In unrelated studies, we noticed that zebrafish log (a null allele for med14) [10] and young (a null allele for brg1) [14] mutants shared a common array of deficiencies in heart, eye, pectoral fin and pigment cell development (Fig. 1a–d and data not shown). This similarity suggested overlapping or common functions for med14 and brg1. To test this possibility, double mutants were generated. The med14; brg1 double mutant embryos displayed a much more severe phenotype compare to single mutants, including a curved body axis, smaller eyes, severe heart edema and loss of pigment (Fig. 1a). To further investigate the role of med14 and brg1 in development, as well as possible functional interactions between the Mediator and BAF complexes, neural crest cell-derived tissues were analyzed in various mutant backgrounds. In med14 and brg1 single mutants, the melanin in melanocytes showed a less even and spiky distribution compared to controls; whereas in med14; brg1 double mutants melanin distribution took on a small, rounded appearance (Fig. 1b–e). Quantification of melanocyte number on the dorsal surface of the trunk revealed no significant differences between controls and mutants (Fig. 1q, P = 0.94).
The development of the neurocranium and viscerocranium were next analyzed through Alcian blue staining at 96 h post-fertilization (hpf). In brg1 mutants, the neurocranium was dismorphic, and the size of Meckel’s cartilage, ceratohyal and palatoquadrate on the viscerocranium were greatly reduced, with the last two branchial arches absent (Fig. 1j–k and n–o). In med14 mutants, trabeculae formed, while most of the ethmoid plate and lateral parts of parachordal plate were not evident (Fig. 1l). Aside from relics of palatoquadrate, viscerocranium structures were absent in med14 mutants (Fig. 1p). In med14; brg1 double mutants, only posterior portions of trabeculae and parachordal plate were apparent, whereas viscerocranium was completely absent (Fig. 1m). It has been reported some posterior elements (part of trabeculae and parachodal plate) are derived from mesoderm [20]. As these elements remained in med14; brg1 double mutants, this suggested med14 and brg1 are only required for neural crest-derived cartilage.
To further explore genetic interactions between med14 and brg1, we examined cartilage defects in brg1−/−; med14+/− and med14−/−; brg1+/− embryos. In either case, further loss of one allele of med14 or brg1 in either brg1 or med14 null mutants resulted in more severe defects in facial cartilage formation as compared to single mutants (Fig. 1r–t). We also observed that med14 and brg1 mutants shared a common array of deficiencies in heart, eye and otic vesicle development. At 48 hpf, eye, otic vesicle and heart defects in med14 or brg1 mutant embryos were similarly exacerbated in med14/brg1 double mutants (data not shown). Taken together, these data showed that zebrafish med14 or brg1 mutants displayed defects in neural crest-derived cells and tissues such like craniofacial cartilage and melanocyte. Furthermore, med14; brg1 double mutants, or single mutants where one allele of the other gene was lost, displayed a more severe phenotype than single mutants, suggesting overlapping functions of the Mediator and BAF complexes.
As the phenotypes observed in med14 and brg1 mutants involved multiple tissues and developmental stages, we subsequently focused our analysis on jaw cartilage development to uncover the mechanisms of med14 and brg1 function in neural crest development. Neural crest specification was first analyzed via expression of foxd3, sox9b and snail1b, which are expressed in pre-migratory neural crest at the dorsal side of neural tube at 14 hpf [21]. No overt difference in expression was apparent between control and mutant embryos (Fig. 2a–l), indicating that neural crest cells were properly specified in med14, brg1 and med14; brg1 mutants. Subsequent migration of neural crest cells was studied via use of a neural crest-specific sox10:EGFP transgenic line [22]. Neural crest cells in all three mutant backgrounds dispersed and migrated around the eye and optic stalk to reach the oral ectoderm and formed primodia of branchial arches by 24 hpf; with the behavior of neural crest cells being similar between mutant and control embryos (Fig. 3a and d). In situ hybridization also showed that markers of migratory facial neural crest, dlx2a and twist1a, were expressed normally in mutants at 18 hpf (Fig. 3b–c).
To determine if postmigratory cranial neural crest was properly maintained in these mutants, we examined expression of sox9a, dlx3b and hand2, which function in neural crest differentiation [23, 24], at 32 hpf. The expression of sox9a and dlx3b were both down-regulated in med14 and brg1 single mutants, and almost abolished in med14; brg1 double mutants (Fig. 3e–f). At 30 hpf, hand2 clearly marks rings of ventral neural crest in the pharyngeal arches (Fig. 3g). In med14 and brg1 mutant embryos, the expression of hand2 was reduced (Fig. 3g). In med14; brg1 double mutants, the expression was largely abolished except for in a small patch posterior to the eyes, which was maintained. Notably, hand2 expression in the heart tube remained in double mutants, indicating that the regulation of hand2 by brg1 and med14 is neural crest-specific (Fig. 3g). During jaw development, neural crest cells undergo extensive proliferation, so it is possible the defects observed in mutants were due to impaired cell proliferation or enhanced cell death. Cell proliferation and cell death were therefore analyzed at 36 hpf, however no obvious differences between control and mutants was observed (Fig. 4, P = 0.94 for cell proliferation and P = 0.95 for cell death). Taken together, this data suggests that in mutants neural crest cells are formed and migrate properly, but are subsequently unable to initiate a differentiation program towards becoming skeletogenic ectomesenchyme.
Neural crest differentiation depends both on intrinsic gene regulatory programs and signals from the surrounding environment [25]. As such, deficiencies either in neural crest cells themselves or other tissues could be responsible for the defects we observed in brg1 and med14 mutants and compound mutants. As signals from the endoderm and notochord are indispensable for the migration and differentiation of pharyngeal neural crest cells [26, 27], we first analyzed expression of foxa1 and shh, which are markers of these two tissues, at 30 hpf. Both genes showed apparently normal expression in mutants and compound mutants, suggesting that these tissues were not grossly abnormal (Fig. 5a–d and e–h). Since endodermal pouches play important role in patterning and differentiation of pharyngeal arches [28], we analyzed expression of nkx2.3 and tbx1, which are expressed in both the pre-pouch endoderm and surrounding mesoderm [29]. At 32 hpf, expression of nkx2.3 and tbx1 appeared normal in both wild type and mutant embryos (Fig. 5 i to l and m to p), suggesting that induction of the endodermal pouches is not dependent on med14 or brg1 function.
To directly examine the cellular autonomy of med14 and brg1 function, we employed a transplantation approach. Cells were first taken from the animal pole of 4 hpf wild type sox10:EGFP donor embryos and transferred to the equivalent location in wild type or mutant 4 hpf host embryos (Fig. 6a). Based on their localization in the host embryo, a proportion of these naïve cells will normally take on a cranial neural crest fate in these experiments, as indicated by donor cell GFP expression. We observed that in all cases wild type donor GFP-positive neural crest cells in either wild type (n = 33) or mutant (med14−/−: n = 26; brg1−/−: n = 24; med14; brg1 double mutant: n = 11) host embryos migrated to oral ectoderm and formed cartilage clusters, (Fig. 6f–i). Strikingly, as assayed by cartilage staining, we found that wild type donor cells could rescue anterior neurocranium cartilage phenotypes in med14 (43 %, n = 34) and med14; brg1 double mutant embryos (31 %, n = 14) (Fig. 6j–m). When sox10:EGFP activity was compared to cartilage staining results from the same embryos, we observed that anterior neurocranium cartilage entirely matched to GFP-positive clusters (Fig. 6g and k), strongly suggesting that wild type donor cells acted as the source of the cartilage.
To further examine the fate of med14 and brg1 mutant neural crest cells, transplantation experiments were next carried out in which wild type or mutant sox10:EGFP donor embryos were injected with rhodamine-dextran to allow observation of all donor cells (regardless of neural crest fate) (Fig. 7a). In wild type hosts, GFP-positive single or double brg1 and med14 mutant donor cells were observed that migrated to oral ectoderm at 24 hpf (Fig. 7 c, g, k and o). At 72 hpf, in 54 % of control (wild type donor, n = 44) transplantation experiments, donor cells were observed contributing to cartilage (Fig. 7d–f). In the case of brg1 mutant donor cells, 11 % of transplants showed a contribution to cartilage (n = 31, Fig. 7h–j), with GFP-positive cells displaying abnormal cell shape (compared to the elongated cuboidal shape observed in control transplants (Fig. 7d and h). Strikingly, in none of the transplants where med14 (n = 37) or med14; brg1 (n = 21) mutant donor cells were used was contribution to cartilage noted (based on GFP expression). However, imaging of the donor cell lineage tracer (rhodamine-dextran) showed that mutant cells still survived in the jaw region of wild type hosts (Fig. 7l–n and p–r). These experiments indicated that both med14 and brg1 function cell autonomously in neural crest cells, to govern proper skeletogenic ectomesenchyme differentiation. Further, the persistence of the lineage label in mutant donor cells, despite the absence of cartilage formation, argues for a model where med14 and brg1 are required subsequent to migration of neural crest cells, where they are necessary to the initiation of chondrocyte differentiation. In the absence of this activity, neural crest differentiation to cartilage, and jaw development, is severely perturbed.