Background Cleft lip and/or palate (CL/P) is the most frequent craniofacial malformation affecting 1 in 700 births. Children born with CL/P have to undergo multiple staged procedures early in their lifetime and may suffer from severe functional sequelae. Surgery remains their only therapeutic option. CL/P can be caused by perturbation of cranial neural crest cell proliferation, migration, and apoptosis in early embryogenesis. Environmental and genetic factors can cause CL/P. We investigated the genetic and developmental basis of craniofacial morphogenesis with the goal to find new therapeutic and potentially preventive measures to mitigate cleft phenotypes. This thesis focused on the functional analysis of IRF6 and SPECC1L, two genes involved in syndromic and nonsyndromic CL/P and oblique facial cleft, respectively.
We used the zebrafish to study gene function in vivo, as this versatile vertebrate animal model allows us to easily manipulate and follow craniofacial developmental processes in real time.
Methods and Results In manuscript 1, we showed that zebrafish and amniote lip/primary palate formation is analogous by fusion of the paired maxillary prominences (MXPs) and the frontonasal prominence (FNP). We suggested that the V-shaped fusion seam between FNP and MXP is analogous to the Y-shaped fusion seam found in humans. Further, we showed that cleft malformation is conserved in zebrafish by creating a dominant negative transgenic line harboring a known human mutation of IRF6 (R84C). Moreover, we described palate extension in detail as a process of morphological change, intercalation, proliferation and integration of cells and showed that wnt9a and irf6 are required for extension and integration, respectively.
In manuscript 2, we described the lineage-tracing method that was used to determine analogy of palate formation between amniotes and zebrafish. We described selection and mounting of embryos, as well as imaging software settings. The three aspects of the protocol (mounting, imaging, software) have wide application, as every part can be used as a guide to confocal microscopy of any tissue.
Manuscript 3 took advantage of knowledge gained in manuscripts 1 and 2.
We investigated SPECC1L, the first gene implicated in ObFC. We showed that specc1lb is the zebrafish ortholog (conserved function) of human SPECC1L and showed expression in epithelia surrounding chondrocytes. We proposed that specc1lb is required for convergence of facial prominences and integration of the FNP and MXPs. In our analysis, migration to pharyngeal arches was not perturbed, although CNCCs contributing to the mandibular prominence failed to form the mandible, and anterior CNCCs did not converge to contribute to their respective facial prominences (FNP, MXP).
Discussion and Conclusions IRF6 has been studied extensively. However, gene expression in different cell types, function of regulatory elements and the irf6 signaling pathways remain to be elucidated.
In order to assess irf6 expression and activity in detail, we are currently working on identifying the irf6 promoter region, as well as regulatory elements upstream of irf6. In addition to investigating the gain of function dominant negative irf6 phenotype described in manuscript 1, we have created CRISPR knockout lines to assess the loss of function genotype. We hope that a combination of stable transgenic lines with irf6 gene expression pattern, as well as irf6 CRISPR lines, will allow us to study gene function and perform genetic and chemical screens to find substrates that can mitigate cleft phenotypes. Although SPECC1L MO knockdown phenotypes in manuscript 3 did not show perturbation of CNCC migration, CRISPR lines that are currently growing up may show NCC migration and delamination defects that were not uncovered by MO. Recent evidence from Co-Immunoprecipitation with a custom made anti-specc1l antibody followed by mass spectrometry provide evidence in support of our hypothesis that SPECC1L plays a role in directional migration during NCC movement. It is possible that SPECC1L is part of cell-cell and cell-ECM junctions, as well as actin and microtubulemembrane junctions to transduce and maybe potentiate mechano-sensitive signals.
Further, it may be able to change the conformation of cell-cell and cell-ECM contacts as well as the actin/ myosin skeleton in response to signals from neighboring cells, the ECM, primary cilia and lamellipodia.
SPECC1L high-throughput screens of potential SPECC1L interactors in the zebrafish animal model will elucidate which regulatory elements of cell movement associate with SPECC1L.
Finally, transgenic irf6 and specc1l zebrafish lines will be used to analyze if the two genes act in the same regulatory gene network.
Potentially, a connection between pathways leading to more or less severe forms of cleft can be made.