ZFIN ID: ZDB-PUB-201120-146
Zebrafish Cdx1b modulates epithalamic asymmetry by regulating ndr2 and lft1 expression
Wu, C.S., Lu, Y.F., Liu, Y.H., Huang, C.J., Hwang, S.L.
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:
  • Animals
  • Body Patterning/genetics*
  • Cell Movement
  • Diencephalon/embryology
  • Diencephalon/metabolism
  • Embryo, Nonmammalian/metabolism
  • Epithalamus/embryology*
  • Epithalamus/metabolism
  • Gene Expression Profiling
  • Gene Expression Regulation, Developmental
  • Gene Knockdown Techniques
  • Habenula/embryology
  • Heart/embryology
  • Homeodomain Proteins/metabolism*
  • Intracellular Signaling Peptides and Proteins/genetics*
  • Intracellular Signaling Peptides and Proteins/metabolism
  • Left-Right Determination Factors/genetics*
  • Left-Right Determination Factors/metabolism
  • Nodal Protein/metabolism
  • Pineal Gland/cytology
  • Pineal Gland/embryology
  • Protein Binding
  • Signal Transduction
  • Zebrafish/embryology*
  • Zebrafish/genetics*
  • Zebrafish/metabolism
  • Zebrafish Proteins/genetics*
  • Zebrafish Proteins/metabolism*
PubMed: 33197427 Full text @ Dev. Biol.
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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.
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