Recombinant Branchiostoma floridae Brachyury protein homolog 2 (BRA-2)

What is the genomic organization of BRA-1 and BRA-2 in Branchiostoma floridae?

BRA-1 and BRA-2 are tandemly duplicated genes located on chromosome 9 (scaffold NC_049987.1) in the B. floridae genome and on scaffold NW_017803491.1 in the B. belcheri genome . These genes exhibit a distinctive head-to-head orientation in the genome, with no other T-box family members present in this genomic region . Molecular phylogenetic analysis using ORTHOSCOPE demonstrates that this duplication is specific to the cephalochordate lineage, as part of the seven T-box subfamily members identified in amphioxus genomes . The genomic arrangement suggests a relatively recent duplication event specific to cephalochordates that resulted in subfunctionalization of these paralogs.

How do the expression patterns of BRA-1 and BRA-2 differ during amphioxus development?

BRA-2 and BRA-1 display substantially different expression patterns during amphioxus development. Single-cell RNA sequencing (scRNA-seq) analysis has revealed that BRA-2 is expressed at significantly higher levels than BRA-1 and in more embryonic regions . Specifically, BRA-2 shows robust expression in the blastopore, notochord, somites, and tail bud, while BRA-1 exhibits much weaker expression primarily restricted to the notochord .

The scRNA-seq analysis classified embryonic cells into 15 clusters across developmental stages from midgastrula to early swimming larva. BRA-2 expression was detected in cells of clusters 4, 5, 8, and 9, whereas BRA-1 expression was limited to clusters 8 and 9 and at lower levels . Temporal analysis showed that BRA-1 expression occurs mainly at late gastrula and early neurula stages in cluster 9 cells, becoming undetectable by late neurula stage, while BRA-2 expression persists across multiple developmental stages .

What evidence suggests that BRA-2 is the ancestral Brachyury gene in amphioxus?

Multiple lines of evidence indicate that BRA-2 is the ancestral Brachyury gene in amphioxus:

• Expression pattern conservation: BRA-2's expression in the blastopore is shared with Brachyury orthologs in other deuterostomes, suggesting retention of ancestral expression domains .

• Expression level: BRA-2 shows significantly higher expression levels than BRA-1, consistent with it maintaining the primary Brachyury function .

• Regulatory element activity: The 5' upstream sequence of BRA-2 contains stronger enhancer activity for both notochord and somites compared to that of BRA-1, suggesting retention of ancestral regulatory functions .

• Expression breadth: BRA-2 is expressed in multiple embryonic tissues (blastopore, notochord, somites, tail bud) that collectively represent the full expression domain of Brachyury in other chordates, while BRA-1 shows a restricted pattern .

These observations support the hypothesis that following duplication, BRA-2 retained most ancestral Brachyury functions, while BRA-1 underwent subfunctionalization, evolving more specialized and restricted roles in notochord and somite formation specific to the Branchiostoma lineage .

What approaches can be used to produce recombinant BRA-2 protein for functional studies?

While the search results don't directly address BRA-2 protein production, established recombinant protein methodologies can be adapted based on approaches used for similar proteins . For BRA-2, a mammalian expression system would likely be optimal given the complex nature of transcription factors.

A comprehensive protocol would include:

• Vector design: Clone the BRA-2 coding sequence into a mammalian expression vector (e.g., pcDNA3.1) with an appropriate purification tag (His, FLAG, or GST) and strong promoter.

• Cell line selection: HEK293 cells are ideal due to their high transfection efficiency and proper post-translational modification capabilities .

• Transfection optimization: Use lipofection methods like Lipofectamine LTX with optimized DNA:lipid ratios for maximum efficiency .

• Stable cell line generation: Transfect the linearized plasmid containing BRA-2 and select with appropriate antibiotics (e.g., G418 at 0.5 mg/mL) to establish stable producer cell lines .

• Protein purification: Implement a two-step purification strategy using affinity chromatography based on the fusion tag, followed by size exclusion chromatography to ensure purity.

• Functional validation: Verify DNA-binding activity of the purified protein using electrophoretic mobility shift assays with known T-box binding elements.

This approach would yield properly folded, functionally active BRA-2 protein suitable for biochemical, structural, and functional studies.

How can single-cell RNA-seq be optimized for studying BRA-1 and BRA-2 expression during amphioxus development?

Based on the successful scRNA-seq analysis described in the search results , several optimization strategies can enhance the study of BRA-1 and BRA-2 expression:

• Developmental stage selection: Include multiple tightly-spaced timepoints from early gastrulation through larval stages to capture dynamic expression changes, as demonstrated in the study that examined midgastrula to early swimming larva stages .

• Cell dissociation protocol: Develop gentle enzymatic dissociation methods specific for amphioxus embryos to maintain cell viability while achieving complete dissociation.

• Transcript detection optimization: Given the sequence similarity between BRA-1 and BRA-2, design sequencing strategies that can distinguish these paralogs, such as targeting unique 3' UTR regions as mentioned in previous whole-mount in situ hybridization approaches .

• Cluster identification: Use established marker genes to accurately identify cell clusters, as demonstrated in the study where muscle-specific genes (myogenic factor genes) were used to identify somite cells in cluster 8 .

• Co-expression analysis: Analyze the co-expression of BRA-1/BRA-2 with toolkit genes like FoxA, Goosecoid, hedgehog (for notochord), Wnt1, Caudal (for blastopore/tail bud), and Hox genes to characterize the developmental context .

• Trajectory analysis: Implement pseudotime analysis to reconstruct developmental trajectories of BRA-1/BRA-2-expressing cells, particularly tracking transitions from blastopore to tail bud and notochord formation.

• Validation: Confirm key findings using complementary approaches such as spatial transcriptomics or multiplex fluorescent in situ hybridization to preserve spatial information lost in dissociated cell preparations.

What techniques can be used to distinguish between BRA-1 and BRA-2 proteins in amphioxus embryos?

Distinguishing between the highly similar BRA-1 and BRA-2 proteins in amphioxus embryos requires specialized approaches:

• Paralog-specific antibodies: Develop antibodies targeting the most divergent regions between BRA-1 and BRA-2, likely in the N- or C-terminal domains outside the highly conserved T-box DNA-binding domain. This requires:

  • Peptide design based on unique epitopes

  • Rigorous validation using recombinant proteins

  • Absorption against the paralog protein to remove cross-reactive antibodies

• Mass spectrometry-based approaches: Utilize targeted proteomics to detect unique peptides from each paralog:

  • Selected Reaction Monitoring (SRM) or Parallel Reaction Monitoring (PRM) targeting paralog-specific peptides

  • Label-free quantification to determine relative abundance

• Reporter constructs: Generate transgenic amphioxus lines with paralog-specific regulatory elements:

  • Use the divergent 5' upstream sequences of BRA-1 and BRA-2 that show different enhancer activities

  • Drive expression of distinct fluorescent proteins to visualize expression patterns in vivo

• RNA-based proxy detection: When protein detection is challenging, use highly specific RNA detection as a proxy:

  • RNAscope multiplex fluorescent in situ hybridization targeting unique untranslated regions

  • Single-molecule FISH to quantify transcript numbers at single-cell resolution

• Functional tagging: Use CRISPR-Cas9 to insert different epitope tags into the endogenous BRA-1 and BRA-2 genes, enabling distinction with commercial tag-specific antibodies.

These approaches, used in combination, would allow researchers to definitively distinguish the expression and localization patterns of these paralogous proteins.

How does BRA-2 contribute to notochord development in amphioxus?

BRA-2 plays a central role in notochord development in amphioxus, as evidenced by its expression pattern and co-expression with other notochord-specific genes. The scRNA-seq analysis identified cluster 9 as comprising notochord cells based on the simultaneous expression of BRA-2 and toolkit genes involved in notochord formation, including FoxA, Goosecoid, and hedgehog .

Notably, while both BRA-1 and BRA-2 are expressed in notochord cells, BRA-2 expression is significantly higher. Cluster 9 contained a larger number of cells with high BRA-2 expression compared to a smaller number of cells with BRA-1 expression . This suggests BRA-2 plays the predominant role in notochord specification and differentiation.

The transcription factor likely functions through similar mechanisms as Brachyury in other chordates, activating genes involved in notochord cell specification, intercalation, and vacuolation. Additionally, the co-expression with hedgehog suggests potential involvement in the notochord's signaling function to adjacent tissues like the neural tube and somites.

The expression of BRA-2 in cluster 9 from early developmental stages, combined with its co-expression with notochord markers, indicates that it functions in both the initial specification of notochord fate and potentially in subsequent differentiation and morphogenesis of this defining chordate structure.

What is the relationship between Sox2-to-Bra ratio and cell behavior during development?

While not specifically addressing amphioxus BRA-2, the search results provide valuable insights into how the ratio between Sox2 and Brachyury (Bra) influences cell behavior during development. In bird embryos, the Sox2-to-Bra ratio has been identified as a critical determinant of cell motility and fate decisions .

Progenitors in the posterior region of bird embryos show high cell-to-cell heterogeneity in Sox2 and Bra expression levels . This heterogeneity serves a functional purpose in guiding cell behavior:

• High Bra levels confer high motility that drives cells to exit the progenitor zone and join the paraxial mesoderm .

• High Sox2 levels inhibit cell movement, causing cells to remain more stationary and integrate into the neural tube .

• The initial heterogeneous expression in progenitors transitions to homogeneous expression in mature tissues .

Experimental evidence demonstrated that overexpression of Bra leads to reduced cell retention in the progenitor zone and increased integration into the paraxial mesoderm, while Sox2 overexpression promotes neural tube integration . Mathematical modeling suggested that the spatial distribution of Sox2/Bra heterogeneity is an important factor regulating morphogenesis .

This principle might extend to amphioxus development, where the relative levels of BRA-2 and neural specifiers could similarly influence cell movements during tissue segregation, particularly during notochord and somite formation.

How do Hox genes interact with BRA-2 during amphioxus development?

The scRNA-seq analysis revealed intriguing temporal coordination between BRA-2 and anterior Hox gene expression during amphioxus development. Specifically, Hox1, Hox3, and Hox4 were found to be highly expressed in the same cell clusters (4, 5, 8, and 9) that express BRA-2 .

This co-expression occurs in a temporally coordinated manner, suggesting potential regulatory interactions between BRA-2 and these Hox genes in the specification of mesodermal organs, including somites, notochord, and tail bud . The coordination is particularly significant because it reveals a potential role for anterior Hox genes in mesodermal specification beyond their well-established function in anteroposterior patterning.

The nature of this interaction could involve:

• BRA-2 potentially regulating Hox gene expression in mesodermal progenitors

• Hox genes possibly modulating BRA-2 activity in a region-specific manner

• Both gene sets being co-regulated by upstream factors in mesodermal lineages

• Physical or functional interactions between BRA-2 and Hox proteins to regulate downstream targets

This relationship between BRA-2 and Hox genes provides insight into how the gene regulatory networks controlling mesoderm development are integrated with those involved in anteroposterior patterning during chordate evolution.

What evidence supports the subfunctionalization of BRA-1 after duplication from the ancestral Brachyury gene?

• Restricted expression pattern: While BRA-2 maintains expression in multiple tissues (blastopore, notochord, somites, tail bud) similar to Brachyury in other deuterostomes, BRA-1 shows a much more restricted expression pattern, being weakly transcribed only in the notochord . This suggests BRA-1 retained only a subset of the ancestral expression domains.

• Reduced expression level: The zygotic expression level of BRA-2 is much higher than that of BRA-1, indicating that BRA-1 has diverged in its transcriptional regulation .

• Reduced enhancer activity: A heterogenic transplantation assay of cis-regulatory sequences demonstrated that the 5' upstream sequence of BRA-1 has lower enhancer activity compared to that of BRA-2 in both notochord and somites . This indicates divergence in the regulatory elements controlling BRA-1 expression.

• Temporal restriction: scRNA-seq analysis showed that BRA-1 expression becomes undetectable by the late neurula stage, while BRA-2 expression persists for longer periods , suggesting temporal subfunctionalization.

• Cell cluster limitation: BRA-1 expression was detected only in clusters 8 and 9 at specific stages, while BRA-2 was expressed in clusters 4, 5, 8, and 9 across multiple stages .

These observations collectively indicate that following duplication, BRA-1 retained only a portion of the ancestral Brachyury function, specifically related to notochord development, while BRA-2 maintained the broader ancestral expression pattern and likely function.

How does BRA-2 function compare with Brachyury in vertebrates?

Comparing BRA-2 function with vertebrate Brachyury reveals both conserved and divergent aspects:

Conserved features:

• Blastopore expression: BRA-2 expression in the amphioxus blastopore mirrors Brachyury expression in the blastopore/primitive streak of vertebrates, reflecting an ancestral role in gastrulation .

• Notochord specification: Both amphioxus BRA-2 and vertebrate Brachyury play essential roles in notochord development, a defining feature of all chordates .

• Posterior growth zone: Expression in the amphioxus tail bud parallels vertebrate Brachyury expression in the tailbud/growth zone, indicating conserved function in posterior axis elongation .

• Co-expressed genes: BRA-2 is co-expressed with toolkit genes like FoxA, Goosecoid, and hedgehog in notochord formation , similar to gene expression networks in vertebrate notochord development.

Divergent features:

• Somite expression: BRA-2 is expressed in amphioxus somites , while vertebrate Brachyury is generally not expressed in somites, reflecting lineage-specific roles in muscle development.

• Interaction with Sox2: In vertebrates, the Sox2-to-Bra ratio influences neural versus mesodermal fate decisions and cell motility . Whether a similar mechanism operates in amphioxus remains to be determined.

• Gene number and organization: Vertebrates typically have a single Brachyury gene, whereas amphioxus has two tandemly duplicated genes (BRA-1 and BRA-2) , representing independent evolutionary trajectories.

These comparisons highlight both the deep conservation of Brachyury function in chordate development and lineage-specific modifications that may contribute to the morphological differences between amphioxus and vertebrates.

What insights does the BRA-1/BRA-2 duplication provide into the evolution of developmental gene regulatory networks?

The duplication and subsequent divergence of BRA-1 and BRA-2 in amphioxus provides valuable insights into developmental gene regulatory network (GRN) evolution:

• Modular GRN architecture: The fact that BRA-1 retained only notochord expression while BRA-2 maintained the ancestral pattern suggests that different aspects of Brachyury function are controlled by modular regulatory elements that can evolve independently after duplication.

• Enhancer evolution as a driver of subfunctionalization: Differential enhancer activity between the regulatory regions of BRA-1 and BRA-2 demonstrates how enhancer evolution can drive functional divergence of duplicated genes without necessarily changing protein-coding sequences.

• Conservation of core GRN components: Despite the duplication, core aspects of Brachyury function in notochord development are conserved across chordates, suggesting that some GRN modules are highly resistant to evolutionary change.

• Paralog deployment in development: The expression of BRA-2 in multiple tissues versus the restricted expression of BRA-1 demonstrates how paralogs can be differently deployed following duplication, potentially allowing for more complex developmental regulation.

• Integration with signaling pathways: The co-expression of BRA-2 with components of major signaling pathways (e.g., hedgehog) indicates that these signaling modules were likely part of the ancestral chordate GRN and have been maintained despite gene duplication events.

These insights reveal how gene duplication provides raw material for the evolution of developmental GRNs, potentially contributing to morphological diversity while maintaining core developmental processes.

What approaches can be used to identify direct transcriptional targets of BRA-2?

Identifying direct transcriptional targets of BRA-2 requires integrative genomic approaches:

• ChIP-seq (Chromatin Immunoprecipitation followed by sequencing):

  • Develop highly specific antibodies for BRA-2 that don't cross-react with BRA-1

  • Perform ChIP-seq at multiple developmental stages focusing on tissues where BRA-2 is expressed

  • Analyze enriched motifs to identify the BRA-2 binding consensus sequence

  • Compare with known T-box binding elements from other species

• CUT&RUN or CUT&Tag:

  • These newer techniques require fewer cells than ChIP-seq and offer improved signal-to-noise ratio

  • Particularly valuable for studying specific cell populations isolated from amphioxus embryos

• RNA-seq following BRA-2 perturbation:

  • Use morpholinos or CRISPR-Cas9 to knock down/out BRA-2

  • Perform RNA-seq to identify differentially expressed genes

  • Cross-reference with ChIP-seq data to distinguish direct from indirect targets

• ATAC-seq combined with expression data:

  • Identify accessible chromatin regions that change following BRA-2 perturbation

  • Integrate with scRNA-seq data to correlate chromatin accessibility with gene expression in BRA-2-positive cells

• HiChIP or Capture-C:

  • Identify long-range chromatin interactions involving BRA-2-bound regions

  • Determine how BRA-2 influences three-dimensional genome organization

• Enhancer reporter assays:

  • Test putative BRA-2 binding sites in reporter constructs

  • Validate the functional significance of identified binding sites

  • Compare activity of enhancers responsive to BRA-1 versus BRA-2

Integration of these complementary approaches would provide a comprehensive map of the BRA-2 transcriptional network, revealing its direct targets and role in gene regulation during amphioxus development.

How might post-translational modifications regulate BRA-2 activity?

Post-translational modifications (PTMs) likely play crucial roles in regulating BRA-2 activity, though specific information about BRA-2 PTMs is not provided in the search results. Based on studies of Brachyury in other organisms, several potential regulatory mechanisms can be proposed:

• Phosphorylation:

  • Key serine/threonine residues may be phosphorylated by kinases downstream of signaling pathways like Wnt, FGF, or BMP

  • Phosphorylation could alter DNA binding affinity, protein-protein interactions, or nuclear localization

  • Different phosphorylation patterns might contribute to context-specific activity in different tissues (blastopore vs. notochord vs. somites)

• SUMOylation:

  • SUMO modification often regulates transcription factor activity and stability

  • SUMOylation could affect BRA-2's repressor vs. activator functions in different contexts

• Ubiquitination:

  • Regulation of BRA-2 protein levels through the ubiquitin-proteasome system

  • Potential for tissue-specific degradation contributing to expression pattern differences between BRA-1 and BRA-2

• Acetylation:

  • Acetylation of lysine residues could influence DNA binding or interactions with cofactors

  • Potential integration with epigenetic regulation through interactions with histone acetyltransferases

• Methylation:

  • Arginine or lysine methylation could modulate BRA-2 activity

  • Potential for crosstalk with DNA methylation machinery

Investigating these potential modifications would require:

• Mass spectrometry analysis of BRA-2 isolated from different embryonic regions

• Mutational analysis of putative modification sites

• Identification of enzymes responsible for adding/removing modifications

• Correlation of modification states with developmental stages and tissues

Understanding the PTM landscape of BRA-2 would provide insight into how this transcription factor achieves context-specific functions during amphioxus development.

What computational approaches can assist in predicting functional differences between BRA-1 and BRA-2?

Advanced computational approaches can help predict functional differences between these highly similar paralogs:

• Comparative sequence analysis:

  • Identify amino acid differences between BRA-1 and BRA-2 and map them onto structural models

  • Assess evolutionary conservation patterns of divergent residues across cephalochordates

  • Use algorithms like PROVEAN or SIFT to predict the functional impact of amino acid differences

• Protein structure prediction and analysis:

  • Generate 3D structural models of both proteins using AlphaFold2 or similar tools

  • Compare electrostatic surface potentials that might influence DNA or protein interactions

  • Identify potential conformational differences that could affect function

• DNA binding specificity prediction:

  • Use algorithms that predict transcription factor binding motifs based on protein sequence

  • Compare predicted binding preferences of BRA-1 versus BRA-2

  • Integrate with amphioxus genomic sequence data to identify potential differential target sites

• Protein-protein interaction prediction:

  • Predict interaction interfaces and potential interaction partners for both proteins

  • Identify differences in predicted protein interaction networks

  • Model how these differences might influence transcriptional complex formation

• Molecular dynamics simulations:

  • Simulate the dynamic behavior of both proteins bound to DNA

  • Analyze potential differences in conformational flexibility or stability

  • Predict how these differences might influence transcriptional activity

• Regulatory network modeling:

  • Integrate expression data from scRNA-seq with predicted binding sites

  • Model potential differences in the gene regulatory networks controlled by each paralog

  • Simulate the effects of perturbations to each network

These computational approaches would generate testable hypotheses about functional differences between BRA-1 and BRA-2, guiding experimental design and helping interpret experimental results.