

Another prominent finding was a lack of continued bone growth at the future coronal but not other sutures in mutants, with the degree of bone stalling predicting synostosis in individuals. We identified Gli1/gli1 and Grem1/grem1a as conserved markers of putative osteoprogenitors in both the growing bone fronts and mature sutures of mice and zebrafish, although both genes display broader expression at early stages of calvarial bone development, including in some periosteal cells. Future lineage tracing experiments should help reveal the extent to which gli1 in fish and Grem1/grem1a in both species mark similar populations of embryonic osteoprogenitors and postnatal sutural stem cells. A previous study had observed reduced expression of Gli1 in the sutures of adult Twist1^+/- mice (Zhao et al., 2015). Here, we extend this finding to embryonic stages when the coronal suture is forming, and uncover the existence of Grem1+ cells in and around the bone fronts of the nascent coronal suture that become depleted in mutant mice. Whether similar osteoprogenitors become exhausted at the developing coronal suture of zebrafish remains to be determined, as it was difficult to precisely quantify the numbers of these progenitors by RNA expression alone. One possibility is that reduced Twist1 and Tcf12 function alters the balance between long-term sutural stem cells (i.e. those marked by Gli1) and proliferative osteoblasts and their immediate progenitors. In such a model, the early increase in osteoblast production would come at the expense of long-term progenitors, thus leading to a later failure of continued bone growth and a loss of the sutural stem cells that would normally separate the skull bones. In addition to further verifying this model, an important next step will be to determine why the fronts of the parietal and frontal bones abutting the future coronal suture are most sensitive to progenitor exhaustion in mutants. We did not observe preferential expression of tcf12 and twist1b at the coronal suture in fish, consistent with similarly broad sutural expression of Twist1 in mice (Rice et al., 2000). Instead, osteoprogenitors in the coronal zone could be fewer in number at initial stages and/or more sensitive to loss of Tcf12 and Twist1, for example due to compensation by related genes at other sutures.

Further support for suture defects arising from much earlier changes in bone growth come from our conditional Twist1 deletion experiments in mouse. Whereas the frontal bone arises from neural crest and the parietal bone from mesoderm, the mesenchyme within the postnatal coronal suture is largely mesoderm-derived (Yen et al., 2010). However, suture loss was only observed upon conditional deletion of one allele of Twist1 from both the embryonic mesoderm and neural crest. This finding is inconsistent with Twist1 functioning solely in the mesoderm-derived postnatal suture mesenchyme for suture patency, instead suggesting that misregulated growth of both the frontal and parietal bones is required to later disrupt the coronal suture in Twist1 mutants.

Our study highlights a selective role for Tcf12 in the later growth of the skull bones and patency of the coronal suture. In contrast to animals lacking Twist1 homologs, zebrafish lacking tcf12 do not display embryonic lethality or defects in ectomesenchyme and facial cartilage formation. Instead, loss of tcf12 partially suppresses the facial cartilage defects and lethality of twist1a; twist1b mutants. Suppression of embryonic defects could be due to loss of competition of Tcf12 with other Twist binding partners, such as Hand2 (Firulli et al., 2005). In this scenario, maternal Twist1a/b and/or other Twist family members (e.g. twist2 and twist3) could compensate for lack of zygotic Twist1a/b. Loss of tcf12 would then allow remaining Twist proteins to more effectively form homodimers or alternate heterodimers important for embryogenesis. Whereas a previous report indicates that Tcf12 and Twist1 can form heterodimers (Connerney et al., 2006), it is also possible that Tcf12 has Twist1-independent functions that antagonize Twist1 during embryogenesis. Future efforts to directly visualize Tcf12-Twist1 complexes will help to resolve how Tcf12 promotes Twist1 function during skull bone growth while counteracting it during embryonic neural crest development.

Development of homologous structures often employs ancestrally conserved gene regulatory networks. For example, a requirement for Pax6 genes in eye development in a wide range of animals has been used to argue for deep homology of eye structures (Gehring and Ikeo, 1999). Here, we show remarkably specific loss of a single anatomical structure, the coronal suture, in zebrafish and mice lacking Tcf12 and Twist1, despite this suture occurring at a mesoderm/mesoderm interface in zebrafish and a neural-crest/mesoderm interface in mice. Hence, sensitivity of the coronal suture in Saethre-Chotzen syndrome is unlikely to be due to its location at a unique neural-crest/mesoderm interface. In addition, there are several other sutures occurring at a neural-crest/mesoderm interface in mice and fish that are not affected by loss of Tcf12 and Twist1. There has been on-going debate, given these distinct tissue boundaries, as to whether coronal sutures are truly homologous across vertebrates (Maddin et al., 2016). Our data indicate that the conserved genetic dependence of the coronal suture in fish and mammals likely reflects a similar sensitivity to early bone growth changes, perhaps owing to similar developmental and anatomical constraints irrespective of the embryonic origins of the bones flanking this suture.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Camilla S Teng

   1. Department of Stem Cell Biology and Regenerative Medicine, University of Southern California, Los Angeles, United States
   2. Department of Biochemistry, Keck School of Medicine, University of Southern California, Los Angeles, United States

   Contribution

   Conceptualization, Investigation, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

2. Man-chun Ting

   Department of Biochemistry, Keck School of Medicine, University of Southern California, Los Angeles, United States

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

3. D'Juan T Farmer

   Department of Stem Cell Biology and Regenerative Medicine, University of Southern California, Los Angeles, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

4. Mia Brockop

   Department of Biochemistry, Keck School of Medicine, University of Southern California, Los Angeles, United States

   Contribution

   Data curation, Formal analysis, Investigation, Visualization, Methodology

   Competing interests

   No competing interests declared

5. Robert E Maxson

   Department of Biochemistry, Keck School of Medicine, University of Southern California, Los Angeles, United States

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing—original draft, Writing—review and editing

   For correspondence

   maxson@usc.edu

   Competing interests

   No competing interests declared

6. J Gage Crump

   Department of Stem Cell Biology and Regenerative Medicine, University of Southern California, Los Angeles, United States

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   gcrump@usc.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3209-0026

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