Finally, to systematically examine how
adamts16 knockdown affected OC gene expression, we used high-throughput sequencing to compare the transcriptomes of 36-hpf
adamts16 MO1- and MO2-morphant eyes with those of control eyes. Data analysis showed that
adamts16 knockdown did not alter OC gene expression drastically; only 54 genes were found to be downregulated, and 47 genes were upregulated in MO1- or MO2-morphant eyes (fold change ≥ 2, probability ≥ 80%,
Fig. 5M;
Supplementary Table S1). KEGG pathway analysis showed that differentially expressed genes (DEGs) in
adamts16-morphant eyes compared with wild-type control eyes were enriched in the following pathways: p53, apoptosis, ribosome biogenesis, FoxO, and Toll-like receptor signaling (
Supplementary Table S2). Ribosome biogenesis pathway enrichment might reflect on the slightly different metabolism status between
adamts16-morphant and wild-type eyes. DEGs contributing to FoxO and Toll-like receptor pathways all overlapped with the p53 or apoptosis pathways (see
Supplementary Table S2 column J for the gene names); this may merely reflect the involvement of these two pathways in cell survival and stress response regulation. Even though p53 pathway components, including
p53, were enriched in the DEG list, MO knockdown of
p53 did not rescue the coloboma phenotype of
adamts16-morphant fish (
Fig. 4G); this suggests that upregulation of the p53 pathway may be due to nonspecific off-target effects of MO1/MO2 injection. In addition to
p53,
caspase8—a core component of the apoptosis cascade pathway—was significantly upregulated in
adamts16-morphant eyes (
Fig. 5M, right), and this upregulation may be responsible for the increased apoptosis activity observed in morphant eyes. Among the significantly downregulated genes, there were several retinal neurogenesis regulatory transcription factors, such as
atoh7,
crx,
rorb,
sox12, and
foxn4, and several crystallin genes, such as
crygm2d2 and
cryaa (
Fig. 5M, left), which may reflect the slightly delayed development of
adamts16-morphant eyes. The cell cycle regulator cdkn1a (p21) was among the significantly upregulated genes (
Fig. 5M, right), possibly leading to the decreased proliferation activity of
adamts16-morphant eyes. Interestingly,
fgf8 was also significantly upregulated in
adamts16-morphant eyes (
Fig. 5M, right, red underline). It has been shown that increased expression of
fgf8 in
aussicht (
aus) mutants disrupts ventral OC development and leads to coloboma,
34 a very similar phenotype to
adamts16-morphants. Therefore, we performed ISH to examine changes in
fgf8 expression. In the developing zebrafish head at 36 hpf,
fgf8 is strongly expressed at the midbrain-hindbrain boundary (
Fig. 5N, red arrowhead) and in the OS (Fig. 5N, red arrow) but can barely be seen in the OC (
Fig. 5N, dashed circle). However, in
adamts16-morphants,
fgf8 was clearly upregulated in the OC (
Fig. 5O, dashed circle). To investigate whether upregulated
fgf8 was responsible for coloboma formation in
adamts16-morphant fish, we coinjected
fgf8 MO
23 and
adamts16 MOs. Indeed, knocking down
fgf8 partially rescued the coloboma phenotype of both MO1- and MO2-morphant fish (
Fig. 5P), suggesting that coloboma formation in
adamts16-morphants was partially mediated by ectopic
fgf8 upregulation. Overall, gene expression analysis and rescue experiments suggest that
adamts16 regulates OF closure through regulation of cell proliferation, survival, and restriction of
fgf8 expression.