We manipulated the expression of Tks5 in zebrafish using morpholino technology. Our studies revealed striking phenotypic differences between control and Tks5 morphants, implicating a role for Tks5 in multiple cell lineages during development. The majority of the defects in Tks5 morphants were in neural crest-derived organs and tissues including the heart, craniofacial structures, the lateral line and pigmentation, suggesting that Tks5 is required for neural crest cell function during embryonic development. While we cannot rule out possible minor gastrulation defects in Tks5 morphants, we believe the phenotype is not solely due to a gastrulation defect, since we observed normal extension of the embryo body and no major reduction in pre-migratory neural crest cells at early stages of development. Furthermore, we demonstrate a role for a Src-Tks5 pathway in development, and the addition of SFK inhibitors post-gastrulation also blocked dorsal-ventral distribution of neural crest cells in the trunk region.
What is the nature of the neural crest defect in Tks5 morphant zebrafish? We have provided several lines of evidence, both in vitro and in vivo, that migration of neural crest and neural crest-derived cells is impaired in the absence of Tks5. Furthermore, our in vitro studies support a cell autonomous requirement for Tks5 in neural crest cell migration in vivo. It is possible that loss of Tks5 also affects neural crest cell proliferation or differentiation. However, we believe that any impact of Tks5 on differentiation is likely to be relatively minor. For instance, in situ hybridization studies using markers for neural crest cells (foxd3 and sox10) demonstrated that there were similar, if not increased, numbers of neural crest cells on the dorsal side of Tks5 morphants at 26 hpf. Furthermore, while markers of neural crest-derived tissues, such as mitf, appeared to be reduced at 26 hpf, Tks5 morphant embryos still had abnormal posterior and ventral migration of mitf-positive cells, suggesting that those cells that have differentiated fail to migrate appropriately in the absence of Tks5. Additionally, neural crest and neural crest-derived cells were present on the dorsal aspect of morphant zebrafish, as well as zebrafish treated with SFK inhibitors. Therefore, while a potential role for Tks5 in differentiation deserves further study, we propose that the primary role of Tks5 is to participate in the directional migration of developmental cell types.
Neural crest progenitors are multipotent cells that are highly migratory during embryonic development, but the actin-cytoskeletal dynamics regulating their migration is relatively unexplored. Here we demonstrate that TGFβ stimulation of neural crest cells in vitro causes the formation of Src and Tks5-dependent podosomes, and that Tks5 and Src are required for neural crest cell migration. In vivo, migrating neural crest cells have been shown to display dendritic-like protrusions with a concentration of actin at their tips, which are dependent on Rho-kinase and myosin II for their formation 
. Furthermore, Src activity is increased 2–7 fold in the dendrites of neural crest-derived melanocytes in vitro 
. We now find that a Src-Tks5-dependent pathway is required for both neural crest cell migration and formation of dendritic-like protrusions in neural crest cells in vivo and in vitro. Might these dendritic-like protrusions be related to podosomes? Podosomes were first discovered by their ability to mediate cell attachment to the ECM 
. The directional migration of neural crest cells is dependent on cell-contact and their attachment to the ECM 
. Therefore, it is intriguing to think that neural crest cell podosomes might regulate ECM contact during migration. Our data suggest this possibility, since there are structural and functional similarities between the Src-Tks5-dependent podosomes that we observed in 2D culture, the elongated actin-rich extensions observed in 3D culture, and the dendritic-like protrusions previously described in neural crest cells in vivo and also visualized in our study. Further analysis is clearly warranted to fully characterize and compare these protrusive structures. The podosomes and invadopodia found in adult cell types are often endowed with the ability to degrade ECM. Neural crest cells also have the capacity to degrade ECM, and they produce proteolytic enzymes, including plasminogen activator and metalloproteinases 
. Additionally, electron microscopy has revealed that the basal lamina around the neural tube is incomplete during neural crest cell emigration and migration 
. Since the ECM plays an important role during development 
, with alterations in expression of collagen and the ECM proteases membrane-type 1 matrix metalloproteinase, ADAM19, and ADAM13 affecting gastrulation, neural crest cell differentiation, and migration 
, it will be important to determine if the dendritic-like protrusions are involved in ECM degradation.
We recently published that mutation of the gene encoding Tks4, which is a family member of Tks5, is a cause of the human developmental disorder, Frank-Ter Haar Syndrome (FTHS), which is characterized by craniofacial and other skeletal abnormalities, as well as eye and heart defects 
. Tks4, like Tks5, is required for invasiveness of Src-transformed cells 
, but the molecular basis of its role in FTHS is not known. The model system we describe here can be used to further dissect the roles and regulation of podosome/invadopodia-associated proteins such as Tks4 and Tks5 during embryonic development, as well as to study the genes involved in other developmental diseases that can be attributed to deficiencies in neural crest cell migration.