Tracking genome-editing and associated molecular perturbations by SWATH mass spectrometry

Lin, Qifeng; Low, Larry W. L.; Lau, Adam; Esther, Chua Wee Ling; Matsuoka, Yuji; Lian, Yilong; Monteiro, Antónia; Tate, Stephen; Gunaratne, Jayantha; Carney, Thomas J.

doi:10.1038/s41598-019-51612-z

Cited by 11 publications

(6 citation statements)

References 28 publications

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“…Unfortunately, we were not able to test this as there are currently no antibodies that uniquely recognize Hmx3a and we would require an antibody that recognizes the N-terminal region of Hmx3a that should be conserved in our single mutant Hmx3a proteins. In addition, we were unable to detect any Hmx3a peptides in a SWATH analysis (data not shown; Hmx3a has also not been detected in other SWATH analyses; Blattmann et al 2019 ; Lin et al 2019 ), presumably because, like many transcription factors, it is expressed in either two few cells and/or at too low a level.…”

Section: Discussionmentioning

confidence: 79%

Hmx3a Has Essential Functions in Zebrafish Spinal Cord, Ear and Lateral Line Development

et al. 2020

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Transcription factors that contain a homeodomain DNA-binding domain have crucial functions in most aspects of cellular function and embryonic development in both animals and plants. Hmx proteins are a sub-family of NK homeodomain-containing proteins that have fundamental roles in development of sensory structures such as the eye and the ear. However, Hmx functions in spinal cord development have not been analyzed. Here we show that zebrafish (Danio rerio) hmx2 and hmx3a are co-expressed in spinal dI2 and V1 interneurons, whereas hmx3b, hmx1 and hmx4 are not expressed in spinal cord. Using mutational analyses, we demonstrate that, in addition to its previously reported role in ear development, hmx3a is required for correct specification of a subset of spinal interneuron neurotransmitter phenotypes, as well as correct lateral line progression and survival to adulthood. Surprisingly, despite similar expression patterns of hmx2 and hmx3a during embryonic development, zebrafish hmx2 mutants are viable and have no obviously abnormal phenotypes in sensory structures or neurons that require hmx3a. In addition, embryos homozygous for deletions of both hmx2 and hmx3a have identical phenotypes to severe hmx3a single mutants. However, mutating hmx2 in hypomorphic hmx3a mutants that usually develop normally, results in abnormal ear and lateral line phenotypes. This suggests that while hmx2 cannot compensate for loss of hmx3a, it does function in these developmental processes, although to a much lesser extent than hmx3a. More surprisingly, our mutational analyses suggest that Hmx3a may not require its homeodomain DNA-binding domain for its roles in viability or embryonic development.

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Section: Discussionmentioning

confidence: 79%

Hmx3a Has Essential Functions in Zebrafish Spinal Cord, Ear and Lateral Line Development

et al. 2020

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“…To demonstrate that, as with other phenotypes, the H 2 O 2 increase in hai1a was due to unrestrained activity of Matriptase1a, we used a matriptase1a mutant allele, st14a sq10 , which prematurely terminates the protein at 156 amino acids ( Figure 2A , Figure 2—figure supplement 1A–C ; Lin et al, 2019 ). Zygotic st14a mutants showed no overt phenotype; however, maternal zygotic mutants lacked ear otoliths ( Figure 2B, C ).…”

Section: Resultsmentioning

confidence: 99%

“…The hai1a/ddf alleles used were hai1a hi2217 , hai1a fr26 , ddf ti251 , and ddf t419 . The st14a sq10 allele was generated previously ( Lin et al, 2019 ). For imaging neutrophils and keratinocytes, the transgenic lines Tg(mpx:EGFP) i114 ( Renshaw et al, 2006 ) and Tg(krtt1c19e:lyn-tdtomato) sq16 ( Lee et al, 2014 ) were used, whilst early leukocytes were imaged with Tg(fli1:EGFP) y1 ( Redd et al, 2006 ).…”

Section: Methodsmentioning

confidence: 99%

Matriptase activation of Gq drives epithelial disruption and inflammation via RSK and DUOX

et al. 2021

eLife

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Epithelial tissues are primed to respond to insults by activating epithelial cell motility and rapid inflammation. Such responses are also elicited upon overexpression of the membrane bound protease, Matriptase, or mutation of its inhibitor, Hai1. Unrestricted Matriptase activity also predisposes to carcinoma. How Matriptase leads to these cellular outcomes is unknown. We demonstrate that zebrafish hai1a mutants show increased H2O2, NfkB signalling, and IP3R -mediated calcium flashes, and that these promote inflammation, but do not generate epithelial cell motility. In contrast, inhibition of the Gq subunit in hai1a mutants rescues both the inflammation and epithelial phenotypes, with the latter recapitulated by the DAG analogue, PMA. We demonstrate that hai1a has elevated MAPK pathway activity, inhibition of which rescues the epidermal defects. Finally, we identify RSK kinases as MAPK targets disrupting adherens junctions in hai1a mutants. Our work maps novel signalling cascades mediating the potent effects of Matriptase on epithelia, with implications for tissue damage response and carcinoma progression.

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“…Overexpression of CD81 partially rescued the infertility phenotype seen in CD9 KO mice [ 39 ] and it was recently demonstrated that CD9 deletion in human melanoma cells was quickly compensated by CD63 expression upregulation [ 40 ]. It would be interesting to perform RNA-seq or SWATH-MS on the cd9 dKOs to identify upregulated RNA and proteins respectively [ 41 ].…”

Section: Discussionmentioning

confidence: 99%

Tetraspanin Cd9b and Cxcl12a/Cxcr4b have a synergistic effect on the control of collective cell migration

et al. 2021

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Collective cell migration is essential for embryonic development and homeostatic processes. During zebrafish development, the posterior lateral line primordium (pLLP) navigates along the embryo flank by collective cell migration. The chemokine receptors, Cxcr4b and Cxcr7b, as well as their cognate ligand, Cxcl12a, are essential for this process. We corroborate that knockdown of the zebrafish cd9 tetraspanin orthologue, cd9b, results in mild pLL abnormalities. Through generation of CRISPR and TALEN mutants, we show that cd9a and cd9b function partially redundantly in pLLP migration, which is delayed in the cd9b single and cd9a; cd9b double mutants. This delay led to a transient reduction in neuromast numbers. Loss of both Cd9a and Cd9b sensitized embryos to reduced Cxcr4b and Cxcl12a levels. Together these results provide evidence that Cd9 modulates collective cell migration of the pLLP during zebrafish development. One interpretation of these observations is that Cd9 contributes to more effective chemokine signalling.

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Tracking genome-editing and associated molecular perturbations by SWATH mass spectrometry

Cited by 11 publications

References 28 publications

Hmx3a Has Essential Functions in Zebrafish Spinal Cord, Ear and Lateral Line Development

Hmx3a Has Essential Functions in Zebrafish Spinal Cord, Ear and Lateral Line Development

Matriptase activation of Gq drives epithelial disruption and inflammation via RSK and DUOX

Tetraspanin Cd9b and Cxcl12a/Cxcr4b have a synergistic effect on the control of collective cell migration

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