Our analysis revealed a 7% mandibular truncation and an abnormal joint phenotype in animals with knockdown of hspg2. Deletion of Hspg2 in mice has been shown to cause truncated snouts, shorter and thicker mandibular structures, and flat faces (13,42,43), but very little has been reported on joint phenotypes and how they pertain to hspg2 function. Although it is relatively novel, the idea of hspg2 mediating the mandibular jaw joint region is not unfounded. Similar to other synovial joints, the mandibular jaw joint contains a synovial capsule, which, in previous cell culture work using synovial cells, has been shown to express and require perlecan for proper development (23,44).
The mechanism by which perlecan mediates joint development is currently unknown. However, as discussed in the introduction, perlecan is a multi-domain protein with side chains that interact with various growth factors like BMP, WNT, and FGF; all of which are essential for neural crest development, chondrogenesis, and joint formation (43,44). Deficiencies or abnormalities in the activation or level of such pathways may account for the decreased number of Sox10+ or Col2a1a+ cells at 4 DPF. Perlecan has also been found to bind to Ihh (Indian hedgehog) through its HS side chains, which in turn mediates the proliferation of chondrocytes (43). It should be noted however, that many of these effects and pathways have predominately been implicated in the joints of the appendicular skeleton and that development of the mandibular jaw joint could be different.
We further demonstrate that knockdown of hspg2 is associated with decreased numbers of Col2a1a+ cells at 4 DPF. These data suggest that hspg2 has a function regulating CNCC differentiation, a finding that is supported by the number of Sox10+ cells at an equivalent time point. Our studies are supported by previous analysis in mice (Hspg2-/-) that demonstrated abnormal arrangement and proliferation of chondrocytes in the appendicular skeleton (13,42). It must be noted however, that although these data support one another, the cells of the appendicular skeleton derive from a different germ layer (the mesoderm) than the cells of the craniofacial skeleton (the ectoderm). Both cell lineages give rise to cartilaginous structures, but the mechanisms by which each population differentiates are likely to be different, prompting further studies. One possible future direction of our work could be to determine the interplay between perlecan and FGF because perlecan binds to FGF-2, which increases the expression of sox9 in vitro (23,45). The protein output of Sox9 in turn is vital to chondrogenesis because it activates Col2a1 expression in mice (46). It is possible however, that there are various mechanisms underlying the function of hspg2 in joint development because the heparin side chains of perlecan are known to bind to collagen II (12) suggesting a direct function for perlecan in chondrogenesis. Interestingly, we also observed an initial increase in the number of Sox10+ cells at 3 DPF, which at the onset seems to counter the results observed at 4 DPF. However, this increase of cells could be due to a period of proliferation in chondrocytes followed by increased cell death between 3 and 4 DPF. Further studies in this area are warranted.
Knockdown of hspg2 was also associated with reduced expression of nkx3.2 at 4 DPF. These results, when understood in the context of the decrease of Col2a1a+ cells found at 4 DPF, are supported by previous results performed in mesenchymal cell culture where nkx3.2 upregulates col2a1 by directly binding to the promoter (47). In this situation, diminished expression of nkx3.2 appears to be directly proportional to a decrease in Col2a1a+ cells and the differentiation of chondrocytes. It is not clear if hspg2 directly modulates nkx3.2 expression or if the decreased expression is simply the result of defects in the mandibular jaw joint, but studies performed in the chick have shown that Nkx3.2 and Sox9 cooperate to promote chondrogenic differentiation and serve as mediators of Sonic Hedgehog (Shh)-induced chondrogenesis (48). This could be one of the mechanisms by which perlecan indirectly mediates the expression of nkx3.2 and it would prove to be a novel discovery. Recently, it was shown that nkx3.2 null animals are viable, making it possible to study this gene in relation to hspg2 without early lethality (49).
In this paper, we demonstrate zebrafish as an alternative animal model to study the role of hspg2 during craniofacial development. Induced knockouts in the murine model have resulted in embryonic lethality from mass hemorrhaging in the pericardial cavity and severe chondrodysplasia, both occurrences which can be temporarily circumvented in the developing zebrafish (31). To circumvent these limitations, three additional mouse models have been produced: the first model lacks exon 3, causing loss of the 3 HS side chains (7), the second model is modeled after an SJS patient mutation where there is a G to an A substitution theorized to cause a misfolded protein (42,50), and the third is a model where early lethality is restored via tissue specific expression of Hspg2 in chondrocytes (51).
While the first two models are viable and can be used to examine adult skeletal phenotypes, the first is centered around exploring the loss of only one domain and the second is mimicking more subtle phenotypes associated with SJS. Our project seeks to understand the role of perlecan in craniofacial development using a null phenotype, a feat not easily performed in a murine model. Zebrafish allow for this type of exploration because unlike mice, they are externally fertilized. This external fertilization enables the study of craniofacial development at early developmental stages, particularly with the use of transgenic fish to target specific genes (52,53). The third model described restores early lethality using a chondrocyte promoter and consequently cannot be utilized to study chondrogenesis or craniofacial development (51). Zebrafish craniofacial development is conserved and the development of the viscerocranium, including the development of the pharyngeal arches, the migration and specification of NCCs, their differentiation, and signaling pathways involved have all been well characterized (29,54). Additionally, because the zebrafish mandibular joint is a synovial joint which develops in a similar fashion to other vertebrate synovial joints, the mechanisms uncovered from this research could be translational to other areas (55).
Zebrafish are remarkably easy to manipulate genetically and have been used with great success in genetic studies. The zebrafish genome shares a high degree of genetic similarity with humans and thus, provides a manner in which to understand gene function and mechanisms (30). 70% of human genes have one zebrafish ortholog and 82% of the genes associated with disease also have at least one zebrafish ortholog (56). CRISPR mutagenesis has emerged as a manner of genetic manipulation readily adaptable to zebrafish (57) and future studies developing a germline non-sense mutant of hspg2 are a critical next step.
All work reported here has been completed by use of a single translation-blocking morpholino. While translation-blocking morpholinos are a simple and effective way in which to knockdown genes of interest, they have been associated with off target effects and non-specific cell death. We did, however, utilize a random control morpholino to account for the possibility of morpholino-induced cell death, an endeavor that proved to be rather successful in previous studies (58,59). And while injection of heparin side chains is a potential rescue for the morphant phenotype we observed, there is the possibility that heparin sulfate co-injection would fail to rescue because a domain outside of domain I is essential for regulation of CNCCs. Therefore, such an experiment is unlikely to demonstrate a full rescue. Collectively, this points out a potential caveat to our work in that we study a morpholino derived phenotype. However, our data with one morpholino is supported by previous studies, including those completed using the murine model (13,42). In these studies, numbers of chondrocytes in the lateral skeleton are depleted, chondrocytes congregate abnormally, and mutant mice exhibit craniofacial abnormalities.
Our work contributes an additional angle to the role of hspg2 in skeletal development by examining the mandibular jaw joint region specifically. We understand that additional morpholinos would help to substantiate our work, but our data is supported by previous studies, suggesting that what we observed is not a consequence of off-target effects. Nevertheless, future studies in a germline zebrafish mutant are required.