Recombinant Danio rerio Nuclear receptor subfamily 2 group F member 1-B (nr2f1b)

What is nr2f1b and what are its alternative names in zebrafish research?

Nr2f1b is a nuclear receptor that functions as a transcription factor in zebrafish (Danio rerio). It is also known as COUP transcription factor 1-B (COUP-TFalpha-B), nr2f1l, zgc:65854, and zgc:77353 . This protein belongs to the nuclear receptor superfamily 2, group F, which includes several related proteins that play critical roles in vertebrate development. Nr2f1b is one of several nr2f genes in zebrafish, with others including nr2f1a, nr2f2, and nr2f5 .

What is the expression pattern of nr2f1b during zebrafish development?

Nr2f1b exhibits a dynamic expression pattern during zebrafish embryonic development:

• At 15 somite stage: Expressed in the telencephalon (t), ventral medial diencephalon (d), hindbrain rhombomeres (h), and lateral plate mesoderm, which contains vascular precursors

• At 20 hpf (~24 somites): Expression appears in developing vessels

• At 24 hpf: Expression in telencephalon, diencephalon, hindbrain, vessels of the trunk, and caudal vein plexus (CVP)

This spatiotemporal expression pattern suggests that nr2f1b functions in both neural and vascular development. The expression has been confirmed through whole-mount in situ hybridization and transverse sectioning of embryos .

What techniques are most effective for manipulating nr2f1b expression in zebrafish?

Multiple complementary approaches have proven effective for studying nr2f1b function:

For Loss-of-Function Studies:

• Morpholino Knockdown

  • Antisense morpholino oligonucleotides targeting nr2f1b translation or splicing

  • Allows for rapid assessment of nr2f1b function during early development

  • Can be combined with other morpholinos for combinatorial knockdown studies

• CRISPR/Cas9 Genome Editing

  • Generation of frameshift mutations in the nr2f1b coding sequence

  • Provides stable genetic models for long-term studies

  • Has revealed roles in telencephalon and eye development

For Gain-of-Function Studies:

• mRNA Overexpression

  • Injection of synthetic nr2f1b mRNA into one-cell stage embryos

  • Dose-dependent effects: high doses disrupt anterior brain development; low doses affect specific gene expression patterns (pax6a in eyes, fgf8a in telencephalon)

• Tissue-Specific Overexpression

  • Using tissue-specific promoters (e.g., fli promoter for vascular expression)

  • Has demonstrated that nr2f1b overexpression increases venous and intersegmental vessel (ISV) endothelial cell numbers

For Functional Analysis:

• Time-lapse Imaging

  • Visualization of angiogenic cell migration and proliferation

  • Assessment of vascular patterning defects in real-time

• Reporter Gene Assays

  • Luciferase assays to assess transcriptional activity

  • Has shown cooperative enhancement of gene expression by Isl2 and Nr2f1b

How can researchers effectively visualize nr2f1b expression and protein activity?

Multiple visualization techniques can be employed to study nr2f1b:

• Whole-mount in situ Hybridization (WISH)

  • Primary method for spatial localization of nr2f1b mRNA

  • Provides tissue-level resolution of expression patterns

  • Can be followed by transverse sectioning for detailed analysis

• Immunohistochemistry/Immunofluorescence

  • Detection of Nr2f1b protein using specific antibodies

  • Can be combined with other markers for co-localization studies

  • Useful for confirming protein expression in specific cell types

• Transgenic Reporter Lines

  • Creation of nr2f1b:GFP transgenic lines to visualize expression in live embryos

  • Enables real-time imaging of expression dynamics

• Protein Interaction Studies

  • Co-immunoprecipitation to detect physical interactions with other proteins

  • Has demonstrated physical interaction between Nr2f1b and Isl2

  • Pull-down assays to identify additional interaction partners

• Chromatin Immunoprecipitation (ChIP)

  • Identification of genomic regions bound by Nr2f1b

  • Elucidation of direct transcriptional targets

What is the role of nr2f1b in zebrafish vascular development?

Nr2f1b plays crucial roles in multiple aspects of vascular development:

• Venous Specification

  • Promotes vein identity during zebrafish development

  • Morpholino knockdown reduces vein cell number and decreases expression of vein-specific markers (flt4 and mrc1)

  • Overexpression increases venous cell numbers

• Angiogenic Patterning

  • Critical for proper intersegmental vessel (ISV) development

  • Nr2f1b morphants show reduced ISV cell numbers and impaired ISV growth

  • Time-lapse imaging reveals that these defects likely result from impaired angiogenic cell migration and/or proliferation

• Tip Cell Identity

  • Promotes tip cell fate during angiogenic sprouting

  • Works in opposition to Notch signaling, which restricts tip cell formation

• Phenotypic Consequences

  • Nr2f1b deficiency leads to pericardial edema and circulation defects

  • Double knockdown of nr2f1b with nr2f2 or isl2 exacerbates vascular defects

The data collectively demonstrate that nr2f1b is both necessary and sufficient for proper venous specification and angiogenic patterning during zebrafish development.

How does nr2f1b function in brain and eye development?

Nr2f1b has significant roles in brain regionalization and eye development:

These roles highlight nr2f1b's multifaceted functions beyond vascular development, emphasizing its importance in coordinating several aspects of embryonic development.

How does nr2f1b interact with Notch signaling in vascular development?

Nr2f1b and Notch signaling have an intricate regulatory relationship in vascular development:

• Reciprocal Regulation

  • Nr2f1b is negatively regulated by Notch activity

  • Nr2f1b expression increases in rbpsuh morphants (where Notch signaling is disrupted)

  • Similarly, embryos treated with DAPT (a γ-secretase inhibitor that blocks Notch signaling) show increased nr2f1b expression

• Opposing Functions

  • Notch promotes arterial fate and suppresses tip cell formation

  • Nr2f1b promotes venous and tip cell identity

  • This opposition establishes the proper balance between arterial and venous fates

• Molecular Mechanism

  • The exact molecular pathway by which Notch represses nr2f1b remains to be fully elucidated

  • This interaction likely involves direct or indirect transcriptional regulation

This antagonistic relationship is crucial for establishing proper vascular patterning and arterial-venous specification during embryonic development.

What is the relationship between Nr2f1b and other transcription factors in vascular development?

Nr2f1b functions within a network of transcription factors to coordinate vascular development:

• Interaction with Islet2 (Isl2)

  • Isl2 and Nr2f1b physically interact, as demonstrated by co-immunoprecipitation

  • They cooperatively enhance gene expression, shown by luciferase assays

  • Isl2 regulates nr2f1b expression, suggesting Isl2 functions upstream of Nr2f1b

  • Double knockdown of isl2/nr2f1b shows enhanced vascular defects compared to single knockdowns

• Functional Relationship with Nr2f2

  • Nr2f2 plays a minor role in vein and ISV growth compared to Nr2f1b

  • Double knockdown of nr2f1b/nr2f2 exhibits enhanced vascular defects, indicating partial functional redundancy

  • Isl2 can regulate the expression of both nr2f1b and nr2f2

• Coordinated Regulation

  • A model has been proposed where Isl2 functions upstream of Nr2f1b and Nr2f2

  • The interaction of Nr2f1b and Nr2f2, in combination with Isl2, coordinates vascular development

This interplay between transcription factors forms a regulatory network that precisely controls venous specification and angiogenic patterning during zebrafish development.

How do nr2f1b and its paralogs differ in their developmental roles?

The nr2f gene family members in zebrafish have overlapping but distinct functions:

The functional differences likely reflect evolutionary divergence following gene duplication events in the teleost lineage .

How does zebrafish nr2f1b function compare to mammalian NR2F orthologs?

Interesting functional divergence exists between zebrafish and mammalian NR2F genes:

• Functional Distribution

  • In mice, Nr2f2 (COUP-TFII) is the major regulator of venous identity and angiogenic growth

  • In contrast, Nr2f1 (COUP-TFI) is critical for CNS and peripheral nervous system development

  • In zebrafish, nr2f1b has adopted vascular functions that are performed by Nr2f2 in mammals

• Evolutionary Implications

  • Zebrafish have four nr2f genes (nr2f1a, nr2f1b, nr2f2, nr2f5) compared to two in mammals (NR2F1, NR2F2)

  • This suggests evolutionary redistribution of functions among paralogs after gene duplication events

  • The vascular function of nr2f1b in zebrafish represents a unique adaptation not documented for NR2F1 in mammals

• Structural Conservation

  • Despite functional divergence, the protein structure of Nr2f family members is highly conserved across vertebrates

  • This structural conservation suggests fundamental importance of these transcription factors in development

This comparative perspective provides valuable insights into the evolution of developmental regulatory networks across vertebrate species.

What downstream genes are regulated by nr2f1b in zebrafish development?

Research has identified several potential target genes regulated by nr2f1b:

• Vascular Genes

  • Vein-specific markers flt4 and mrc1 are positively regulated by nr2f1b

  • Knockdown of nr2f1b reduces their expression, while overexpression enhances venous identity

• Brain and Eye Development Genes

  • Overexpression of nr2f1b affects pax6a expression in the eyes

  • Also impacts fgf8a expression in the telencephalon

  • These effects may be direct or indirect regulatory relationships

• Genome-wide Approaches

  • Microarray analysis has been employed to identify nr2f1b-regulated genes in the vasculature

  • This has revealed potential downstream targets in vascular development pathways

Future research using ChIP-seq and RNA-seq would provide more comprehensive identification of direct Nr2f1b targets across different developmental contexts.

What are promising future research directions for nr2f1b studies?

Several promising avenues for future nr2f1b research include:

• Single-cell Transcriptomics

  • Characterization of nr2f1b expression at single-cell resolution

  • Identification of cell-type specific regulatory networks

  • Temporal dynamics of nr2f1b activity during development

• Chromatin Architecture

  • Investigation of how Nr2f1b influences chromatin organization

  • Mapping enhancer-promoter interactions regulated by Nr2f1b

  • Epigenetic mechanisms controlling nr2f1b expression

• Structure-Function Analysis

  • Detailed analysis of Nr2f1b protein domains and their specific functions

  • Identification of critical residues for DNA binding, protein interactions, and transcriptional activity

  • Development of domain-specific inhibitors or activators

• Disease Relevance

  • Exploration of human NR2F1 mutations in vascular disorders

  • Potential therapeutic applications based on Nr2f1b pathways

  • Connection to Bosch-Boonstra-Schaaf optic atrophy syndrome, which is linked to human NR2F1

• Translational Applications

  • Development of nr2f1b-based models for studying vascular disorders

  • Screening platforms for compounds that modulate Nr2f1b activity

  • Regenerative medicine applications targeting Nr2f1b pathways

These research directions will provide deeper mechanistic understanding of nr2f1b function and potential therapeutic applications.