PUBLICATION

The zebrafish paralog six2b is required for early proximal pronephros morphogenesis

Authors

Belcher, B., Vestal, J., Lane, S., Kell, M., Smith, L., Camarata, T.

ID

ZDB-PUB-231112-12

Date

2023

Source

Scientific Reports 13: 1969919699 (Journal)

Registered Authors

Camarata, Troy

Keywords

none

MeSH Terms

Zebrafish Proteins/genetics
Zebrafish Proteins/metabolism
Morphogenesis/genetics
Humans
Animals
Nephrons/metabolism
Zebrafish*/genetics
Zebrafish*/metabolism
Mice
Pronephros*/metabolism

(all 10)

PubMed

37952044 Full text @ Sci. Rep.

Abstract

The transcription factor Six2 plays a crucial role in maintaining self-renewing nephron progenitor cap mesenchyme (CM) during metanephric kidney development. In mouse and human, expression at single-cell resolution has detected Six2 in cells as they leave the CM pool and differentiate. The role Six2 may play in these cells as they differentiate remains unknown. Here, we took advantage of the zebrafish pronephric kidney which forms directly from intermediate mesoderm to test six2b function during pronephric tubule development and differentiation. Expression of six2b during early zebrafish development was consistent with a role in pronephros formation. Using morpholino knock-down and CRISPR/Cas9 mutagenesis, we show a functional role for six2b in the development of proximal elements of the pronephros. By 48 h post-fertilization, six2b morphants and mutants showed disrupted pronephric tubule morphogenesis. We observed a lower-than-expected frequency of phenotypes in six2b stable genetic mutants suggesting compensation. Supporting this, we detected increased expression of six2a in six2b stable mutant embryos. To further confirm six2b function, F₀ crispant embryos were analyzed and displayed similar phenotypes as morphants and stable mutants. Together our data suggests a conserved role for Six2 during nephrogenesis and a role in the morphogenesis of the proximal tubule.

Genes / Markers

Marker	Marker Type	Name
cdh17	GENE	cadherin 17, LI cadherin (liver-intestine)
nphs1	GENE	NPHS1 adhesion molecule, nephrin
nphs2	GENE	NPHS2 stomatin family member, podocin
pax2a	GENE	paired box 2a
six2a	GENE	SIX homeobox 2a
six2b	GENE	SIX homeobox 2b
slc20a1a	GENE	solute carrier family 20 member 1a
trpm7	GENE	transient receptor potential cation channel, subfamily M, member 7
wt1a	GENE	WT1 transcription factor a

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Figures

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Expression

Gene	Fish	Conditions	Stage	Anatomy	Assay	Figure
cdh17	AB	control	Long-pec	pronephric proximal convoluted tubule	ISH	Figure 4
cdh17	AB	control	Long-pec	pronephric proximal convoluted tubule	ISH	Figure 2
cdh17	AB	control	Long-pec	pronephric tubule epithelium	ISH	Figure 2
cdh17	AB	control	Prim-5	pronephric tubule epithelium	ISH	Figure 4
cdh17	AB	control	Long-pec	pronephric tubule epithelium	ISH	Figure 4
cdh17	AB	control	Prim-5	pronephric tubule epithelium	ISH	Figure 2
cdh17	AB + CRISPR1-six2b	control	Long-pec	pronephric proximal convoluted tubule	ISH	Figure 4
cdh17	AB + CRISPR1-six2b	control	Long-pec	pronephric tubule epithelium	ISH	Figure 4
cdh17	AB + CRISPR1-six2b	control	Prim-5	pronephric tubule epithelium	ISH	Figure 4
cdh17	AB + MO1-six2b	control	Long-pec	pronephric proximal convoluted tubule	ISH	Figure 2

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Phenotype

Fish	Conditions	Stage	Phenotype	Figure
AB + CRISPR1-six2b	control	Long-pec	pronephric glomerulus nphs1 expression increased distribution, abnormal	Figure 4
AB + CRISPR1-six2b	control	Long-pec	pronephric glomerulus nphs1 expression spatial pattern, abnormal	Figure 4
AB + CRISPR1-six2b	control	Long-pec	pronephric glomerulus nphs2 expression increased distribution, abnormal	Figure 4
AB + CRISPR1-six2b	control	Long-pec	pronephric glomerulus nphs2 expression spatial pattern, abnormal	Figure 4
AB + CRISPR1-six2b	control	Long-pec	pronephric glomerulus wt1a expression increased distribution, abnormal	Figure 4
AB + CRISPR1-six2b	control	Long-pec	pronephric glomerulus wt1a expression spatial pattern, abnormal	Figure 4
AB + CRISPR1-six2b	control	Long-pec	pronephric proximal convoluted tubule cdh17 expression decreased distribution, abnormal	Figure 4
AB + CRISPR1-six2b	control	Long-pec	pronephric proximal convoluted tubule slc20a1a expression spatial pattern, abnormal	Figure 4
AB + CRISPR1-six2b	control	Long-pec	pronephric tubule pax2a expression spatial pattern, abnormal	Figure 4
AB + MO1-six2b	control	Long-pec	pronephric glomerulus nphs1 expression increased distribution, abnormal	Figure 2

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
bhs1		Indel	six2b

Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
six2b	CRISPR1-six2b	CRISPR
six2b	MO1-six2b	MRPHLNO
six2b	MO2-six2b	MRPHLNO

Fish

Fish
AB
AB + CRISPR1-six2b
AB + MO1-six2b
six2b^bhs1/bhs1 (AB)
six2b^bhs1/bhs1 (AB)

Antibodies

Orthology

Engineered Foreign Genes

Mapping

Belcher et al., 2023 ZDB-PUB-231112-12 PMID:37952044

The zebrafish paralog six2b is required for early proximal pronephros morphogenesis

Belcher et al., 2023

ZDB-PUB-231112-12
PMID:37952044