FIGURE SUMMARY
Title

Mitf-family transcription factor function is required within cranial neural crest cells to promote choroid fissure closure

Authors
Sinagoga, K.L., Larimer-Picciani, A.M., George, S.M., Spencer, S.A., Lister, J.A., Gross, J.M.
Source
Full text @ Development
Fig. 1

Zebrafish mitfa;tfec mutants phenocopy human colobomas. (A) At 4 dpf, mitfa−/−;tfec−/− mutants possess colobomas (outlined in red), with a range of severity (B). (C) At 48 hpf, serial sections through the distal (D) to proximal (P) axis of the CF reveal persistent expression of laminin in the mitfa−/−;tfec−/− mutant CF. The boxes indicate the areas enlarged in the insets showing laminin staining within the CF. Dorsal is upwards in all images. Scale bars: 50 µm.
Fig. 2

mitfa−/−;tfec−/− mutants display RPE and cNCC phenotypes. (A) At 48 hpf, mitf−/−;tfec+−/− and mitfa−/−;tfec−/− mutants display mild to severe hypopigmentation, respectively. The RPE eventually becomes normally pigmented by 4 dpf in both genotypes. (B) At 24 hpf, fewer cNCCs are present around the eye of mitfa−/−;tfec−/−;mitfa:GFP embryos than in the control (1) (***P=0.0007). However, mitfa−/−;tfec−/− mutants contain cNCCs posterior to the optic field (2). (C) Serial sections of mitfa−/−;tfec+/+;mitfa:GFP and mitfa−/−;tfec−/−;mitfa:GFP embryos at 48 hpf demonstrate that mitfa−/−;tfec−/− mutants possess fewer cNCCs in the POM surrounding the eyes. Dorsal is upwards in the images in B,C. Data are mean±s.e.m. Scale bars: 100 μm in A; 50 μm in B,C.
Fig. 3

mitfa and tfec are required within cNCCs to promote CF closure. (A) NCC transplantations. At 6 hpf, wild-type dextran-labeled cells are transplanted into the cNCC-fated region of a mitfa−/−;tfec−/− embryo. At 24 hpf, transplanted cells are detected in the cNCC population. (B) Example of 4 dpf wild-type (WT) cNCC transplanted mitfa−/−;tfec−/− embryo in which the coloboma has been rescued. Transplanted cNCCs are detected in and around the eye and rescue cNCC-derived pigmented melanocytes (red arrow) confirming successful transplantation. Scale bar: 25 µm. (C) Retina/RPE transplants. At 6 hpf, wild-type dextran-labeled cells are transplanted into the RPE/retinal-fated region of a mitfa−/−;tfec−/− mutant. At 24 hpf, transplanted cells are detected within the eye, indicating successful transplantation. (D) At 4 dpf, transplanted embryos still possess colobomas. Arrow indicates a coloboma in the retina/RPE transplanted eye. (E) Quantification of colobomas (largest angle of CF opening) in rescue experiments. CF opening is significantly reduced in mitfa−/−;tfec−/− embryos transplanted with wild-type cNCCs (P=0.0004, n=14 eyes). However, colobomas are not resolved in embryos transplanted with wild-type retina/RPE cells and are actually larger than in non-transplanted control mitfa−/−;tfec−/− mutants (P=0.037, n=6 eyes). tfec cDNA injection also rescues colobomas (P=0.033, n=14 eyes). (F) pDestTol2pA2-sox10:mCherry-nostop-t2A-FLAG-tfec-pA injection reduces CF opening in mitfa−/−;tfec−/− mutants. Arrows indicate mCherry+ (rescued) neural crest cells. Quantification of sox10:eGFP+ cells shows rescue of cNCCs within the eye field (P=0.023, n=20 mCherry+, n=8 mCherry− eyes). *P<0.05, ***P<0.001. Dorsal is upwards in all images. Data are mean±s.e.m. Scale bars: 25 μm (B,D); 50 μm (F).
Fig. S1

tfec-/- single mutants do not possess colobomas. Whole mount and transverse sections of 4dpf tfec -/- mutants reveals mild microphthalmia and pigmentation defects. No colobomas are detected. Dorsal is up in all images. Scale bar = 100μm.
Fig. S3

Loss of cNCCs in the POM is not the result cell death. A) Whole-mount images of mitfa -/-;tfec +/- and mitfa -/-;tfec -/- embryos reveals persistent loss of cNCC within the POM at 48hpf and 72hpf. B) Quantification of TUNEL+ cNCCs. While a trend of increased cell death is present, loss of cNCC numbers cannot be attributed solely to apoptosis at 24hpf (p=0.512, n=5 embryos).
Fig. S4

Transplantation of wild-type cells does not affect eye development. A)Wild-type cells transplanted into the region of cNCC origin in a mitfa -/-;tfec +/- embryo shows no effect on eye development. B) Similarly, transplanted wild-types cells targeting the retina/RPE in mitfa -/-;tfec +/+ embryos show no effect on eye development. C) Serial sections of representative retina/RPE and cNCC transplanted embryos. CF closure phenotypes are only detected in retina/RPE cell transplanted embryos. Dorsal is up in all images. Scale bar = 100μm.
Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Development
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