Tryptophan 2,3-dioxygenase (TDO) is a heme-dependent oxidoreductase that catalyzes the first committed step in ommochrome pigment biosynthesis in D. pealeii, converting L-tryptophan to N-formylkynurenine via oxidative cleavage of the indole ring. TDO is expressed in the retina and chromatophore layer of the skin, the two principal ommochrome-pigmented tissues. Crawford et al. (2020) demonstrated that CRISPR-Cas9 knockout of TDO in D. pealeii embryos efficiently eliminated pigmentation in both eyes and chromatophores with >90% disruption efficiency, while ink sac melanin pigmentation was unaffected. This was the first gene knockout achieved in any cephalopod. TDO inhibition with 680C91 also completely blocked pigmentation, confirming the enzyme is required for ommochrome synthesis. Notably, D. pealeii lacks an IDO (indoleamine 2,3-dioxygenase) ortholog, unlike Euprymna berryi where IDO can compensate for TDO loss. TDO belongs to the PANTHER family PTHR10138 (Tryptophan 2,3-dioxygenase), is predicted to function as a homotetramer (dimer of dimers), and contains a single heme cofactor per subunit. The gene spans 13 exons over 120 kb of genomic DNA.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
|
GO:0004833
L-tryptophan 2,3-dioxygenase activity
|
IEA
GO_REF:0000120 |
ACCEPT |
Summary: L-tryptophan 2,3-dioxygenase activity (EC 1.13.11.11) is the defining enzymatic function of TDO. This IEA annotation is based on combined automated methods (InterPro, ARBA, PANTHER, UniRule). In D. pealeii, Crawford et al. (2020) demonstrated that CRISPR-Cas9 knockout of TDO eliminated ommochrome pigmentation, and pharmacological inhibition with 680C91 (a TDO-selective inhibitor) blocked pigmentation in both eyes and chromatophores [PMID:32735817]. The UniProt entry confirms TDO catalyzes L-tryptophan + O2 = N-formylkynurenine (Rhea:RHEA:24536). Phylogenetic analysis confirmed this gene as a bona fide TDO (distinct from IDO) [PMID:32735817].
Reason: This is the core enzymatic function of TDO, supported by sequence homology, PANTHER family assignment (PTHR10138), HAMAP rule MF_03020, and functional knockout data in this species showing loss of ommochrome pigmentation.
Supporting Evidence:
PMID:32735817
In invertebrates, Tryptophan, 2,3 Dioxygenase (TDO) catalyzes the first committed step in ommochrome biosynthesis, converting Tryptophan to N-Formylkyneurenine.
PMID:32735817
A phylogenetic comparison of this sequence with diverse TDOs, and indolamine-2,3-dioxygenases (a different enzyme that catalyzes the same reaction), supported its identity as TDO.
|
|
GO:0006569
L-tryptophan catabolic process
|
IEA
GO_REF:0000117 |
ACCEPT |
Summary: L-tryptophan catabolic process is annotated via ARBA machine learning. TDO catalyzes the oxidative cleavage of L-tryptophan as the first step in the kynurenine pathway. Crawford et al. (2020) showed that TDO knockout eliminates ommochrome biosynthesis from tryptophan in D. pealeii [PMID:32735817]. This term is correct but could be more specific. The UniProt record lists both GO:0019441 (L-tryptophan catabolic process to kynurenine) and GO:0019442 (L-tryptophan catabolic process to acetyl-CoA), and GO:0019441 more precisely captures TDO function as it catalyzes the first step to kynurenine specifically.
Reason: L-tryptophan catabolic process correctly describes the biological process TDO participates in. While a more specific term like L-tryptophan catabolic process to kynurenine (GO:0019441) would be ideal, that term is now obsolete in GO. GO:0006569 is the most appropriate active term for this function. Crawford et al. (2020) showed TDO knockout eliminates the tryptophan-to-ommochrome pathway in D. pealeii [PMID:32735817].
Supporting Evidence:
PMID:32735817
TDO catalyzes the first committed step in ommochrome biosynthesis, converting Tryptophan to N-Formylkyneurenine.
|
|
GO:0020037
heme binding
|
IEA
GO_REF:0000120 |
ACCEPT |
Summary: Heme binding is annotated by combined IEA methods (InterPro, ARBA, PANTHER, UniRule). The UniProt record explicitly states: "Binds 1 heme group per subunit" (HAMAP-Rule:MF_03020), and lists heme as a cofactor (ChEBI:30413). TDO is a heme-dependent dioxygenase, and heme is essential for its catalytic mechanism. This is well-established for TDO family members across all species.
Reason: Heme binding is integral to TDO catalytic function. The enzyme requires bound heme to catalyze oxidative cleavage of the tryptophan indole ring. This is confirmed by UniProt cofactor annotation and is a defining feature of the TDO family (HAMAP MF_03020, Pfam PF03301 Trp_dioxygenase).
Supporting Evidence:
PMID:32735817
we determined whether a TDO-selective inhibitor (680C91) impeded pigmentation in developing embryos.
|
|
GO:0046872
metal ion binding
|
IEA
GO_REF:0000002 |
MARK AS OVER ANNOTATED |
Summary: Metal ion binding is annotated via InterPro2GO mapping (IPR037217). This is correct as TDO binds iron through its heme prosthetic group. However, this term is very generic and redundant with the more informative heme binding (GO:0020037) annotation already present, which more precisely captures the nature of the metal cofactor interaction.
Reason: While technically correct (TDO binds iron via heme), GO:0046872 (metal ion binding) is too generic to be informative. The heme binding annotation (GO:0020037) already captures this function at a more appropriate level of specificity. The metal ion binding term does not add useful functional information beyond what heme binding conveys.
|
|
GO:0006727
ommochrome biosynthetic process
|
IDA
PMID:32735817 Highly Efficient Knockout of a Squid Pigmentation Gene |
NEW |
Summary: NEW annotation. Crawford et al. (2020) demonstrated by CRISPR-Cas9 knockout that TDO is required for ommochrome biosynthesis in D. pealeii. TDO knockout eliminated pigmentation in both chromatophores and eyes, which contain ommochrome pigments. The TDO-selective inhibitor 680C91 also blocked pigmentation. In situ hybridization confirmed TDO expression in chromatophore precursors and eyes. This is the most biologically meaningful annotation for this gene in cephalopods, as ommochrome pigmentation is the primary downstream biological process served by TDO in this organism.
Reason: Ommochrome biosynthesis is the key biological process requiring TDO in cephalopods. CRISPR knockout and pharmacological inhibition both demonstrate TDO is essential for ommochrome production. This annotation is not present in the existing GOA set but is strongly supported by direct experimental evidence in this species.
Supporting Evidence:
PMID:32735817
In cephalopods, the pigments in the eyes and chromatophores are ommochromes, a derivative of tryptophan
PMID:32735817
Knocking out TDO in squid embryos efficiently eliminated pigmentation. By precisely timing CRISPR-Cas9 delivery during early development, the degree of pigmentation could be finely controlled. Genotyping revealed knockout efficiencies routinely greater than 90%.
PMID:32735817
We added 680C91 to developing embryos at stage 20, and it clearly blocked pigmentation in both the eyes and chromatophores, with animals developing normally otherwise.
PMID:29989284
Ommochromes are widely occurring coloured molecules of invertebrates, arising from tryptophan catabolism through the so-called Tryptophan → Ommochrome pathway.
|
|
GO:0043473
pigmentation
|
IMP
PMID:32735817 Highly Efficient Knockout of a Squid Pigmentation Gene |
NEW |
Summary: NEW annotation. Crawford et al. (2020) showed that TDO knockout by CRISPR-Cas9 resulted in loss of pigmentation in D. pealeii embryos, with chromatophore and eye pigmentation eliminated. Ink sac melanin was unaffected, demonstrating TDO specifically contributes to ommochrome-based pigmentation. Ahuja et al. (2023) further showed in E. berryi that TDO knockout delays pigmentation and TDO+IDO double knockout produces complete albinism [PMID:37343558].
Reason: Pigmentation is the primary phenotype affected by TDO loss of function. The knockout phenotype directly demonstrates involvement in pigmentation. This is a core biological role for TDO in cephalopods, where ommochrome pigments color the chromatophores and retina.
Supporting Evidence:
PMID:32735817
pigmentation is completely absent from the chromatophores and is minimal in the eyes which are light red
PMID:37343558
The knockout of tdo in D. pealeii completely inhibited pigmentation but had little to no effect in E. berryi.
|
Q: Does D. pealeii TDO have any role in tryptophan catabolism beyond ommochrome biosynthesis (e.g. kynurenine pathway metabolites for immune or neural signaling)?
Suggested experts: Rosenthal JJC, Albertin CB
Q: What are the consequences of partial TDO loss on chromatophore function and dynamic color change in adult squid?
Suggested experts: Crawford K
Q: Is there any post-translational regulation of TDO activity in cephalopods, as seen with substrate activation in mammalian TDO?
Experiment: Characterize the enzymatic kinetics of recombinant D. pealeii TDO, including heme stoichiometry and substrate specificity, to confirm in vitro activity.
Hypothesis: D. pealeii TDO has similar catalytic properties to mammalian TDO, with strict specificity for L-tryptophan over other indoleamine substrates.
Type: biochemistry
Experiment: Use single-cell RNA-seq to map TDO expression across chromatophore subtypes and developmental stages in D. pealeii embryos.
Hypothesis: TDO expression is restricted to ommochrome-producing cell types (chromatophores and retinal pigment cells) and onset correlates with pigmentation timing.
Type: transcriptomics
Experiment: Measure kynurenine pathway metabolite levels (N-formylkynurenine, kynurenine, 3-hydroxykynurenine, xanthurenic acid) in TDO knockout vs wildtype D. pealeii embryos by LC-MS/MS.
Hypothesis: TDO knockout eliminates kynurenine pathway flux entirely in D. pealeii due to the absence of IDO, unlike in E. berryi where IDO provides compensation.
Type: metabolomics
Experiment: Test whether partial TDO knockout in adult squid affects chromatophore expansion dynamics and camouflage behavior using video analysis.
Hypothesis: Reduced ommochrome content from partial TDO loss impairs chromatophore opacity and dynamic range of color change.
Type: behavioral/physiological
provider: falcon
model: Edison Scientific Literature
cached: false
start_time: '2026-05-02T17:11:54.029198'
end_time: '2026-05-02T17:20:36.588688'
duration_seconds: 522.56
template_file: templates/gene_research_go_focused.md
template_variables:
organism: DORPE
gene_id: TDO
gene_symbol: TDO
uniprot_accession: A0A7G4RN94
protein_description: 'RecName: Full=Tryptophan 2,3-dioxygenase {ECO:0000256|HAMAP-Rule:MF_03020};
Short=TDO {ECO:0000256|HAMAP-Rule:MF_03020}; EC=1.13.11.11 {ECO:0000256|HAMAP-Rule:MF_03020};
AltName: Full=Tryptamin 2,3-dioxygenase {ECO:0000256|HAMAP-Rule:MF_03020}; AltName:
Full=Tryptophan oxygenase {ECO:0000256|HAMAP-Rule:MF_03020}; Short=TO {ECO:0000256|HAMAP-Rule:MF_03020};
Short=TRPO {ECO:0000256|HAMAP-Rule:MF_03020}; AltName: Full=Tryptophan pyrrolase
{ECO:0000256|HAMAP-Rule:MF_03020}; AltName: Full=Tryptophanase {ECO:0000256|HAMAP-Rule:MF_03020};'
gene_info: Name=TDO {ECO:0000313|EMBL:QMT62772.1};
organism_full: Doryteuthis pealeii (Longfin inshore squid) (Loligo pealeii).
protein_family: Belongs to the tryptophan 2,3-dioxygenase family.
protein_domains: Trp/Indoleamine_2_3_dOase-like. (IPR037217); Trp_2_3_dOase. (IPR004981);
Trp_dioxygenase (PF03301)
provider_config:
timeout: 600
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citation_count: 25
BEFORE YOU BEGIN RESEARCH: You MUST verify you are researching the CORRECT gene/protein. Gene symbols can be ambiguous, especially for less well-characterized genes from non-model organisms.
DO NOT PROCEED WITH RESEARCH ON A DIFFERENT GENE. Instead:
- State clearly: "The gene symbol 'TDO' is ambiguous or literature is limited for this specific protein"
- Explain what you found (e.g., "Found extensive literature on a different gene with the same symbol in a different organism")
- Describe the protein based ONLY on the UniProt information provided above
- Suggest that the protein function can be inferred from domain/family information
Please provide a comprehensive research report on the gene TDO (gene ID: TDO, UniProt: A0A7G4RN94) in DORPE.
The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.
You should prioritize authoritative reviews and primary scientific literature when conducting research. You can supplement
this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.
We are specifically interested in the primary function of the gene - for enzymes, what reaction is catalyzed, and what is the substrate specificity? For transporters, what is the substrate? For structural proteins or adapters, what is the broader structural role? For signaling molecules, what is the role in the pathway.
We are interested in where in or outside the cell the gene product carries out its function.
We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.
Include evidence where possible. We are interested in both experimental evidence as well as inference from structure, evolution, or bioinformatic analysis. Precise studies should be prioritized over high-throughput, where available.
Question: You are an expert researcher providing comprehensive, well-cited information.
Provide detailed information focusing on:
1. Key concepts and definitions with current understanding
2. Recent developments and latest research (prioritize 2023-2024 sources)
3. Current applications and real-world implementations
4. Expert opinions and analysis from authoritative sources
5. Relevant statistics and data from recent studies
Format as a comprehensive research report with proper citations. Include URLs and publication dates where available.
Always prioritize recent, authoritative sources and provide specific citations for all major claims.
BEFORE YOU BEGIN RESEARCH: You MUST verify you are researching the CORRECT gene/protein. Gene symbols can be ambiguous, especially for less well-characterized genes from non-model organisms.
DO NOT PROCEED WITH RESEARCH ON A DIFFERENT GENE. Instead:
- State clearly: "The gene symbol 'TDO' is ambiguous or literature is limited for this specific protein"
- Explain what you found (e.g., "Found extensive literature on a different gene with the same symbol in a different organism")
- Describe the protein based ONLY on the UniProt information provided above
- Suggest that the protein function can be inferred from domain/family information
Please provide a comprehensive research report on the gene TDO (gene ID: TDO, UniProt: A0A7G4RN94) in DORPE.
The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.
You should prioritize authoritative reviews and primary scientific literature when conducting research. You can supplement
this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.
We are specifically interested in the primary function of the gene - for enzymes, what reaction is catalyzed, and what is the substrate specificity? For transporters, what is the substrate? For structural proteins or adapters, what is the broader structural role? For signaling molecules, what is the role in the pathway.
We are interested in where in or outside the cell the gene product carries out its function.
We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.
Include evidence where possible. We are interested in both experimental evidence as well as inference from structure, evolution, or bioinformatic analysis. Precise studies should be prioritized over high-throughput, where available.
The gene symbol TDO is ambiguous across taxa (e.g., vertebrate TDO2), but the Doryteuthis pealeii target relevant to UniProt A0A7G4RN94 is strongly supported as a tryptophan 2,3-dioxygenase family enzyme by: (i) a squid-specific locus identified by BLAST against known TDO/IDO sequences and placed in a phylogeny to distinguish TDO from related dioxygenases (e.g., IDO), (ii) mapping to a single genomic locus reported as 13 exons spanning >120 kb and deposited as GenBank MT648678, and (iii) a causal pigmentation phenotype following inhibition or CRISPR disruption of this locus in D. pealeii embryos/hatchlings. (crawford2020highlyefficientknockout pages 8-10, crawford2020highlyefficientknockout pages 1-3)
Tryptophan 2,3-dioxygenase (TDO; EC 1.13.11.11) is a heme-containing dioxygenase that catalyzes oxidative cleavage of the indole ring of L-tryptophan to yield N-formyl-L-kynurenine (NFK)—the first (often rate-limiting) committed step of the kynurenine pathway (KP). (alves2024thebiologyand pages 2-5)
Although TDO and indoleamine 2,3-dioxygenases (IDO1/IDO2) perform the same overall initial KP chemistry, they are described as differing in structure, substrate specificity, and tissue distribution, which affects biological roles across organisms. (alves2024thebiologyand pages 2-5)
A 2024 KP-focused review summarizes that (for human TDO as a well-studied reference) TDO shows high physiological specificity for L-tryptophan (with much lower activity on D-tryptophan), supporting the common annotation of TDO enzymes as primarily L-Trp oxygenases. (alves2024thebiologyand pages 5-6)
Given that D. pealeii TDO is experimentally tied to ommochrome pigmentation derived from tryptophan catabolism (Section 3), the most parsimonious functional annotation for UniProt A0A7G4RN94 is an L-tryptophan dioxygenase entering the kynurenine/ommochrome branch. (crawford2020highlyefficientknockout pages 1-3, alves2024thebiologyand pages 2-5)
In invertebrates, tryptophan catabolism through the Tryptophan → Ommochrome pathway produces ommochrome pigments (e.g., xanthommatin/ommatins) used in coloration and vision-related tissues. An authoritative synthesis in Biological Reviews identifies TDO (EC 1.13.11.11) as the enzyme catalyzing the first step of this pathway and lists heme as its cofactor in ommochrome biosynthesis contexts. (figon2019ommochromesininvertebrates pages 8-9, figon2019ommochromesininvertebrates pages 9-10)
In D. pealeii embryos, fluorescent in situ hybridization detected TDO mRNA in the developing eyes/retina and in punctate signals consistent with developing chromatophores on the mantle/arms. (crawford2020highlyefficientknockout pages 3-5, crawford2020highlyefficientknockout media 7a0753cd)
Complementing embryo spatial expression, adult tissue RNA-seq (as analyzed in the same study) indicated highest expression in retina and in the chromatophore layer of the skin, consistent with a role in local pigment precursor production. (crawford2020highlyefficientknockout pages 3-5)
Treatment of embryos with the TDO-selective inhibitor 680C91 (reported regimen: 3.15 mM, from stage 20–27) “clearly blocked pigmentation” in both eyes and chromatophores, supporting that enzymatic TDO activity is upstream of ommochrome deposition in these tissues. (crawford2020highlyefficientknockout pages 1-3, crawford2020highlyefficientknockout pages 10-12)
CRISPR-Cas9 disruption of D. pealeii tdo produced a strong and reproducible loss-of-pigmentation phenotype in hatchlings, with near-complete loss of pigmentation in eyes and chromatophores, while the ink sac (melanin-based pigment) was unaffected—supporting specificity for tryptophan-derived ommochromes rather than melanin. (crawford2020highlyefficientknockout pages 1-3, crawford2020highlyefficientknockout pages 3-5, crawford2020highlyefficientknockout media 7a0753cd)
A peer-reviewed research highlight independently summarizes the same core observation: CRISPR targeting of TDO in D. pealeii yields marked loss/reduction of pigment in eyes and chromatophores, with reported knockout efficiencies >90% in genotyped embryos. (neff2020crisprincephalopods pages 1-1)
Amplicon sequencing around CRISPR cut sites showed high on-target editing with quantitative metrics including:
- Individuals with >90% disruption at the tdo locus; across specimens, cumulative disruption about 30%–95%. (crawford2020highlyefficientknockout pages 5-7)
- Many distinct indel outcomes per embryo (mean 38 ± 13 events), with no single dominant allele (most frequent event around 15% ± 8%). (crawford2020highlyefficientknockout pages 5-7)
- Indel size distributions: sgRNA1 median deletion 9 nt / median insertion 2 bp; sgRNA2 median deletion 7 nt / median insertion 3 bp. (crawford2020highlyefficientknockout pages 5-7)
These quantitative results strengthen the genotype–phenotype link supporting TDO’s functional annotation in squid pigmentation. (crawford2020highlyefficientknockout pages 5-7, crawford2020highlyefficientknockout pages 1-3)
Direct subcellular localization of squid TDO protein (e.g., cytosolic vs organellar) was not established by protein-level localization in the retrieved D. pealeii sources; however, within the ommochrome pathway literature (in insects as the best-established reference), TDO is discussed as acting early in tryptophan catabolism and is contrasted with downstream steps having mitochondrial association (e.g., kynurenine monooxygenase in some systems). (cordell2021biosynthesisofthe pages 4-6)
Therefore, for UniProt A0A7G4RN94, the most supportable localization statement from available evidence is tissue-level localization to retina and chromatophore-bearing skin, with subcellular localization remaining an inference rather than squid-specific experimental fact. (crawford2020highlyefficientknockout pages 3-5, crawford2020highlyefficientknockout media 7a0753cd)
A 2024 review in International Journal of Molecular Sciences (Aug 2024) explicitly defines TDO (EC 1.13.11.11) as heme-containing and producing N-formyl-L-kynurenine, and emphasizes differences vs IDO enzymes—helpful for avoiding misannotation and for predicting substrate specificity (L-Trp preference) in non-model species. URL: https://doi.org/10.3390/ijms25169082 (alves2024thebiologyand pages 2-5, alves2024thebiologyand pages 5-6)
A 2024 primary research article in JACS Au (Oct 2024) reports gram-scale biomimetic synthesis and characterization of ommatins (xanthommatin family) and compares antioxidant activities among ommatin derivatives; while not a gene study, it underscores continued growth of cephalopod-linked ommochrome chemistry and quantification of functional properties of pathway products. URL: https://doi.org/10.1021/jacsau.4c00667 (paper retrieved; see listing in search results)
A recent preprint posted 2024-10-04 (bioRxiv record published Oct 2025) describes growth-coupled microbial biosynthesis of xanthommatin and uses a minimal two-gene module (TDO + kynurenine formamidase) as part of the conversion from L-tryptophan toward pigment precursors, illustrating practical implementation of the pathway for production. URL: https://doi.org/10.1101/2024.10.04.616593 (bushin2025growthcoupledmicrobialbiosynthesis pages 3-6)
The D. pealeii work demonstrates an operational, high-efficiency CRISPR workflow in cephalopods using TDO pigmentation loss as a visible readout, enabling real-world deployment of genome editing in squid embryos for functional studies. (crawford2020highlyefficientknockout pages 1-3, neff2020crisprincephalopods pages 1-1)
Ommochrome pigments (derived from the kynurenine branch initiated by TDO) are increasingly targeted for (i) synthetic access and characterization of optical/redox properties and (ii) biomanufacturing approaches for pigments, where upstream enzymes such as TDO define entry into the pathway. (bushin2025growthcoupledmicrobialbiosynthesis pages 3-6)
An authoritative review of invertebrate ommochromes emphasizes that ommochromes arise from tryptophan catabolism through the “Tryptophan → Ommochrome pathway” and highlights TDO as the entry enzyme that opens the indole ring and governs flux into pigment biogenesis, as well as documenting cellular biology of ommochrome-producing organelles (ommochromasomes) and methodological advances enabling pathway dissection. URL: https://doi.org/10.1111/brv.12441 (figon2019ommochromesininvertebrates pages 8-9, figon2019ommochromesininvertebrates pages 9-10)
The retrieved figure panels directly support the two most important organism-specific claims:
- TDO expression in squid embryonic retina and chromatophores by FISH. (crawford2020highlyefficientknockout media 7a0753cd)
- Wild-type vs TDO knockout pigmentation loss in eyes/chromatophores. (crawford2020highlyefficientknockout media 12743825)
| Evidence type | Key findings | Source (author year journal) | URL | Publication date | Notes linking to UniProt A0A7G4RN94 and ommochrome/kynurenine pathway |
|---|---|---|---|---|---|
| Sequence/phylogeny | D. pealeii tdo was identified by BLASTP using human TDO/IDO queries, aligned with MUSCLE, and placed by approximate maximum-likelihood phylogeny to distinguish TDO from related dioxygenases; locus mapped to the squid genome; authors report a single TDO gene with 13 exons spanning >120 kb and deposited the locus as GenBank MT648678 (crawford2020highlyefficientknockout pages 8-10, crawford2020highlyefficientknockout pages 1-3) | Crawford et al. 2020, Current Biology | https://doi.org/10.1016/j.cub.2020.06.099 | Sep 2020 | Strongly matches UniProt A0A7G4RN94 annotation as tryptophan 2,3-dioxygenase (EC 1.13.11.11) in Doryteuthis pealeii; phylogeny helps avoid confusion with vertebrate TDO2 or IDO paralogs (crawford2020highlyefficientknockout pages 8-10, crawford2020highlyefficientknockout pages 1-3) |
| Expression | Fluorescent in situ hybridization in stage 22 embryos showed TDO transcript in developing eyes/retina and punctate signal in chromatophores on mantle and arms; adult RNA-seq showed highest expression in retina and the chromatophore layer of the skin (crawford2020highlyefficientknockout pages 3-5, crawford2020highlyefficientknockout media 7a0753cd) | Crawford et al. 2020, Current Biology | https://doi.org/10.1016/j.cub.2020.06.099 | Sep 2020 | Expression pattern fits a role in local ommochrome pigment production rather than general melanin synthesis, consistent with the squid UniProt family/domain assignment (crawford2020highlyefficientknockout pages 3-5, crawford2020highlyefficientknockout media 7a0753cd) |
| Pharmacology | Treatment with the TDO-selective inhibitor 680C91 at 3.15 mM from stage 20 to stage 27 blocked pigmentation in both eyes and chromatophores (methods and summarized results reported) (crawford2020highlyefficientknockout pages 1-3, crawford2020highlyefficientknockout pages 10-12) | Crawford et al. 2020, Current Biology | https://doi.org/10.1016/j.cub.2020.06.099 | Sep 2020 | Pharmacologic phenocopy supports that squid TDO activity is required upstream of ommochrome deposition; this is consistent with TDO catalyzing entry into the kynurenine/ommochrome branch from tryptophan (crawford2020highlyefficientknockout pages 1-3, crawford2020highlyefficientknockout pages 10-12) |
| CRISPR phenotype | CRISPR-Cas9 knockout of tdo in G0 embryos caused near-complete loss of pigmentation in hatchlings, with greatly reduced or absent pigment in eyes and chromatophores; earlier injections produced stronger whole-animal effects, later injections mosaic phenotypes; ink sac pigmentation was unaffected (crawford2020highlyefficientknockout pages 1-3, crawford2020highlyefficientknockout pages 3-5, crawford2020highlyefficientknockout media 7a0753cd) | Crawford et al. 2020, Current Biology; Neff 2020, Lab Animal | https://doi.org/10.1016/j.cub.2020.06.099 ; https://doi.org/10.1038/s41684-020-0655-5 | Sep 2020; Sep 2020 | This is the most direct functional evidence that the D. pealeii protein corresponding to UniProt A0A7G4RN94 is required for ommochrome-based pigmentation in chromatophores/eyes, not melanin in the ink sac (crawford2020highlyefficientknockout pages 1-3, crawford2020highlyefficientknockout pages 3-5, neff2020crisprincephalopods pages 1-1) |
| Genotyping stats | Edited embryos showed >90% disruption in some individuals; cumulative tdo disruption ranged about 30%–95%; mean 38 ± 13 distinct indel events per animal; most frequent single event only about 15% ± 8%; median deletion/insertion sizes: sgRNA1 9 nt / 2 bp, sgRNA2 7 nt / 3 bp (crawford2020highlyefficientknockout pages 5-7, crawford2020highlyefficientknockout pages 3-5) | Crawford et al. 2020, Current Biology | https://doi.org/10.1016/j.cub.2020.06.099 | Sep 2020 | High on-target editing efficiency links genotype to pigment-loss phenotype and supports confident functional annotation of the squid TDO locus represented by UniProt A0A7G4RN94 (crawford2020highlyefficientknockout pages 5-7, crawford2020highlyefficientknockout pages 3-5) |
| Pathway context/biochemistry | TDO is a heme-containing enzyme, EC 1.13.11.11, that catalyzes oxidative opening of the indole ring of L-tryptophan to N-formyl-L-kynurenine, the first and often rate-limiting step of the kynurenine pathway; TDO and IDO catalyze the same first reaction, but TDO is structurally distinct and generally more substrate-specific for L-tryptophan (alves2024thebiologyand pages 2-5, alves2024thebiologyand pages 5-6, figon2019ommochromesininvertebrates pages 9-10) | de Fátima Alves et al. 2024, Int J Mol Sci; Figon & Casas 2019, Biological Reviews | https://doi.org/10.3390/ijms25169082 ; https://doi.org/10.1111/brv.12441 | Aug 2024; Jul 2019 | For cephalopod pigmentation, this reaction supplies the kynurenine-derived precursors that feed the ommochrome pathway; thus the UniProt annotation of A0A7G4RN94 as TDO is biochemically coherent with the squid pigmentation phenotypes (alves2024thebiologyand pages 2-5, figon2019ommochromesininvertebrates pages 8-9, figon2019ommochromesininvertebrates pages 9-10) |
Table: This table summarizes the main lines of evidence supporting functional annotation of Doryteuthis pealeii TDO, including identity verification, tissue expression, perturbation phenotypes, and conserved biochemistry. It is useful for linking UniProt A0A7G4RN94 to the kynurenine/ommochrome pigmentation pathway with quantitative support.
Primary molecular function: Heme-dependent dioxygenase catalyzing oxidative cleavage of L-tryptophan → N-formyl-L-kynurenine (EC 1.13.11.11), initiating kynurenine-pathway flux that supplies precursors for ommochrome pigment synthesis. (alves2024thebiologyand pages 2-5, figon2019ommochromesininvertebrates pages 8-9, crawford2020highlyefficientknockout pages 1-3)
Biological process (squid evidence): Required for pigment formation in eyes/retina and chromatophores; disruption causes near-complete loss of pigmentation without affecting melanin-based ink pigment. (crawford2020highlyefficientknockout pages 1-3, crawford2020highlyefficientknockout pages 3-5, crawford2020highlyefficientknockout media 7a0753cd)
Localization (supported level): Enriched expression in pigment tissues (retina; chromatophore skin layer; embryonic chromatophores), consistent with localized pigment precursor biosynthesis. (crawford2020highlyefficientknockout pages 3-5, crawford2020highlyefficientknockout media 7a0753cd)
References
(crawford2020highlyefficientknockout pages 8-10): Karen Crawford, Juan F. Diaz Quiroz, Kristen M. Koenig, Namrata Ahuja, Caroline B. Albertin, and Joshua J.C. Rosenthal. Highly efficient knockout of a squid pigmentation gene. Current Biology, 30:3484-3490.e4, Sep 2020. URL: https://doi.org/10.1016/j.cub.2020.06.099, doi:10.1016/j.cub.2020.06.099. This article has 83 citations and is from a highest quality peer-reviewed journal.
(crawford2020highlyefficientknockout pages 1-3): Karen Crawford, Juan F. Diaz Quiroz, Kristen M. Koenig, Namrata Ahuja, Caroline B. Albertin, and Joshua J.C. Rosenthal. Highly efficient knockout of a squid pigmentation gene. Current Biology, 30:3484-3490.e4, Sep 2020. URL: https://doi.org/10.1016/j.cub.2020.06.099, doi:10.1016/j.cub.2020.06.099. This article has 83 citations and is from a highest quality peer-reviewed journal.
(alves2024thebiologyand pages 2-5): Luana de Fátima Alves, J. B. Moore, and D. Kell. The biology and biochemistry of kynurenic acid, a potential nutraceutical with multiple biological effects. International Journal of Molecular Sciences, Aug 2024. URL: https://doi.org/10.3390/ijms25169082, doi:10.3390/ijms25169082. This article has 31 citations.
(alves2024thebiologyand pages 5-6): Luana de Fátima Alves, J. B. Moore, and D. Kell. The biology and biochemistry of kynurenic acid, a potential nutraceutical with multiple biological effects. International Journal of Molecular Sciences, Aug 2024. URL: https://doi.org/10.3390/ijms25169082, doi:10.3390/ijms25169082. This article has 31 citations.
(figon2019ommochromesininvertebrates pages 8-9): Florent Figon and Jérôme Casas. Ommochromes in invertebrates: biochemistry and cell biology. Biological Reviews, 94:156-183, Jul 2019. URL: https://doi.org/10.1111/brv.12441, doi:10.1111/brv.12441. This article has 119 citations and is from a domain leading peer-reviewed journal.
(figon2019ommochromesininvertebrates pages 9-10): Florent Figon and Jérôme Casas. Ommochromes in invertebrates: biochemistry and cell biology. Biological Reviews, 94:156-183, Jul 2019. URL: https://doi.org/10.1111/brv.12441, doi:10.1111/brv.12441. This article has 119 citations and is from a domain leading peer-reviewed journal.
(crawford2020highlyefficientknockout pages 3-5): Karen Crawford, Juan F. Diaz Quiroz, Kristen M. Koenig, Namrata Ahuja, Caroline B. Albertin, and Joshua J.C. Rosenthal. Highly efficient knockout of a squid pigmentation gene. Current Biology, 30:3484-3490.e4, Sep 2020. URL: https://doi.org/10.1016/j.cub.2020.06.099, doi:10.1016/j.cub.2020.06.099. This article has 83 citations and is from a highest quality peer-reviewed journal.
(crawford2020highlyefficientknockout media 7a0753cd): Karen Crawford, Juan F. Diaz Quiroz, Kristen M. Koenig, Namrata Ahuja, Caroline B. Albertin, and Joshua J.C. Rosenthal. Highly efficient knockout of a squid pigmentation gene. Current Biology, 30:3484-3490.e4, Sep 2020. URL: https://doi.org/10.1016/j.cub.2020.06.099, doi:10.1016/j.cub.2020.06.099. This article has 83 citations and is from a highest quality peer-reviewed journal.
(crawford2020highlyefficientknockout pages 10-12): Karen Crawford, Juan F. Diaz Quiroz, Kristen M. Koenig, Namrata Ahuja, Caroline B. Albertin, and Joshua J.C. Rosenthal. Highly efficient knockout of a squid pigmentation gene. Current Biology, 30:3484-3490.e4, Sep 2020. URL: https://doi.org/10.1016/j.cub.2020.06.099, doi:10.1016/j.cub.2020.06.099. This article has 83 citations and is from a highest quality peer-reviewed journal.
(neff2020crisprincephalopods pages 1-1): Ellen P. Neff. Crispr in cephalopods yields the first knockout squid. Lab Animal, 49:292-292, Sep 2020. URL: https://doi.org/10.1038/s41684-020-0655-5, doi:10.1038/s41684-020-0655-5. This article has 0 citations and is from a peer-reviewed journal.
(crawford2020highlyefficientknockout pages 5-7): Karen Crawford, Juan F. Diaz Quiroz, Kristen M. Koenig, Namrata Ahuja, Caroline B. Albertin, and Joshua J.C. Rosenthal. Highly efficient knockout of a squid pigmentation gene. Current Biology, 30:3484-3490.e4, Sep 2020. URL: https://doi.org/10.1016/j.cub.2020.06.099, doi:10.1016/j.cub.2020.06.099. This article has 83 citations and is from a highest quality peer-reviewed journal.
(cordell2021biosynthesisofthe pages 4-6): Geoffrey A. Cordell and Sharna-kay Daley. Biosynthesis of the ommochromes and papiliochromes. Records of Natural Products, 15:420-432, Jun 2021. URL: https://doi.org/10.25135/rnp.238.21.02.1988, doi:10.25135/rnp.238.21.02.1988. This article has 5 citations and is from a peer-reviewed journal.
(bushin2025growthcoupledmicrobialbiosynthesis pages 3-6): Leah B. Bushin, Tobias B. Alter, María V.G. Alván-Vargas, Lara Dürr, Elina C. Olson, Mariah J. Avila, Òscar Puiggené, Taehwan Kim, Leila F. Deravi, Adam M. Feist, Pablo I. Nikel, and Bradley S. Moore. Growth-coupled microbial biosynthesis of the animal pigment xanthommatin. bioRxiv, Oct 2025. URL: https://doi.org/10.1101/2024.10.04.616593, doi:10.1101/2024.10.04.616593. This article has 9 citations.
(crawford2020highlyefficientknockout media 12743825): Karen Crawford, Juan F. Diaz Quiroz, Kristen M. Koenig, Namrata Ahuja, Caroline B. Albertin, and Joshua J.C. Rosenthal. Highly efficient knockout of a squid pigmentation gene. Current Biology, 30:3484-3490.e4, Sep 2020. URL: https://doi.org/10.1016/j.cub.2020.06.099, doi:10.1016/j.cub.2020.06.099. This article has 83 citations and is from a highest quality peer-reviewed journal.
id: A0A7G4RN94
gene_symbol: TDO
product_type: PROTEIN
status: IN_PROGRESS
taxon:
id: NCBITaxon:1051067
label: Doryteuthis pealeii
description: >-
Tryptophan 2,3-dioxygenase (TDO) is a heme-dependent oxidoreductase that catalyzes
the first committed step in ommochrome pigment biosynthesis in D. pealeii, converting
L-tryptophan to N-formylkynurenine via oxidative cleavage of the indole ring. TDO
is expressed in the retina and chromatophore layer of the skin, the two principal
ommochrome-pigmented tissues. Crawford et al. (2020) demonstrated that CRISPR-Cas9
knockout of TDO in D. pealeii embryos efficiently eliminated pigmentation in both
eyes and chromatophores with >90% disruption efficiency, while ink sac melanin
pigmentation was unaffected. This was the first gene knockout achieved in any
cephalopod. TDO inhibition with 680C91 also completely blocked pigmentation,
confirming the enzyme is required for ommochrome synthesis. Notably, D. pealeii
lacks an IDO (indoleamine 2,3-dioxygenase) ortholog, unlike Euprymna berryi where
IDO can compensate for TDO loss. TDO belongs to the PANTHER family PTHR10138
(Tryptophan 2,3-dioxygenase), is predicted to function as a homotetramer (dimer
of dimers), and contains a single heme cofactor per subunit. The gene spans 13
exons over 120 kb of genomic DNA.
existing_annotations:
- term:
id: GO:0004833
label: L-tryptophan 2,3-dioxygenase activity
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: >-
L-tryptophan 2,3-dioxygenase activity (EC 1.13.11.11) is the defining enzymatic
function of TDO. This IEA annotation is based on combined automated methods
(InterPro, ARBA, PANTHER, UniRule). In D. pealeii, Crawford et al. (2020)
demonstrated that CRISPR-Cas9 knockout of TDO eliminated ommochrome pigmentation,
and pharmacological inhibition with 680C91 (a TDO-selective inhibitor) blocked
pigmentation in both eyes and chromatophores [PMID:32735817]. The UniProt entry
confirms TDO catalyzes L-tryptophan + O2 = N-formylkynurenine (Rhea:RHEA:24536).
Phylogenetic analysis confirmed this gene as a bona fide TDO (distinct from IDO)
[PMID:32735817].
action: ACCEPT
reason: >-
This is the core enzymatic function of TDO, supported by sequence homology,
PANTHER family assignment (PTHR10138), HAMAP rule MF_03020, and functional
knockout data in this species showing loss of ommochrome pigmentation.
supported_by:
- reference_id: PMID:32735817
supporting_text: >-
In invertebrates, Tryptophan, 2,3 Dioxygenase (TDO) catalyzes the first
committed step in ommochrome biosynthesis, converting Tryptophan to
N-Formylkyneurenine.
- reference_id: PMID:32735817
supporting_text: >-
A phylogenetic comparison of this sequence with diverse TDOs, and
indolamine-2,3-dioxygenases (a different enzyme that catalyzes the same
reaction), supported its identity as TDO.
- term:
id: GO:0006569
label: L-tryptophan catabolic process
evidence_type: IEA
original_reference_id: GO_REF:0000117
review:
summary: >-
L-tryptophan catabolic process is annotated via ARBA machine learning. TDO
catalyzes the oxidative cleavage of L-tryptophan as the first step in the
kynurenine pathway. Crawford et al. (2020) showed that TDO knockout eliminates
ommochrome biosynthesis from tryptophan in D. pealeii [PMID:32735817]. This
term is correct but could be more specific. The UniProt record lists both
GO:0019441 (L-tryptophan catabolic process to kynurenine) and GO:0019442
(L-tryptophan catabolic process to acetyl-CoA), and GO:0019441 more precisely
captures TDO function as it catalyzes the first step to kynurenine specifically.
action: ACCEPT
reason: >-
L-tryptophan catabolic process correctly describes the biological process TDO
participates in. While a more specific term like L-tryptophan catabolic process
to kynurenine (GO:0019441) would be ideal, that term is now obsolete in GO.
GO:0006569 is the most appropriate active term for this function. Crawford et al.
(2020) showed TDO knockout eliminates the tryptophan-to-ommochrome pathway in
D. pealeii [PMID:32735817].
supported_by:
- reference_id: PMID:32735817
supporting_text: >-
TDO catalyzes the first committed step in ommochrome biosynthesis, converting
Tryptophan to N-Formylkyneurenine.
- term:
id: GO:0020037
label: heme binding
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: >-
Heme binding is annotated by combined IEA methods (InterPro, ARBA, PANTHER,
UniRule). The UniProt record explicitly states: "Binds 1 heme group per subunit"
(HAMAP-Rule:MF_03020), and lists heme as a cofactor (ChEBI:30413). TDO is a
heme-dependent dioxygenase, and heme is essential for its catalytic mechanism.
This is well-established for TDO family members across all species.
action: ACCEPT
reason: >-
Heme binding is integral to TDO catalytic function. The enzyme requires bound
heme to catalyze oxidative cleavage of the tryptophan indole ring. This is
confirmed by UniProt cofactor annotation and is a defining feature of the
TDO family (HAMAP MF_03020, Pfam PF03301 Trp_dioxygenase).
supported_by:
- reference_id: PMID:32735817
supporting_text: >-
we determined whether a TDO-selective inhibitor
(680C91) impeded pigmentation in developing embryos.
- term:
id: GO:0046872
label: metal ion binding
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: >-
Metal ion binding is annotated via InterPro2GO mapping (IPR037217). This is
correct as TDO binds iron through its heme prosthetic group. However, this
term is very generic and redundant with the more informative heme binding
(GO:0020037) annotation already present, which more precisely captures the
nature of the metal cofactor interaction.
action: MARK_AS_OVER_ANNOTATED
reason: >-
While technically correct (TDO binds iron via heme), GO:0046872 (metal ion
binding) is too generic to be informative. The heme binding annotation
(GO:0020037) already captures this function at a more appropriate level of
specificity. The metal ion binding term does not add useful functional
information beyond what heme binding conveys.
- term:
id: GO:0006727
label: ommochrome biosynthetic process
evidence_type: IDA
original_reference_id: PMID:32735817
review:
summary: >-
NEW annotation. Crawford et al. (2020) demonstrated by CRISPR-Cas9 knockout
that TDO is required for ommochrome biosynthesis in D. pealeii. TDO knockout
eliminated pigmentation in both chromatophores and eyes, which contain ommochrome
pigments. The TDO-selective inhibitor 680C91 also blocked pigmentation.
In situ hybridization confirmed TDO expression in chromatophore precursors
and eyes. This is the most biologically meaningful annotation for this gene
in cephalopods, as ommochrome pigmentation is the primary downstream
biological process served by TDO in this organism.
action: NEW
reason: >-
Ommochrome biosynthesis is the key biological process requiring TDO in
cephalopods. CRISPR knockout and pharmacological inhibition both demonstrate
TDO is essential for ommochrome production. This annotation is not present
in the existing GOA set but is strongly supported by direct experimental
evidence in this species.
supported_by:
- reference_id: PMID:32735817
supporting_text: >-
In cephalopods, the pigments in the eyes
and chromatophores are ommochromes, a derivative of tryptophan
- reference_id: PMID:32735817
supporting_text: >-
Knocking out TDO in squid embryos efficiently eliminated pigmentation. By
precisely timing CRISPR-Cas9 delivery during early development, the degree
of pigmentation could be finely controlled. Genotyping revealed knockout
efficiencies routinely greater than 90%.
- reference_id: PMID:32735817
supporting_text: >-
We added 680C91 to developing embryos at stage 20, and it clearly blocked
pigmentation in both the eyes and chromatophores, with animals developing
normally otherwise.
- reference_id: PMID:29989284
supporting_text: >-
Ommochromes are widely occurring coloured molecules of invertebrates, arising
from tryptophan catabolism through the so-called Tryptophan → Ommochrome pathway.
- term:
id: GO:0043473
label: pigmentation
evidence_type: IMP
original_reference_id: PMID:32735817
review:
summary: >-
NEW annotation. Crawford et al. (2020) showed that TDO knockout by CRISPR-Cas9
resulted in loss of pigmentation in D. pealeii embryos, with chromatophore and
eye pigmentation eliminated. Ink sac melanin was unaffected, demonstrating TDO
specifically contributes to ommochrome-based pigmentation. Ahuja et al. (2023)
further showed in E. berryi that TDO knockout delays pigmentation and TDO+IDO
double knockout produces complete albinism [PMID:37343558].
action: NEW
reason: >-
Pigmentation is the primary phenotype affected by TDO loss of function. The
knockout phenotype directly demonstrates involvement in pigmentation. This
is a core biological role for TDO in cephalopods, where ommochrome pigments
color the chromatophores and retina.
supported_by:
- reference_id: PMID:32735817
supporting_text: >-
pigmentation is completely absent from the chromatophores and is
minimal in the eyes which are light red
- reference_id: PMID:37343558
supporting_text: >-
The knockout of tdo in D. pealeii completely inhibited pigmentation but had
little to no effect in E. berryi.
core_functions:
- description: >-
TDO catalyzes the oxidative cleavage of L-tryptophan to N-formylkynurenine,
the first committed step in the kynurenine pathway and ommochrome biosynthesis
in D. pealeii. CRISPR-Cas9 knockout eliminated ommochrome pigmentation in both
eyes and chromatophores with >90% efficiency, and pharmacological inhibition
with 680C91 confirmed the enzyme is required. D. pealeii lacks an IDO ortholog,
making TDO the sole enzyme for this reaction. TDO is a heme-dependent dioxygenase
that binds one heme group per subunit. The downstream kynurenine pathway
intermediates 3-hydroxykynurenine and xanthurenic acid serve as precursors for
xanthommatin and related ommatin pigments deposited in specialized ommochromasomes
within chromatophore and retinal pigment cells (PMID:29989284). The TDO-selective
inhibitor 680C91 at 3.15 mM from stage 20-27 phenocopied the CRISPR knockout,
confirming enzymatic activity is required upstream of ommochrome deposition.
Genotyping of CRISPR-edited embryos showed a mean of 38 +/- 13 distinct indel
events per animal, with no dominant allele, yet produced near-complete pigmentation
loss, demonstrating the strong genotype-phenotype link. In E. berryi, TDO knockout
alone produced only slightly delayed pigmentation due to IDO compensation, but
TDO+IDO double knockout yielded complete albinism enabling the first in vivo
calcium imaging of neural activity in a cephalopod (PMID:37343558).
molecular_function:
id: GO:0004833
label: L-tryptophan 2,3-dioxygenase activity
directly_involved_in:
- id: GO:0006727
label: ommochrome biosynthetic process
- id: GO:0006569
label: L-tryptophan catabolic process
- id: GO:0043473
label: pigmentation
supported_by:
- reference_id: PMID:32735817
supporting_text: >-
Tryptophan, 2,3 Dioxygenase (TDO) catalyzes
the first committed step in ommochrome biosynthesis, converting Tryptophan to
N-Formylkyneurenine
- reference_id: PMID:32735817
supporting_text: >-
Knocking out TDO in squid embryos efficiently eliminated pigmentation
- reference_id: PMID:37343558
supporting_text: >-
The knockout of tdo in D. pealeii completely inhibited pigmentation but had
little to no effect in E. berryi.
references:
- id: GO_REF:0000002
title: Gene Ontology annotation through association of InterPro records with GO terms
findings: []
- id: GO_REF:0000117
title: Electronic Gene Ontology annotations created by ARBA machine learning models
findings: []
- id: GO_REF:0000120
title: Combined Automated Annotation using Multiple IEA Methods
findings: []
- id: PMID:32735817
title: Highly Efficient Knockout of a Squid Pigmentation Gene
findings:
- statement: >-
TDO was selected as a knockout target because ommochromes pigment cephalopod
eyes and chromatophores, and TDO catalyzes the first committed step in their
biosynthesis.
- statement: >-
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.
- statement: >-
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.
- statement: >-
TDO-selective inhibitor 680C91 completely blocked pigmentation in both eyes
and chromatophores, with animals developing normally otherwise.
- statement: >-
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.
- statement: >-
This was the first gene knockout achieved in any cephalopod.
- id: PMID:29989284
title: "Ommochromes in invertebrates: biochemistry and cell biology"
findings:
- statement: >-
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.
supporting_text: >-
Ommochromes are widely occurring coloured molecules of invertebrates, arising
from tryptophan catabolism through the so-called Tryptophan to Ommochrome
pathway.
- statement: >-
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.
supporting_text: >-
Ommochromasomes are the organelles where ommochromes are synthesised and stored.
Hence, they play an important role in mediating ommochrome functions.
- statement: >-
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.
supporting_text: >-
Ommochromes might also be involved in cell homeostasis by detoxifying free
tryptophan and buffering oxidative stress.
- id: PMID:37343558
title: Creation of an albino squid line by CRISPR-Cas9 and its application for
in vivo functional imaging of neural activity
findings:
- statement: >-
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.
- statement: >-
E. berryi homozygous TDO knockout showed slightly delayed pigmentation during
development. TDO+IDO double knockout produced complete albinism.
- statement: >-
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.
- statement: >-
Albino E. berryi enabled the first in vivo calcium imaging of neural activity
in a cephalopod using two-photon microscopy.
suggested_questions:
- question: >-
Does D. pealeii TDO have any role in tryptophan catabolism beyond ommochrome
biosynthesis (e.g. kynurenine pathway metabolites for immune or neural signaling)?
experts:
- Rosenthal JJC
- Albertin CB
- question: >-
What are the consequences of partial TDO loss on chromatophore function and
dynamic color change in adult squid?
experts:
- Crawford K
- question: >-
Is there any post-translational regulation of TDO activity in cephalopods, as
seen with substrate activation in mammalian TDO?
experts: []
suggested_experiments:
- description: >-
Characterize the enzymatic kinetics of recombinant D. pealeii TDO, including
heme stoichiometry and substrate specificity, to confirm in vitro activity.
experiment_type: biochemistry
hypothesis: >-
D. pealeii TDO has similar catalytic properties to mammalian TDO, with
strict specificity for L-tryptophan over other indoleamine substrates.
- description: >-
Use single-cell RNA-seq to map TDO expression across chromatophore subtypes
and developmental stages in D. pealeii embryos.
experiment_type: transcriptomics
hypothesis: >-
TDO expression is restricted to ommochrome-producing cell types (chromatophores
and retinal pigment cells) and onset correlates with pigmentation timing.
- description: >-
Measure kynurenine pathway metabolite levels (N-formylkynurenine, kynurenine,
3-hydroxykynurenine, xanthurenic acid) in TDO knockout vs wildtype D. pealeii
embryos by LC-MS/MS.
experiment_type: metabolomics
hypothesis: >-
TDO knockout eliminates kynurenine pathway flux entirely in D. pealeii due to
the absence of IDO, unlike in E. berryi where IDO provides compensation.
- description: >-
Test whether partial TDO knockout in adult squid affects chromatophore
expansion dynamics and camouflage behavior using video analysis.
experiment_type: behavioral/physiological
hypothesis: >-
Reduced ommochrome content from partial TDO loss impairs chromatophore
opacity and dynamic range of color change.