PUBLICATION

nr3c1 null mutant zebrafish are viable and reveal DNA-binding-independent activities of the glucocorticoid receptor

Authors: Facchinello, N., Skobo, T., Meneghetti, G., Colletti, E., Dinarello, A., Tiso, N., Costa, R., Gioacchini, G., Carnevali, O., Argenton, F., Colombo, L., Dalla Valle, L.
ID: ZDB-PUB-170701-5
Date: 2017
Source: Scientific Reports 7: 4371 (Journal)
Registered Authors: Argenton, Francesco, Carnevali, Oliana, Colletti, Elisa, Costa, Roberto, Dalla Valle, Luisa, Facchinello, Nicola, Skobo, Tatjana, Tiso, Natascia
Keywords: Adrenal cortex hormones, Disease model, Endocrine system and metabolic diseases
MeSH Terms: none
PubMed: 28663543 Full text @ Sci. Rep.

Abstract

Glucocorticoids (GCs) play important roles in developmental and physiological processes through the transcriptional activity of their cognate receptor (Gr). Using CRISPR/Cas9 technology, we established a zebrafish null Gr mutant line and compared its phenotypes with wild type and a zebrafish line with partially silenced gr (gr ^s357/s357 ). Homozygous gr ^-/- larvae are morphologically inconspicuous and, in contrast to GR ^-/- knockout mice, viable through adulthood, although with reduced fitness and early life survival. Mutants gr ^-/- are fertile, but their reproductive capabilities fall at around 10 months of age, when, together with cardiac and intestinal abnormalities already visible at earlier stages, increased fat deposits are also observed. Mutants show higher levels of whole-body cortisol associated with overstimulated basal levels of crh and pomca transcripts along the HPI axis, which is unresponsive to a mechanical stressor. Transcriptional activity linked to immune response is also hampered in the gr ^-/- line: after intestinal damage by dextran sodium sulphate exposure, there are neither inflammatory nor anti-inflammatory cytokine gene responses, substantiating the hypothesis of a dual-action of the GC-GR complex on the immune system. Hence, the zebrafish gr mutant line appears as a useful tool to investigate Gr functions in an integrated in vivo model.

Genes / Markers

Marker	Marker Type	Name
crhb	GENE	corticotropin releasing hormone b
cxcl8a	GENE	chemokine (C-X-C motif) ligand 8a
fkbp5	GENE	FKBP prolyl isomerase 5
foxo3b	GENE	forkhead box O3b
hsd11b2	GENE	hydroxysteroid (11-beta) dehydrogenase 2
il1b	GENE	interleukin 1, beta
il6	GENE	interleukin 6 (interferon, beta 2)
mmp13a.1	GENE	matrix metallopeptidase 13a, tandem duplicate 1
mmp9	GENE	matrix metallopeptidase 9
nr3c1	GENE	nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor)

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Figures

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Expression

Phenotype

Fish	Conditions	Stage	Phenotype	Figure
WT	chemical treatment: dextran sulfate	Day 5	whole organism cxcl8a expression increased amount, abnormal	Fig. 6
WT	chemical treatment: dextran sulfate	Day 5	whole organism il1b expression increased amount, abnormal	Fig. 6
WT	chemical treatment: dextran sulfate	Day 5	whole organism il6 expression increased amount, abnormal	Fig. 6
WT	chemical treatment: dextran sulfate	Day 5	whole organism mmp13a.1 expression increased amount, abnormal	Fig. 6
WT	chemical treatment: dexamethasone, chemical treatment: dextran sulfate	Day 5	whole organism il1b expression increased amount, abnormal	Fig. 6
WT	chemical treatment: dexamethasone, chemical treatment: dextran sulfate	Day 5	whole organism il6 expression increased amount, abnormal	Fig. 6
WT	chemical treatment: dexamethasone, chemical treatment: dextran sulfate	Day 5	whole organism mmp13a.1 expression increased amount, abnormal	Fig. 6
WT	mechanical stress	Day 5	whole organism crhb expression increased amount, abnormal	Fig. 4
WT	mechanical stress	Day 5	whole organism fkbp5 expression increased amount, abnormal	Fig. 4
WT	mechanical stress	Day 5	whole organism pomca expression increased amount, abnormal	Fig. 4

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
ia20Tg	Tg(9xGCRE-HSV.Ul23:EGFP)	Transgenic Insertion
ia30		Insertion	nr3c1
s357		Point Mutation	nr3c1

Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
nr3c1	CRISPR1-nr3c1	CRISPR

Fish

Fish
ia20Tg
nr3c1^ia30/ia30
nr3c1^ia30/ia30
nr3c1^ia30/ia30; ia20Tg
nr3c1^ia30/ia30; ia20Tg
nr3c1^s357/s357
nr3c1^s357/s357
nr3c1^s357/s357; ia20Tg
nr3c1^s357/s357; ia20Tg
WT

Antibodies

Name	Type	Antigen Genes	Isotypes	Host Organism
Ab1-nr3c1	polyclonal		IgG	Rabbit

Orthology

Engineered Foreign Genes

Marker	Marker Type	Name
EGFP	EFG	EGFP

Mapping

Facchinello et al., 2017 ZDB-PUB-170701-5 PMID:28663543

nr3c1 null mutant zebrafish are viable and reveal DNA-binding-independent activities of the glucocorticoid receptor

Facchinello et al., 2017

ZDB-PUB-170701-5
PMID:28663543