A common developmental mode employed by many embryos is a segmentation clock that oscillates within a posterior growth zone; with each cycle, a segment forms. This stratagem has evolved independently within the three major bilaterian clades (annelids, arthropods, and chordates) as well as in plants (Clark et al., 2019; Richmond and Oates, 2012; Chipman, 2010; Ten Tusscher, 2020; Balavoine, 2014). In chordates, a segmentation clock oscillates in the presomitic mesoderm (PSM); with each cycle, a pair of somites form flanking the neural tube (Pourquié, 2011; Hubaud and Pourquié, 2014). Somites differentiate into dermis, skeletal muscle, tendons, as well as the vertebral column, which retains the segmented attribute of the somites (Christ et al., 2007).

In vertebrates, genes with an oscillatory pattern within the PSM include those encoding components or targets of the FGF, WNT, and Notch signaling pathways (Hubaud and Pourquié, 2014; Krol et al., 2011; Dequéant et al., 2006). However, which individual genes oscillate differs among species, with the exception of the Notch-responsive HES/HER transcription factors, suggesting that Notch signaling is at the core of the somitogenesis clock (Krol et al., 2011). Supporting this idea, genetic and pharmacological manipulations demonstrate that Notch pathway oscillation in the mouse embryo is essential for somitogenesis (Ferjentsik et al., 2009). In the mouse, oscillatory waves of the Notch1 receptor and Delta-like 1 (Dll1) ligand expression, as well as the activated Notch receptor (cleaved Notch intracellular domain, NICD) sweep from the posterior to anterior, where oscillations arrest (Bone et al., 2014; Shimojo et al., 2016). These oscillations are established through negative-feedback loops of Notch components such as the transcriptional repressor, HES7 (Niwa et al., 2011), and the glycosyltransferase, LFNG (Morimoto et al., 2005), both of which are encoded by oscillating genes under Notch pathway regulation (Niwa et al., 2011). Notch oscillations arrest in the anterior PSM, where NICD cooperates with TBX6 to periodically activate Mesp2 expression (Oginuma et al., 2008). MESP2 then regulates formation of the nascent somite as well as its rostro-caudal patterning, which is essential for normal patterning of the subsequent vertebral column (Takahashi et al., 2000; Saga et al., 1997).

Mutations in nearly all of the aforementioned Notch pathway genes have been identified in human patients where defective somitogenesis is thought to be the cause of Segmentation Defects of the Vertebrae (SDV) (Eckalbar et al., 2012; Giampietro et al., 2009). For example, frequently recessive mutations in DLL3, HES7, MESP2 and LFNG and a dominant mutation in TBX6 (Sparrow et al., 2013; Lefebvre et al., 2017) have been identified in patients with spondylocostal dysostosis (SCDO), which is characterized by severe vertebral malformations that include hemivertebrae, vertebral loss and fusion along the length of the axis (Takeda et al., 2018). Whereas SCDO is relatively rare, congenital scoliosis (CS), defined as a lateral curvature of the spine exceeding 10%, is much more common, with a frequency of 1:1000, which is suspected to be an underestimation because asymptomatic individuals do not seek medical care (Giampietro et al., 2013). Mutated alleles of HES7, LFNG, MESP2, and TBX6 are all associated with CS (Lefebvre et al., 2017; Takeda et al., 2018; Giampietro et al., 2013; Sparrow et al., 2012).

The position in the anterior PSM where Notch oscillations arrest and Mesp2 expression establishes the future somite boundary is called the determination front (Morimoto et al., 2005; Saga et al., 1997; Dubrulle et al., 2001). This region is also the anterior limit of the wavefront in the classical clock-and-wavefront, a theoretical model proposed over 40 years ago to explain the precipitous and periodic formation of segments in the PSM (Cooke and Zeeman, 1976). In this model, wavefront activity prevents the PSM from responding to the segmentation clock; hence somites form only anteriorly, where the wavefront ends. In chick or zebrafish embryos, exogenously added FGF protein or pharmacological inhibition of FGF signaling will shift the determination front rostrally or caudally, respectively. In the mouse, Cre-mediated inactivation of Fgf4 and Fgf8 specifically in the PSM results in an initial expansion of Mesp2 expression, followed by the premature expression of somite markers throughout the PSM (Naiche et al., 2011). Canonical WNT signaling is also a wavefront candidate, mostly because ectopic activation of βcatenin in the PSM results in an expansion of this tissue and a rostral shift of Mesp2 expression (Aulehla et al., 2008; Dunty et al., 2008). However, this expansion observed in βcatenin ‘gain-of-function’ mutants requires FGF4 and FGF8 activity, suggesting that these FGF signals are synonymous with the wavefront (Naiche et al., 2011).

In addition to its role in preventing somite differentiation, FGF signaling is also implicated in the regulation of key Notch components of the segmentation clock. Several studies have demonstrated that pharmacological inhibition of FGF disrupts Notch oscillations (Wahl et al., 2007; Niwa et al., 2007). In mouse embryos with PSM-specific loss of FGF receptor one or both Fgf4 and Fgf8, Notch pathway genes such as Hes7, are downregulated, although this analysis can be complicated due to the loss of PSM tissue in these mutants (Naiche et al., 2011; Niwa et al., 2007). Here, we focus on FGF-Notch interactions by analyzing mouse mutants that lack only one of the wavefront Fgf genes, Fgf8 or Fgf4. An indispensable technique in our analysis is whole mount in situ hybridization chain reaction (HCR), which allows us to multiplex different gene expression domains and quantify mRNA levels within specific embryonic tissues (Choi et al., 2014; Choi et al., 2018; Trivedi et al., 2018). We demonstrate that, while Fgf8 is not required for somitogenesis, Fgf4 is required for normal Notch oscillatory patterns and patterning of the vertebral column.

We describe mutants with only Fgf4 or Fgf8 inactivated specifically in the primitive streak and PSM. In previous work, we reported that somites form in such mutants (Naiche et al., 2011; Perantoni et al., 2005); here we examine the patterning of these somites, the Notch pathway oscillations that control their formation, as well as the vertebral elements that they eventually form. We found that Fgf4 mutants display a range of cervical and thoracic vertebral defects that are caused by aberrant Notch oscillations during somitogenesis. In contrast, the vertebral columns of Fgf8 mutants are normally segmented, with about 30% displaying minor homeotic transformations; such alternations in vertebral identity are not likely due to defects in somitogenesis per se. Previously, a role for FGF signaling in wavefront activity in chick and zebrafish embryos was supported by pharmacological manipulation (Dubrulle et al., 2001; Sawada et al., 2001) and in mouse mutants where both Fgf4 and Fgf8 are simultaneously inactivated using the same TCre activity that we use in this study (Naiche et al., 2011). These double Fgf4/Fgf8 mutants as well as mutants with tissue-specific inactivation of Fgfr1 support a role for FGF signaling in clock oscillations, but these conclusions were complicated by a loss of PSM tissue due to potential wavefront defects (Oginuma et al., 2008; Naiche et al., 2011; Wahl et al., 2007; Niwa et al., 2007). Compared to these Fgf pathway mutants, the phenotype of Fgf4 mutants is relatively subtle, with no wavefront defect, as indicated by no loss of caudal vertebrae (Anderson et al., 2016a) and no quantitative change in wavefront gene markers (Figure 2). This lack of any overt axis extension defect allows us to unambiguously identify Fgf4 as an Fgf ligand gene required for a normal segmentation clock. This insight is supported by recently published work showing that FGF signaling regulates the dynamics of gene expression oscillations in PSM cells cultured in vitro (Diaz-Cuadros et al., 2020).

Our mutants model human Segmentation Defects of the Vertebrae (SDV); Fgf4 mutants resemble CS and the synergistic phenotype resulting from the additional removal of one Hes7 copy in these mutants resembles human SCDO. Both of these diseases are caused by mutations in Notch signaling components that we find are misexpressed in Fgf4 mutants, such as HES7, MESP2, and LFNG (Eckalbar et al., 2012; Giampietro et al., 2009). With regard to the human FGF pathway and SDV, a straightforward gene-disorder relationship has not been uncovered, probably because of the pleiotropic phenotypes of such putative mutations would preclude embryonic survival. Sparrow et al found that gestational hypoxia in mice results in an increase in the severity and penetrance of CS in Notch1, Mesp2, and Hes7 heterozygotes (Sparrow et al., 2012). Intriguingly, CS is more severe in children living at high altitudes (Hou et al., 2018), suggesting a similar environmental effect may affect human development. In mice, hypoxia was found to diminish FGF signaling components, but expression of Fgf8 was unchanged, and Fgf4 was unexamined (Sparrow et al., 2012). Our data suggest that FGF4 may be part of the system that is responsive to this environmental insult. However, if this is the case, reduced FGF4 activity cannot be the only response to reduced oxygen levels because gestational hypoxia reduces expression of the FGF target gene Spry4 in the PSM, whereas we found no change in expression in this or other such canonical FGF target genes in Fgf4 mutants.

Rather, as is the case for hypoxia-treated embryos (Sparrow et al., 2012), Hes7 expression is reduced in Fgf4 mutants and we propose that this leads to aberrant segmentation. An indispensable tool to this insight was the use of multiplex HCR imaging (Choi et al., 2014; Choi et al., 2018; Trivedi et al., 2018). The simultaneous fluorescent imaging of multiple mRNA domains with this technique is particularly useful in a complex embryonic process, such as somitogenesis, where many complex and dynamic gene expression patterns need to be analyzed. HCR is a relatively new tool for mutant embryo analysis, just beginning to be used to analyze mutant embryos (Lignell et al., 2017; Kim et al., 2019; Attardi et al., 2018; McKinney et al., 2016). We used HCR to examine the expression of multiple genes in a single tissue by combining it with Imaris image analysis software. This combination of HCR and Imaris-modeling is broadly applicable for in situ quantification of gene expression within any complex embryonic or clinical sample. With distinct molecular markers, one can use this approach to quantify gene expression in any cell population with a precision that heretofore was not possible and still retain the intact whole mount embryo or tissue. Here, we generated and analyzed Imaris-based volumetric models of the PSM, based on Tbx6 expression, and determined that Hes7 levels are reduced by about 40–50% specifically in the PSM of Fgf4 mutants during early somite stages when the progenitors of aberrant vertebrae are segmenting.

We propose that this reduction in Hes7 levels is the primary defect in Fgf4 mutants, and subsequently causes an irregular activated Notch (NICD) pattern, misexpression of Mesp2, and ultimately leads to vertebral defects. A reduction in Hes7 levels, due to heterozygosity or hypomorphic mutations, has been shown to cause vertebral defects (Sparrow et al., 2012; Fujimuro et al., 2015; Stauber et al., 2009). The concept that Hes7 is generally regulated by FGF signaling is supported by numerous studies (Naiche et al., 2011; Niwa et al., 2007; Harima and Kageyama, 2013). Future work will determine whether Hes7 is a direct FGF4 signaling target, possibly through putative ETS binding sites in the Hes7 promoter (González et al., 2013). Alternatively, FGF4 may indirectly regulate Hes7 transcription by affecting the activity of TBX6, NICD or MESOGENIN, all shown to regulate Hes7 expression in the PSM (González et al., 2013; Hayashi et al., 2018). If so, we speculate such regulation would be post-transcriptional because we find no significant difference in levels of the genes encoding these factors. Additionally, it is possible that FGF4 signaling is interfacing with WNT signaling, which also has been shown to regulate Hes7 Aulehla et al., 2008; Dunty et al., 2008; González et al., 2013. Given the molecular feedbacks during genetic oscillations in the PSM, we considered whether FGF4 might have a primary role on Lfng expression, whose pattern is abnormal in our mutants. However, we suggest this is not the case, because, although embryos completely lacking Lfng also display aberrant Hes7 oscillations (Niwa et al., 2007; Stauber et al., 2009), overall Hes7 mRNA levels are not reduced (Ferjentsik et al., 2009), unlike the case in our Fgf4 mutants.

We support our proposal that the observed 50% reduction in Hes7 is the cause of the Fgf4 mutant with the synergistic worsening of the vertebral segmentation defect that occurs when we further reduce Hes7 levels by removal of one wildtype allele. Notably, such synergy in defects does not occur when a copy of Hes7 is removed from Fgf8 mutants (Figure 7—figure supplement 1). From another perspective, the mild CS caused by loss of one Hes7 allele in a wildtype background (Sparrow et al., 2012) is worsened 9-fold by removing Fgf4 (Figure 7). Therefore, Fgf4 is a robustness-conferring gene that buffers somitogenesis against a perturbation in Hes7 gene dosage (Félix and Barkoulas, 2015).

We observed that in Fgf4 mutants Hes7 mRNA levels are reduced during rostral somitogenesis at E8.5 (5–6 somite stages), when defective somitogenesis occurs. At about E9.5 (24–26 somite stages), Hes7 levels are unaffected, and the Fgf4 mutant embryo is beginning to generate correctly patterned somites that will differentiate into normally patterned vertebrae. Tam, 1981 showed that between these two stages of mouse development, somites more than double in size. Such an increase in segment size can be due to a slower segmentation clock and/or faster regression of the determination front (Oates et al., 2012). Gomez et al., 2008 found that the caudal movement of the wavefront does not significantly change at these stages, suggesting a slowing clock between E8.5 and E9.5. Consistent with a slower clock, we observed at least 2 and frequently 3 bands of Hes7 expression at the Fgf4-sensitive stages (5–6 somite stages; Figures 4 and 5), but only 1 to 2 bands when Fgf4 loss has no effect on Hes7 expression (24–26 somite stages; Figure 5—figure supplement 5). In future experiments, we will test this hypothesis directly by measuring the clock rate, using recently generated fluorescent transgenic reporters of Hes7 (Yoshioka-Kobayashi et al., 2020). Thus, it appears that FGF4 activity is required to maintain Hes7 mRNA levels above a certain threshold when the segmentation clock is faster; when Notch oscillations slow, they may become FGF-independent, or other FGFs may be at play. If this is the case in all vertebrates, we might expect embryos with faster segmentation clocks, such as snakes (Gomez et al., 2008), to have a longer window of FGF4-dependence for normal somitogenesis.

This study and past work indicate that FGF4 and FGF8 have both shared and unique roles in the vertebrate embryo’s posterior growth zone. They redundantly encode wavefront activities, preventing PSM differentiation (Naiche et al., 2011; Boulet and Capecchi, 2012). In this capacity, when signaling together, they activate downstream canonical FGF target genes, such as Spry2, Spry4, and Etv4 (Naiche et al., 2011). Here, we show the role that FGF4 signals play in maintaining Hes7 mRNA levels cannot be replaced by FGF8. Future efforts will explore how the signaling machinery within the PSM discriminates between these two FGF ligands. For example, our work argues for a reassessment of FGFR function in the PSM; prior work, in which the same Cre driver used here (TCre) inactivates Fgfr1 (Wahl et al., 2007), thought to be the only FGF receptor gene expressed in the PSM, does not exactly phenocopy the defects due to TCre-mediated loss of either Fgf4 (this study) or Fgf4/Fgf8 (Naiche et al., 2011).

Our insights regarding the roles of FGF4 and FGF8 in the caudal embryonic axis are intriguing in the context of the evolution of the vertebrate body plan. Vertebrates, together with Cephalochordates and Urochordates make up the phylum Chordata (Putnam et al., 2008). Somitogenesis predates vertebrate evolution as it occurs in Cephalochordata, the most basal living chordates (Holland and Onai, 2012). In both Cephalochordates and Urochordates, the FGF essential for embryonic axis extension is an Fgf8 ortholog (Bertrand et al., 2015; Pasini et al., 2012), suggesting that this Fgf (‘Fgf8/17/18’) may be the ancestral gene in this process. However, Fgf8/17/18 activity in these invertebrate chordates does not control gene oscillations as these embryos apparently lack a segmentation clock (Bertrand et al., 2015; Onai et al., 2015). Therefore, we speculate that the recruitment of an Fgf4 role during the evolution of vertebrate axis extension may have coincided with the development of gene oscillations that control somitogenesis.

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

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Author details

1. Matthew J Anderson

   Genetics of Vertebrate Development Section, Cancer and Developmental Biology Laboratory, National Cancer Institute, National Institutes of Health, Frederick, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, 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:" 0000-0001-9387-5743

2. Valentin Magidson

   Optical Microscopy and Analysis Laboratory, Frederick National Laboratory for Cancer Research, Frederick, United States

   Contribution

   Resources, Writing - review and editing

   Competing interests

   No competing interests declared

3. Ryoichiro Kageyama

   Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan

   Contribution

   Resources, Writing - review and editing

   Competing interests

   No competing interests declared

4. Mark Lewandoski

   Genetics of Vertebrate Development Section, Cancer and Developmental Biology Laboratory, National Cancer Institute, National Institutes of Health, Frederick, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   lewandom@mail.nih.gov

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1066-3735

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