Recombinant Macaca fascicularis Transcription factor SOX-14 (SOX14)

What is SOX14 and what functional role does it serve in Macaca fascicularis?

SOX14 is a member of the SOXB2 subgroup of transcription factors that plays critical roles in neural development. Although SOX14 is one of the most conserved SOX proteins across species, its specific functions in Macaca fascicularis neural development remain partially characterized. SOX14 has been implicated in neuronal differentiation and may participate in regulating the expression of other SOX family members during development . The gene functions similarly to human SOX14, with involvement in neurodevelopmental processes and potential roles in determining cell fate decisions in neural progenitor cells.

How is SOX14 expression regulated during neural development?

SOX14 expression is regulated through multiple mechanisms during neural development. Research has demonstrated that Sonic hedgehog (SHH) regulates SOX14 gene expression in spinal cord explants in a dose-dependent manner, suggesting SHH acts as a key regulator of Sox14-expressing interneuron differentiation . Additionally, the transcription factor FOXA2 has been identified as an important upregulator of SOX14 expression. Functional analysis has shown that mutation in the FOXA2 binding site reduces SOX14 reporter construct activity, while FOXA2 overexpression increases endogenous SOX14 protein expression . Furthermore, human SOX14 expression is GLI1-dependent in U87MG cells and SHH-N dependent in both U87MG and HepG2 cell lines, with FOXA2 serving as a mediator of SHH-dependent SOX14 activation .

How does SOX14 interact with other members of the SOX family?

SOX14 demonstrates complex interactions with other SOX family members, particularly those in the SOXB1 subgroup. Studies in human NT2/D1 and mouse P19 embryonal carcinoma cells have shown that upregulation of SOX14 during neural differentiation is accompanied by dynamic changes in SOXB1 member expression patterns . Specifically, increased SOX14 expression correlates with downregulation of SOX1 and SOX3 and upregulation of SOX2 at 4 weeks of retinoic acid treatment in NT2/D1 cells. In P19 cells, increased SOX14 expression correlates with downregulation of SOX1 and upregulation of SOX2 and SOX3 at the final phase of retinoic acid induction . Importantly, ectopic expression of SOX14 has been shown to repress SOX1 expression in HeLa cells, suggesting a potential regulatory relationship between these transcription factors .

What is the temporal and spatial expression pattern of SOX14 during neural differentiation?

SOX14 expression exhibits specific temporal patterns during neural differentiation. In in vitro models of neural differentiation using human NT2/D1 and mouse P19 pluripotent embryonal carcinoma cells, SOX14 expression is upregulated during retinoic acid-induced neural differentiation . Studies have shown that SOX14 protein levels increase progressively over 4 weeks of retinoic acid treatment in NT2/D1 cells, with the highest expression observed at 4 weeks. Similarly, in P19 cells, SOX14 expression increases during differentiation, with elevated levels in embryoid bodies and the neuronal population . Contrary to previous assumptions that SOX14 is exclusively a neuronal marker, immunocytochemistry has revealed that SOX14 is expressed in both neuronal and non-neuronal differentiated derivatives, suggesting broader functions than initially proposed .

What methods are most effective for detecting SOX14 expression in Macaca fascicularis tissues?

The most effective methods for detecting SOX14 expression in Macaca fascicularis tissues include:

• Western blot analysis using specific anti-SOX14 antibodies that recognize the Macaca fascicularis protein

• Quantitative real-time PCR (qRT-PCR) using primers specific for Macaca fascicularis SOX14 mRNA

• Immunohistochemistry/immunocytochemistry for spatial localization of SOX14 protein expression

• RNA-seq for transcriptome-wide analysis of SOX14 expression patterns

When performing these analyses, it's important to include appropriate controls. For Western blot and qRT-PCR, researchers typically use GAPDH as a loading control or reference gene, respectively . For immunohistochemistry, researchers often include neural differentiation markers such as β-III Tubulin (neuronal marker) and GFAP (glial marker) to correlate SOX14 expression with specific cell types .

What factors influence SOX14 expression levels in experimental settings?

Several factors have been shown to influence SOX14 expression levels in experimental settings:

Researchers should carefully control these factors when designing experiments involving SOX14 expression analysis .

What expression systems are optimal for producing recombinant Macaca fascicularis SOX14?

For producing recombinant Macaca fascicularis SOX14, several expression systems may be considered, with each offering distinct advantages:

Success with recombinant SOX14 production may require optimization of expression constructs. Research has shown that while SOX14 mRNA expression can be detected following transfection in various cell lines (NT2/D1, P19, HeLa), increased SOX14 protein levels are more readily detected in HeLa cells compared to NT2/D1 and P19 cells . This suggests cell-type specific factors may influence the stability or translation efficiency of SOX14.

What purification strategies yield the highest purity and activity for recombinant SOX14?

Effective purification of recombinant SOX14 typically involves a multi-step approach:

• Affinity chromatography: Using epitope tags (His-tag, FLAG-tag, or GST-tag) enables efficient initial capture of the recombinant protein.

• Ion exchange chromatography: This helps remove contaminants based on charge differences.

• Size exclusion chromatography: As a polishing step, this separates any remaining contaminants, aggregates, or degradation products based on size.

For maintaining SOX14 activity during purification, consider these critical factors:

• Include protease inhibitors throughout the purification process to prevent degradation

• Maintain appropriate buffer conditions (pH 7.4-8.0) to preserve protein structure

• Include reducing agents (DTT or β-mercaptoethanol) to maintain the native state of cysteine residues

• Use glycerol (10-20%) in storage buffers to enhance stability

• Store purified protein at -80°C with minimal freeze-thaw cycles

DNA-binding activity of purified SOX14 can be verified using electrophoretic mobility shift assays (EMSA) with oligonucleotides containing SOX binding motifs.

How can researchers verify the functional activity of recombinant SOX14?

Verification of recombinant SOX14 functional activity can be achieved through multiple complementary approaches:

• DNA binding assays: Electrophoretic mobility shift assays (EMSA) can determine if the recombinant SOX14 binds to its consensus DNA sequences. Chromatin immunoprecipitation (ChIP) assays can identify genomic binding sites in cellular contexts.

• Reporter gene assays: Luciferase or GFP reporter constructs containing SOX14 binding sites can assess transcriptional activity. Studies have used SOX14 reporter constructs to demonstrate that mutation in the FOXA2 binding site reduces SOX14 reporter activity .

• Gene expression analysis: Overexpression of recombinant SOX14 followed by analysis of known target genes can confirm functional activity. For example, ectopic expression of SOX14 has been shown to repress SOX1 expression in HeLa cells .

• Protein-protein interaction studies: Co-immunoprecipitation or pull-down assays can verify interactions with known partner proteins, such as other transcription factors in the SOX family.

• Functional rescue experiments: Introduction of recombinant SOX14 into systems where endogenous SOX14 has been knocked down can demonstrate restoration of normal function.

How does SOX14 contribute to neural differentiation and specification?

SOX14 plays multiple roles in neural differentiation and specification processes:

• Temporal regulation: SOX14 expression increases during neural differentiation, suggesting a role in the progression of neural progenitors toward differentiated states. In NT2/D1 and P19 embryonal carcinoma cells, SOX14 expression is upregulated during retinoic acid-induced neural differentiation .

• Cross-regulation with SOXB1 factors: SOX14 appears to participate in a regulatory network with SOXB1 transcription factors (SOX1, SOX2, SOX3). The upregulation of SOX14 during neural differentiation coincides with dynamic changes in SOXB1 expression patterns, with SOX14 potentially repressing SOX1 expression . This suggests SOX14 may help fine-tune the balance of SOX factors required for proper neural development.

• Integration with signaling pathways: SOX14 expression is regulated by the Sonic hedgehog (SHH) signaling pathway in a dose-dependent manner, indicating it functions as a downstream effector of this critical neurodevelopmental pathway . This suggests SOX14 may be particularly important in ventral neural tube development, where SHH signaling is prominent.

• Non-exclusive neural expression: Contrary to earlier assumptions, SOX14 is expressed in both neuronal and non-neuronal differentiated derivatives, suggesting it may have broader functions in determining cell fates beyond strictly neuronal lineages .

What phenotypes result from SOX14 knockdown or overexpression in neural development models?

Manipulation of SOX14 expression levels produces specific phenotypes in neural development models:

SOX14 knockdown effects:

• Potentially disrupts the proper specification of certain interneuron populations, as Sox14 expression has been linked to interneuron differentiation regulated by Sonic hedgehog

• May lead to altered expression of SOXB1 members, particularly increased SOX1 expression, based on the observed repressive effect of SOX14 on SOX1

SOX14 overexpression effects:

• Represses SOX1 expression, potentially altering the balance of SOX factors required for proper neural development

• May influence the timing or efficiency of neural differentiation in embryonal carcinoma cells and potentially in neural progenitors

• Could affect the proportion of cells adopting neuronal versus non-neuronal fates

These effects highlight the importance of precise SOX14 dosage control during neural development, similar to the dosage sensitivity observed with other SOX family members like SOX2 .

How do SOX14 functions compare between Macaca fascicularis and other model organisms?

SOX14 functions show both conservation and divergence across species:

• Conservation of developmental roles: SOX14 is one of the most conserved SOX proteins across vertebrates, suggesting fundamental roles in development that are likely preserved in Macaca fascicularis. The basic function in neural development appears consistent across mammals.

• Regulation by SHH signaling: The regulation of SOX14 by Sonic hedgehog signaling has been observed across different model systems, suggesting this regulatory relationship is evolutionarily conserved and likely present in Macaca fascicularis as well .

• Interaction with SOXB1 factors: The relationship between SOX14 and SOXB1 factors (SOX1, SOX2, SOX3) appears to be conserved between human and mouse models, with similar expression dynamics during neural differentiation . This suggests comparable interactions may exist in Macaca fascicularis.

• Species-specific differences: While core functions are conserved, species-specific differences may exist in the precise timing, spatial expression patterns, or regulatory mechanisms of SOX14. These differences might reflect adaptations in brain development and organization between species.

• Experimental considerations: When extending findings from human or mouse studies to Macaca fascicularis, researchers should consider potential differences in developmental timing, regulatory sequences, and protein-protein interactions that might affect SOX14 function.

What are the most reliable antibodies and detection systems for SOX14 in Macaca fascicularis samples?

For reliable detection of SOX14 in Macaca fascicularis samples, researchers should consider:

Primary antibodies:

• Polyclonal antibodies: Often provide good sensitivity but may have cross-reactivity issues

• Monoclonal antibodies: Offer higher specificity but sometimes lower sensitivity

• Antibodies validated for cross-reactivity with Macaca fascicularis SOX14 or raised against conserved epitopes

Detection systems:

• Western blot: Proteins separated by SDS-PAGE and transferred to membranes allow for size verification (approximately 20-30 kDa for SOX14)

• Immunofluorescence: Enables visualization of subcellular localization, typically nuclear for SOX14

• Immunohistochemistry: For tissue sections, allows assessment of spatial expression patterns

Control recommendations:

• Positive controls: Human or mouse samples with known SOX14 expression

• Negative controls: Samples from tissues not expressing SOX14

• Blocking peptide controls: To confirm antibody specificity

• siRNA knockdown controls: To validate signal specificity

Western blot detection of SOX14 has been successfully performed in NT2/D1 and P19 cells, with GAPDH serving as a loading control .

What cell culture models best represent SOX14 expression and function in Macaca fascicularis?

Several cell culture models can effectively represent SOX14 expression and function relevant to Macaca fascicularis:

• Primary neural progenitor cells: Isolated from Macaca fascicularis embryonic or fetal CNS tissue, these provide the most physiologically relevant model but are challenging to obtain and maintain.

• Induced pluripotent stem cells (iPSCs): Generated from Macaca fascicularis somatic cells and differentiated toward neural lineages, these can recapitulate aspects of neural development.

• Human and mouse embryonal carcinoma cells: NT2/D1 (human) and P19 (mouse) cells undergo neural differentiation upon retinoic acid treatment and show dynamic SOX14 expression . These established models may provide insights applicable to Macaca fascicularis.

• Neuroblastoma or glioblastoma cell lines: Cell lines like U87MG have been used to study SOX14 regulation by SHH signaling and FOXA2 .

Culture conditions that influence SOX14 expression include:

• Retinoic acid treatment: Induces neural differentiation and increases SOX14 expression in NT2/D1 and P19 cells

• SHH pathway activation: Regulates SOX14 expression in a dose-dependent manner

• Growth factor composition: May affect neural progenitor differentiation and SOX14 expression

What genomic and proteomic techniques are most informative for studying SOX14 regulatory networks?

Several advanced techniques provide valuable insights into SOX14 regulatory networks:

Genomic techniques:

• Chromatin Immunoprecipitation sequencing (ChIP-seq): Identifies genome-wide binding sites of SOX14, revealing direct target genes.

• ATAC-seq: Maps open chromatin regions, potentially identifying accessible SOX14 binding sites.

• CUT&RUN or CUT&Tag: Provides higher resolution mapping of transcription factor binding sites with lower background than traditional ChIP-seq.

• HiChIP or ChIA-PET: Identifies long-range chromatin interactions involving SOX14 binding sites.

Transcriptomic techniques:

• RNA-seq following SOX14 modulation: Reveals genes responsive to changes in SOX14 levels.

• Single-cell RNA-seq: Captures cell-type specific effects of SOX14 in heterogeneous populations.

• SLAM-seq or other metabolic labeling approaches: Distinguishes direct from indirect transcriptional effects.

Proteomic techniques:

• Immunoprecipitation coupled with mass spectrometry (IP-MS): Identifies SOX14 protein interaction partners.

• Proximity labeling (BioID, APEX): Maps the protein neighborhood of SOX14 in living cells.

• Protein crosslinking approaches: Captures transient interactions between SOX14 and other proteins.

Integration of these multi-omic approaches can provide a comprehensive understanding of SOX14 function and regulation in neural development contexts.

How does SOX14 cooperate with or antagonize other transcription factors in gene regulatory networks?

SOX14 operates within complex transcriptional networks through several mechanisms:

• Interactions with SOXB1 factors: SOX14 (a SOXB2 member) shows a complex relationship with SOXB1 factors (SOX1, SOX2, SOX3). Upregulation of SOX14 during neural differentiation correlates with changes in SOXB1 expression patterns, and ectopic SOX14 expression represses SOX1 . This suggests SOX14 may antagonize certain SOXB1 functions, potentially by competing for binding sites or co-factors.

• Regulation by upstream factors: FOXA2 has been identified as an upstream regulator that can increase SOX14 expression. Mutation in the FOXA2 binding site reduces SOX14 reporter construct activity, while FOXA2 overexpression increases endogenous SOX14 protein expression . This places SOX14 downstream in a transcriptional cascade initiated by FOXA2.

• Integration with signaling pathways: SOX14 expression is regulated by Sonic hedgehog (SHH) signaling, partially through FOXA2-mediated mechanisms. Human SOX14 expression is also GLI1-dependent in certain cellular contexts . This positions SOX14 as an integrator of developmental signaling information.

• Potential dosage-dependent effects: Like other SOX factors, SOX14 likely functions in a dosage-dependent manner within gene regulatory networks. SOX2's effects on self-renewal and differentiation of ESCs are highly dosage-dependent , suggesting similar principles may apply to SOX14's functions.

What epigenetic mechanisms regulate SOX14 expression during neural development?

Epigenetic mechanisms likely play crucial roles in regulating SOX14 expression during neural development:

• Chromatin modifications: The SOX14 promoter and enhancer regions likely undergo dynamic changes in histone modifications during neural development. Activating marks (H3K4me3, H3K27ac) may increase at the SOX14 locus during neural differentiation as expression is upregulated.

• DNA methylation: Changes in DNA methylation status, particularly at CpG islands near the SOX14 promoter, may correlate with its developmental expression patterns. Demethylation could contribute to increased SOX14 expression during neural differentiation.

• Pioneer factor activity: FOXA2, identified as an upstream regulator of SOX14 , is known to function as a pioneer factor that can bind to condensed chromatin and facilitate its opening. This suggests FOXA2 may promote SOX14 expression partly through epigenetic mechanisms by enhancing chromatin accessibility.

• Polycomb and Trithorax regulation: SOX factors like SOX2 interact with these chromatin modifying complexes to regulate target genes . SOX14 may similarly participate in recruiting or antagonizing these complexes at specific genomic loci.

• Long non-coding RNAs: These may participate in regulating SOX14 expression through various mechanisms including recruitment of chromatin modifiers or transcriptional machinery.

What are the implications of SOX14 research for understanding neurodevelopmental disorders in primates?

SOX14 research has several important implications for understanding neurodevelopmental disorders in primates:

• Evolutionary insights: Studying SOX14 in Macaca fascicularis provides an intermediate evolutionary perspective between rodent models and humans, potentially revealing primate-specific aspects of neural development relevant to human disorders.

• Interneuron development: Given SOX14's role in interneuron specification regulated by Sonic hedgehog , abnormalities in SOX14 expression or function could contribute to disorders involving interneuron dysfunction, such as epilepsy, autism spectrum disorders, or schizophrenia.

• Regulation of neural progenitor balance: The interactions between SOX14 and SOXB1 factors suggest it participates in regulating the balance between neural progenitor maintenance and differentiation . Disruptions to this balance are implicated in various neurodevelopmental disorders.

• Integration with known pathways: SOX14's regulation by SHH signaling connects it to a pathway with established roles in neurodevelopmental disorders. Mutations affecting this regulatory relationship could contribute to developmental abnormalities.

• Therapeutic considerations: Understanding SOX14's role in neural development could inform potential therapeutic strategies for neurodevelopmental disorders, particularly those aiming to restore proper neural circuit formation or function through targeted manipulation of specific interneuron populations.

How might single-cell technologies advance our understanding of SOX14 function in neural development?

Single-cell technologies offer powerful approaches to advance SOX14 research:

• Cell-type specific expression patterns: Single-cell RNA sequencing (scRNA-seq) can reveal the precise cell populations expressing SOX14 during neural development, beyond the broad categorization of "neuronal" versus "non-neuronal" cells . This may identify specific progenitor states or differentiation trajectories associated with SOX14 expression.

• Temporal dynamics: Single-cell technologies applied across developmental timepoints can capture the dynamic expression changes of SOX14 and related factors, revealing the precise timing of expression changes relative to cell fate decisions.

• Regulatory network reconstruction: By correlating SOX14 expression with other transcription factors and signaling components at single-cell resolution, researchers can infer regulatory relationships and construct more accurate gene regulatory networks governing neural development.

• Spatial context: Spatial transcriptomics or multiplexed in situ hybridization techniques can map SOX14 expression patterns in intact tissue, preserving information about spatial relationships between SOX14-expressing cells and their microenvironment.

• Functional heterogeneity: Single-cell approaches may reveal functional heterogeneity among SOX14-expressing cells, potentially identifying distinct subpopulations with different developmental potentials or functions.

• Lineage tracing: Combining single-cell transcriptomics with lineage tracing can determine the developmental trajectories and ultimate fates of SOX14-expressing progenitors.

What are the most promising future research directions for SOX14 in Macaca fascicularis?

The most promising future research directions for SOX14 in Macaca fascicularis include:

• Comprehensive characterization of SOX14 expression patterns throughout Macaca fascicularis neural development using advanced imaging and single-cell technologies.

• Detailed analysis of SOX14 genomic binding sites and the genes it regulates specifically in Macaca fascicularis neural tissues using ChIP-seq and related technologies.

• Comparative studies examining similarities and differences in SOX14 function between Macaca fascicularis, humans, and other model organisms to identify conserved and divergent aspects.

• Investigation of SOX14's role in specific interneuron populations and their circuit integration in the primate nervous system.

• Examination of potential links between SOX14 variation or dysfunction and neurodevelopmental disorders in Macaca fascicularis models.

• Development of improved tools for manipulating SOX14 expression in Macaca fascicularis cells and tissues, including conditional knockout or knockin approaches.