The function of
ZFRP2 was studied by morpholino knockdown. The main finding in
ZFRP2 morphants was that the eyes were smaller than in controls at both 24 hpf and 72 hpf, with the latter showing both defective retinal lamination and abnormal photoreceptors, lacking outer segments, which appeared by 60 hpf (
Figs. 4A,
4B).
29 The defects in retinal development were further investigated by staining with acridine orange, which stains apoptotic but not necrotic cells.
19,23,24,27 The number of apoptotic retinal neurons was significantly increased in
ZFRP2-depleted morphants (
Fig. 5). Cell death did not appear to affect other ocular structures, suggesting that it might be a consequence of abnormal retinal differentiation. Defects in retinal lamination or differentiation with increased cell death have been observed in the context of genetic defects affecting transcription factors (e.g., math5, NR2E3),
30 –32 cell cycle regulators (e.g., Cdkn1b/c, cdk5),
33 and the intraflagellar transport protein IFT88.
34 Mutations in the nuclear receptor factor, NR2E3, result in abnormal retinal lamination and differentiation.
31,32 A homozygous mutation in the mouse
IFT88 gene causes abnormal photoreceptor differentiation, with reduced and disorganized outer segment but normal lamination.
34 Cell death resulting from
ZFRP2 knockdown could result from abnormal retinal development, as seen in
Nr2e3 31,32 or
Sdkn1b/c mutants.
33 Alternatively, abnormal retinal development could itself result from severe and early retinal degeneration, as appears likely with
smarca5 and
IFT88 mutations.
34,35 The small eye phenotype of
ZFRP2-depleted morphants appears to be caused by both abnormal retinal development and extensive retinal cell death.