PUBLICATION

Zebrafish Cdx1b modulates epithalamic asymmetry by regulating ndr2 and lft1 expression

Authors

Wu, C.S., Lu, Y.F., Liu, Y.H., Huang, C.J., Hwang, S.L.

ID

ZDB-PUB-201120-146

Date

2020

Source

Developmental Biology 470: 21-36 (Journal)

Registered Authors

Huang, Chang-Jen, Hwang, Sheng-Ping L.

Keywords

Epithalamic laterality, Gene expression, Left-right patterning, Zebrafish, cdx1b, lft1, ndr2

MeSH Terms

Embryo, Nonmammalian/metabolism
Heart/embryology
Epithalamus/embryology*
Epithalamus/metabolism
Signal Transduction
Left-Right Determination Factors/genetics*
Left-Right Determination Factors/metabolism
Gene Knockdown Techniques
Habenula/embryology
Body Patterning/genetics*
Nodal Protein/metabolism
Intracellular Signaling Peptides and Proteins/genetics*
Intracellular Signaling Peptides and Proteins/metabolism
Protein Binding
Zebrafish Proteins/genetics*
Zebrafish Proteins/metabolism*
Pineal Gland/cytology
Pineal Gland/embryology
Homeodomain Proteins/metabolism*
Cell Movement
Zebrafish/embryology*
Zebrafish/genetics*
Zebrafish/metabolism
Animals
Diencephalon/embryology
Diencephalon/metabolism
Gene Expression Profiling
Gene Expression Regulation, Developmental

(all 28)

PubMed

33197427 Full text @ Dev. Biol.

Abstract

Nodal signaling is essential for mesoderm and endoderm formation, as well as neural plate induction and establishment of left-right asymmetry. However, the mechanisms controlling expression of Nodal pathway genes in these contexts are not fully known. Previously, we showed that Cdx1b induces expression of downstream Nodal signaling factors during early endoderm formation. In this study, we show that Cdx1b also regulates epithalamic asymmetry in zebrafish embryos by modulating expression of ndr2 and lft1. We first knocked down cdx1b with translation-blocking and splicing-blocking morpholinos (MOs). Most embryos injected with translation-blocking MOs showed absent ndr2, lft1 and pitx2c expression in the left dorsal diencephalon during segmentation and pharyngula stages accompanied by aberrant parapineal migration and habenular laterality at 72 h post fertilization (hpf). These defects were less frequent in embryos injected with splicing-blocking MO. To confirm the morphant phenotype, we next generated both zygotic (Z)cdx1b^-/- and maternal zygotic (MZ)cdx1b^-/- mutants by CRISPR-Cas9 mutagenesis. Expression of ndr2, lft1 and pitx2c was absent in the left dorsal diencephalon of a high proportion of MZcdx1b^-/- mutants; however, aberrant dorsal diencephalic pitx2c expression patterns were observed at low frequency in Zcdx1b^-/- mutant embryos. Correspondingly, dysregulated parapineal migration and habenular laterality were also observed in MZcdx1b^-/- mutant embryos at 72 hpf. On the other hand, Kupffer's vesicle cilia length and number, expression pattern of spaw in the lateral plate mesoderm and pitx2c in the gut as well as left-right patterning of various visceral organs were not altered in MZcdx1b^-/- mutants compared to wild-type embryos. Chromatin immunoprecipitation revealed that Cdx1b directly regulates ndr2 and lft1 expression. Furthermore, injection of cdx1b-vivo MO1 but not cdx1b-vivo 4 mm MO1 in the forebrain ventricle at 18 hpf significantly downregulated lft1 expression in the left dorsal diencephalon at 23-24 s stages. Together, our results suggest that Cdx1b regulates transcription of ndr2 and lft1 to maintain proper Nodal activity in the dorsal diencephalon and epithalamic asymmetry in zebrafish embryos.

Genes / Markers

Figures

Show all Figures

Expression

Phenotype

Fish	Conditions	Stage	Phenotype	Figure
AB + MO1-cdx1b	control	20-25 somites	diencephalon dorsal region lft1 expression absent, abnormal	Table 1
AB + MO1-cdx1b	control	20-25 somites	diencephalon dorsal region lft1 expression mislocalised, abnormal	Fig. 7
AB + MO1-cdx1b	control	20-25 somites	diencephalon dorsal region ndr2 expression absent, abnormal	Table 1
AB + MO1-cdx1b	control	Prim-5	diencephalon dorsal region pitx2 expression absent, abnormal	Table 1
AB + MO2-cdx1b	control	20-25 somites	diencephalon dorsal region lft1 expression mislocalised, abnormal	Table 1
AB + MO2-cdx1b	control	20-25 somites	diencephalon dorsal region ndr2 expression mislocalised, abnormal	Table 1
AB + MO2-cdx1b	control	Prim-5	diencephalon dorsal region pitx2 expression mislocalised, abnormal	Table 1
AB + MO4-cdx1b	control	20-25 somites	diencephalon dorsal region lft1 expression absent, abnormal	Table 1
AB + MO4-cdx1b	control	20-25 somites	diencephalon dorsal region ndr2 expression absent, abnormal	Table 1
AB + MO4-cdx1b	control	Prim-5	diencephalon dorsal region pitx2 expression absent, abnormal	Table 1

1 - 10 of 27

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Mutations / Transgenics

Allele	Construct	Type	Affected Genomic Region
as39		Complex	cdx1b

Human Disease / Model

Sequence Targeting Reagents

Target	Reagent	Reagent Type
cdx1b	CRISPR1-cdx1b	CRISPR
cdx1b	MO1-cdx1b	MRPHLNO
cdx1b	MO2-cdx1b	MRPHLNO
cdx1b	MO4-cdx1b	MRPHLNO

Fish

Fish
AB
AB + MO1-cdx1b
AB + MO2-cdx1b
AB + MO4-cdx1b
cdx1b^as39/as39 (AB)
cdx1b^as39/as39 (AB)
cdx1b^as39/+ (AB)

Antibodies

Orthology

Engineered Foreign Genes

Mapping

Wu et al., 2020 ZDB-PUB-201120-146 PMID:33197427

Zebrafish Cdx1b modulates epithalamic asymmetry by regulating ndr2 and lft1 expression

Wu et al., 2020

ZDB-PUB-201120-146
PMID:33197427