PUBLICATION

Functional Verification of Novel ELMO1 Variants by Live Imaging in Zebrafish

Authors: Xue, R., Wang, Y., Wang, T., Lyu, M., Mo, G., Fan, X., Li, J., Yen, K., Yu, S., Liu, Q., Xu, J.
ID: ZDB-PUB-220108-4
Date: 2021
Source: Frontiers in cell and developmental biology 9: 723804 (Journal)
Registered Authors: Xu, Jin
Keywords: ELMO1, cell motility, neutrophil, variants, zebrafish
MeSH Terms: none
PubMed: 34993193 Full text @ Front Cell Dev Biol

Abstract

ELMO1 (Engulfment and Cell Motility1) is a gene involved in regulating cell motility through the ELMO1-DOCK2-RAC complex. Contrary to DOCK2 (Dedicator of Cytokinesis 2) deficiency, which has been reported to be associated with immunodeficiency diseases, variants of ELMO1 have been associated with autoimmune diseases, such as diabetes and rheumatoid arthritis (RA). To explore the function of ELMO1 in immune cells and to verify the functions of novel ELMO1 variants in vivo, we established a zebrafish elmo1 mutant model. Live imaging revealed that, similar to mammals, the motility of neutrophils and T-cells was largely attenuated in zebrafish mutants. Consequently, the response of neutrophils to injury or bacterial infection was significantly reduced in the mutants. Furthermore, the reduced mobility of neutrophils could be rescued by the expression of constitutively activated Rac proteins, suggesting that zebrafish elmo1 mutant functions via a conserved mechanism. With this mutant, three novel human ELMO1 variants were transiently and specifically expressed in zebrafish neutrophils. Two variants, p.E90K (c.268G>A) and p.D194G (c.581A>G), could efficiently recover the motility defect of neutrophils in the elmo1 mutant; however, the p.R354X (c.1060C>T) variant failed to rescue the mutant. Based on those results, we identified that zebrafish elmo1 plays conserved roles in cell motility, similar to higher vertebrates. Using the transient-expression assay, zebrafish elmo1 mutants could serve as an effective model for human variant verification in vivo.

Genes / Markers

Figures

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Expression

Gene	Fish	Conditions	Stage	Anatomy	Assay	Figure
DsRed2	elmo1^{szy103/szy103}; nz50Tg/nz50Tg	standard conditions	Protruding-mouth	yolk syncytial layer neutrophil	IFL	FIGURE 3
DsRedx	elmo1^{szy103/szy103}; hkz04tTg/hkz04tTg; nz50Tg/nz50Tg	standard conditions	Day 5	thymus T cell	IFL	FIGURE 2
EGFP	elmo1^{szy103/szy103}; hkz016tTg/hkz016tTg	standard conditions	Day 5	thymus T cell	IFL	FIGURE 2
EGFP	elmo1^{szy103/szy103}; hkz04tTg/hkz04tTg; nz50Tg/nz50Tg	standard conditions	Day 5	thymus leukocyte	IFL	FIGURE 2
elmo1	elmo1^szy103/+	standard conditions	Protruding-mouth	whole organism	ISH	FIGURE 1
elmo1	elmo1^szy103/+	standard conditions	Protruding-mouth	whole organism	RTPCR	FIGURE 1
elmo1	elmo1^{szy103/szy103}	standard conditions	Protruding-mouth	whole organism	ISH	FIGURE 1
elmo1	elmo1^{szy103/szy103}	standard conditions	Protruding-mouth	whole organism	RTPCR	FIGURE 1
lyz	elmo1^{szy103/szy103}	standard conditions	Protruding-mouth	neutrophil	ISH	Fig. S2
mpeg1.1	elmo1^{szy103/szy103}	standard conditions	Protruding-mouth	macrophage	ISH	Fig. S2

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Phenotype

Fish	Conditions	Stage	Phenotype	Figure
elmo1^szy103/+	standard conditions	Protruding-mouth	whole organism elmo1 expression decreased amount, abnormal	FIGURE 1
elmo1^szy103/+	standard conditions	Protruding-mouth	whole organism elmo1 expression decreased amount, abnormal	FIGURE 1
elmo1^{szy103/szy103}	standard conditions	Protruding-mouth	neutrophil regulation of Rac protein signal transduction decreased process quality, abnormal	FIGURE 5
elmo1^{szy103/szy103}	standard conditions	Protruding-mouth	thymus T cell migration decreased rate, abnormal	FIGURE 4
elmo1^{szy103/szy103}	standard conditions	Protruding-mouth	whole organism elmo1 expression decreased amount, abnormal	FIGURE 1
elmo1^{szy103/szy103}	standard conditions	Protruding-mouth	whole organism elmo1 expression decreased amount, abnormal	FIGURE 1
elmo1^{szy103/szy103}	standard conditions	Protruding-mouth	yolk syncytial layer leukocyte migration decreased process quality, abnormal	FIGURE 4
elmo1^{szy103/szy103}	standard conditions	Protruding-mouth	yolk syncytial layer neutrophil migration decreased process quality, abnormal	FIGURE 5
elmo1^{szy103/szy103}	standard conditions	Protruding-mouth	yolk syncytial layer neutrophil migration decreased process quality, abnormal	FIGURE 6
elmo1^{szy103/szy103}	standard conditions	Day 5	thymus T cell rag1 expression decreased amount, abnormal	FIGURE 2

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
hkz04tTg	Tg(coro1a:EGFP)	Transgenic Insertion
hkz015tTg	Tg(mpeg1.1:LOXP-DsRedx-LOXP-EGFP)	Transgenic Insertion
hkz016tTg	Tg(lck:LOXP-DsRedx-LOXP-EGFP)	Transgenic Insertion
hkz017tTg	Tg(LCR:LOXP-DsRedx-LOXP-EGFP)	Transgenic Insertion
nz50Tg	Tg(lyz:DsRed2)	Transgenic Insertion
szy103		Small Deletion	elmo1

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Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
elmo1	TALEN2-elmo1	TALEN

Fish

Fish
elmo1^szy103/+
elmo1^{szy103/szy103}
elmo1^{szy103/szy103}; hkz04tTg/hkz04tTg; nz50Tg/nz50Tg
elmo1^{szy103/szy103}; hkz016tTg/hkz016tTg
elmo1^{szy103/szy103}; nz50Tg/nz50Tg

Antibodies

Name	Type	Antigen Genes	Isotypes	Host Organism
Ab3-elmo1	polyclonal		IgG	Rabbit

Orthology

Engineered Foreign Genes

Marker	Marker Type	Name
DsRed2	EFG	DsRed2
DsRedx	EFG	DsRedx
EGFP	EFG	EGFP

Mapping

Xue et al., 2021 ZDB-PUB-220108-4 PMID:34993193

Functional Verification of Novel ELMO1 Variants by Live Imaging in Zebrafish

Xue et al., 2021

ZDB-PUB-220108-4
PMID:34993193