Gene Ontology annotation through association of InterPro records with GO terms
Electronic Gene Ontology annotations created by ARBA machine learning models
Combined Automated Annotation using Multiple IEA Methods
Highly Efficient Knockout of a Squid Pigmentation Gene
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TDO was selected as a knockout target because ommochromes pigment cephalopod eyes and chromatophores, and TDO catalyzes the first committed step in their biosynthesis.
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A single TDO gene was identified in the D. pealeii genome, spanning 13 exons over 120 kb. Phylogenetic analysis confirmed the gene as a bona fide TDO distinct from IDO.
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In situ hybridization showed TDO expression in chromatophore precursors on arms and mantle, and in eyes; RNAseq confirmed highest expression in retina and chromatophore layer of skin.
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TDO-selective inhibitor 680C91 completely blocked pigmentation in both eyes and chromatophores, with animals developing normally otherwise.
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CRISPR-Cas9 knockout with two sgRNAs targeting exons 1 and 2 eliminated pigmentation with >90% disruption efficiency. Ink sac melanin was unaffected, confirming specificity for the ommochrome pathway.
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This was the first gene knockout achieved in any cephalopod.
Ommochromes in invertebrates: biochemistry and cell biology
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Ommochromes are widely occurring coloured molecules of invertebrates, arising from tryptophan catabolism through the Tryptophan → Ommochrome pathway. They are mainly known to mediate compound eye vision, as well as reversible and irreversible colour patterning.
"Ommochromes are widely occurring coloured molecules of invertebrates, arising from tryptophan catabolism through the so-called Tryptophan to Ommochrome pathway."
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Ommochromasomes are the organelles where ommochromes are synthesised and stored, playing an important role in mediating ommochrome functions. They are part of the lysosome-related organelles (LROs) family, which includes vertebrate melanosomes.
"Ommochromasomes are the organelles where ommochromes are synthesised and stored. Hence, they play an important role in mediating ommochrome functions."
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Ommochromes may also be involved in cell homeostasis by detoxifying free tryptophan and buffering oxidative stress, in addition to their roles in pigmentation and vision.
"Ommochromes might also be involved in cell homeostasis by detoxifying free tryptophan and buffering oxidative stress."
Creation of an albino squid line by CRISPR-Cas9 and its application for in vivo functional imaging of neural activity
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In E. berryi, TDO knockout alone did not eliminate pigmentation despite >90% disruption efficiency, due to compensation by IDO. D. pealeii genome lacks IDO orthologs, explaining why TDO knockout alone is sufficient in that species.
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E. berryi homozygous TDO knockout showed slightly delayed pigmentation during development. TDO+IDO double knockout produced complete albinism.
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TDO expression in E. berryi is approximately 20-fold higher than IDO in stage 24 embryos. TDO is expressed in eyes and chromatophores, similar to D. pealeii.
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Albino E. berryi enabled the first in vivo calcium imaging of neural activity in a cephalopod using two-photon microscopy.