Recombinant Xenopus tropicalis Dehydrogenase/reductase SDR family member 7C (dhrs7c)

What is Xenopus tropicalis DHRS7C and why is it used in research?

DHRS7C (Dehydrogenase/reductase SDR family member 7C) belongs to the short-chain dehydrogenase/reductase (SDR) family, classified as SDR32C2. It is a strongly conserved protein in vertebrates that localizes to the endo/sarcoplasmic reticulum. Research interest in DHRS7C has grown significantly because:

• It shows tissue-specific expression patterns, with highest levels in heart and skeletal muscle followed by skin

• It exhibits inverse correlation with adrenergic stimulation and heart failure development

• Xenopus tropicalis provides an ideal model for studying this protein due to its diploid genome (unlike the tetraploid X. laevis), faster development time, and amenability to genetic manipulation

The expression profile of dhrs7c in X. tropicalis spans from NF stage 12.5 to adult frog stage, making it valuable for developmental biology research .

How does the expression pattern of dhrs7c in Xenopus tropicalis compare to mammals?

DHRS7C expression patterns show notable similarities and differences between X. tropicalis and mammals:

The conserved expression in cardiac tissue across species suggests evolutionary conservation of DHRS7C function in heart development and physiology, making X. tropicalis dhrs7c a valuable model for studying cardiac-related functions of this protein .

What experimental approaches can be used to study dhrs7c expression in Xenopus tropicalis?

Multiple experimental techniques can be applied to study dhrs7c expression in X. tropicalis:

• RNA-Seq and Transcriptomics:

  • RNA-Seq data for dhrs7c expression is available across developmental stages

  • Transcriptomic analysis can reveal stage-specific and tissue-specific expression patterns

• In situ Hybridization:

  • Standard X. laevis whole-mount in situ hybridization protocols work effectively on X. tropicalis without modification

  • This technique visualizes spatial expression patterns in intact embryos

• RT-PCR and qPCR:

  • For quantitative analysis of dhrs7c expression levels during development or in different tissues

• Immunohistochemistry:

  • Antibodies against X. laevis proteins often cross-react with X. tropicalis homologs

  • This allows protein-level detection of DHRS7C

• Microarray Analysis:

  • Data from platforms like Yanai et al. 2011 provide expression profiles across developmental stages

Importantly, the genetic tools developed for X. tropicalis enable more sophisticated expression analyses than were previously possible in amphibian models .

What genetic manipulation techniques can be applied to study dhrs7c function in Xenopus tropicalis?

X. tropicalis offers several genetic manipulation approaches for studying dhrs7c function:

• Morpholino-based Knockdown:

  • Antisense morpholino oligonucleotides (MOs) can be designed to target dhrs7c mRNA

  • MOs function effectively in X. tropicalis to inhibit translation or modify splicing

• CRISPR/Cas9 Genome Editing:

  • Can generate targeted mutations in the dhrs7c gene

  • The diploid genome of X. tropicalis makes genetic editing more straightforward than in X. laevis

• Transgenesis:

  • A simplified method of generating transgenic Xenopus can be applied to X. tropicalis

  • This allows for overexpression studies or rescue experiments

• Gynogenesis for Recessive Mutation Analysis:

  • Gynogenetic screening facilitates the identification of recessive phenotypes in a single generation

  • This technique has been adapted specifically for X. tropicalis

• Cell Line-based Studies:

  • Immortal cell lines derived from X. tropicalis embryos can be used for in vitro studies of dhrs7c

  • These cell lines can be maintained at 30°C in simple L-15 medium containing fetal bovine serum

Each approach offers unique advantages for dissecting dhrs7c function, with the choice depending on specific research questions and experimental goals.

How can Xenopus tropicalis models help understand the role of DHRS7C in heart development and disease?

X. tropicalis offers several advantages for investigating DHRS7C's role in cardiac development and disease:

• Evolutionary Conservation:

  • DHRS7C is strongly conserved in vertebrates with similar expression patterns in heart tissue

  • X. tropicalis heart development follows comparable pathways to mammals

• Experimental Accessibility:

  • Transparent embryos allow direct observation of heart development

  • Cardiac function can be assessed in living embryos

• Disease Modeling:

  • DHRS7C expression is down-regulated in heart failure models and in response to adrenergic stimulation

  • X. tropicalis embryos can be exposed to suspected cardiotoxins to unravel environmental risk factors for congenital heart defects

• Genetic Approaches:

  • The diploid genome of X. tropicalis facilitates genetic analysis of cardiac defects

  • DHRS7C mutations can be generated and their effects on heart development and function assessed

• Drug Screening:

  • Controlled exposure of genetically modified X. tropicalis embryos can be valuable in screening for drugs that suppress cardiac defects

These approaches provide a comprehensive framework for understanding DHRS7C's role in normal heart development and potential contributions to cardiac pathology.

What is the genomic organization of the dhrs7c gene in Xenopus tropicalis?

The genomic organization of dhrs7c in X. tropicalis has several notable features:

• Chromosomal Location:

  • Located within the well-annotated genome assembly of X. tropicalis

  • Can be precisely mapped due to the diploid nature of the genome (unlike the pseudotetraploid X. laevis)

• Gene Structure:

  • The gene contains specific regulatory regions that can be identified through BAC library analysis

  • Promoter regions can be analyzed using the highly efficient transgenic system in Xenopus

• Evolutionary Conservation:

  • Exhibits strong synteny with mammalian genomes, often in stretches of a hundred genes or more

  • This conservation facilitates comparative genomic analysis between amphibians and mammals

• Three-dimensional Organization:

  • May be organized within specific topologically associating domains (TADs) that influence its expression

  • TAD structures in X. tropicalis form similarly to those in mice and flies and continue to refine during development

Understanding the genomic context of dhrs7c provides insights into its regulation and evolutionary history, potentially revealing mechanisms controlling its tissue-specific expression patterns.

How does microRNA regulation affect dhrs7c expression in Xenopus tropicalis?

MicroRNA regulation of dhrs7c in X. tropicalis presents an important regulatory layer:

• MicroRNA Landscape in X. tropicalis:

  • More than 300 genes encoding 142 X. tropicalis miRNAs have been identified

  • Approximately 77% of miRNAs are found within introns of RNA Pol II-transcribed genes

• Potential Regulatory Mechanisms:

  • MiRNAs expressed in heart and skeletal muscle tissue may target dhrs7c mRNA

  • This regulation could contribute to the tissue-specific expression pattern of dhrs7c

• Experimental Approaches:

  • Northern blot analysis can confirm the expression of miRNAs in both X. tropicalis and X. laevis

  • Luciferase reporter assays can validate specific miRNA-dhrs7c interactions

• Developmental Dynamics:

  • Differential expression of miRNAs during development may influence temporal expression of dhrs7c

  • This could help explain the stage-specific expression patterns observed from NF stage 12.5 to adult stages

• Comparative Analysis:

  • Comparison between X. tropicalis and mammalian miRNA regulatory networks may reveal conserved mechanisms controlling DHRS7C expression

This regulatory layer adds complexity to understanding dhrs7c expression patterns and may provide novel therapeutic targets for conditions involving DHRS7C dysregulation.

What protein-protein interactions are known for DHRS7C in Xenopus tropicalis?

The protein interaction network of DHRS7C in X. tropicalis remains partially characterized:

• Subcellular Localization Context:

  • DHRS7C localizes to the endo/sarcoplasmic reticulum

  • This localization suggests potential interactions with other ER/SR proteins

• Enzymatic Function Partners:

  • As a member of the short-chain dehydrogenase/reductase family, DHRS7C likely interacts with:

    • Substrate molecules (not yet definitively identified)

    • Cofactors such as NAD(P)H

    • Potential electron transfer partners

• Experimental Approaches for Identification:

  • Immunoprecipitation followed by mass spectrometry using X. tropicalis tissue

  • Yeast two-hybrid screening using DHRS7C as bait

  • Proximity labeling approaches in X. tropicalis cell lines

• Computational Predictions:

  • Based on sequence homology with other SDR family members

  • Structural modeling to predict interaction interfaces

• Comparative Analysis:

  • Interactions identified in mammalian systems may provide clues about conserved interaction partners in X. tropicalis

Characterizing these interactions will be crucial for understanding DHRS7C's molecular function and its role in cellular processes, particularly in cardiac and muscle tissues where it is highly expressed.

How can chromosome conformation capture techniques be applied to study dhrs7c regulation in Xenopus tropicalis?

Chromosome conformation capture techniques offer powerful approaches to understand the 3D regulatory landscape of dhrs7c:

• TAD Organization:

  • High-throughput chromosome conformation capture (Hi-C) has revealed that X. tropicalis chromosomes are organized into topologically associating domains (TADs)

  • Understanding how dhrs7c is positioned within these TADs may reveal long-range regulatory interactions

• Experimental Procedures:

  • Hi-C can map genome-wide chromatin interactions

  • 4C-seq (Circular Chromosome Conformation Capture) can identify all regions interacting with the dhrs7c locus

  • 5C (Chromosome Conformation Capture Carbon Copy) can analyze interactions across specific genomic regions containing dhrs7c

• Developmental Dynamics:

  • TAD establishment in X. tropicalis is similar to that in mice and flies

  • Changes in chromatin interactions during development may explain stage-specific expression of dhrs7c

• Tissue-Specific Differences:

  • TAD structures vary between different tissues in X. tropicalis

  • This may contribute to the tissue-specific expression pattern of dhrs7c

• Role of Architectural Proteins:

  • Higher self-interaction frequencies at TAD boundaries are associated with higher DNA occupancy of architectural proteins CTCF and Rad21

  • These proteins may influence dhrs7c expression through chromatin organization

These approaches can reveal how three-dimensional genome organization contributes to the regulation of dhrs7c expression in different tissues and developmental stages.

What methodologies can be used to produce recombinant Xenopus tropicalis DHRS7C protein for functional studies?

Several approaches can be employed to produce recombinant X. tropicalis DHRS7C:

• Expression Systems:

  • Bacterial Expression: E. coli systems (BL21(DE3), Rosetta) for high yield

  • Insect Cell Expression: Baculovirus systems for eukaryotic post-translational modifications

  • Mammalian Cell Expression: HEK293 or CHO cells for proper folding and modifications

  • X. tropicalis Cell Lines: Recently developed immortal cell lines from X. tropicalis embryos

• Protein Purification Strategy:

  Step	Technique	Purpose
  Extraction	Detergent solubilization	Release membrane-associated DHRS7C from ER/SR
  Initial Capture	Affinity chromatography (His-tag, GST-tag)	Selective binding of tagged DHRS7C
  Intermediate Purification	Ion exchange chromatography	Remove contaminants based on charge
  Polishing	Size exclusion chromatography	Obtain homogeneous protein preparation
  Quality Control	SDS-PAGE, Western blot, Mass spectrometry	Verify purity and identity

• Functional Assays:

  • Enzymatic activity assays using potential substrates

  • Structural studies (X-ray crystallography, cryo-EM)

  • Protein-protein interaction studies

• Challenges and Solutions:

  • As an ER/SR-localized protein, DHRS7C may require detergents or amphipols for stability

  • Codon optimization for the expression system of choice may improve yield

  • Co-expression with chaperones may enhance proper folding

• Application in Research:

  • Recombinant protein can be used to generate specific antibodies

  • In vitro substrate screening to identify physiological substrates

  • Structure-function relationship studies

These methodologies provide a comprehensive approach to producing functional recombinant DHRS7C protein for detailed biochemical and structural characterization.

How does chromatin remodeling affect dhrs7c expression during Xenopus tropicalis development?

Chromatin remodeling plays a crucial role in regulating dhrs7c expression during development:

• Chromatin Remodeling Factors:

  • ISWI (Imitation SWItch) is required for de novo TAD formation in X. tropicalis

  • This suggests chromatin remodeling may be essential for establishing proper dhrs7c expression domains

• Developmental Dynamics:

  • TAD establishment is followed by refinements in active and repressive chromatin compartments

  • These refinements likely influence dhrs7c expression at different developmental stages

• Experimental Approaches:

  • ChIP-seq for histone modifications around the dhrs7c locus

  • ATAC-seq to assess chromatin accessibility

  • CUT&RUN for precise mapping of transcription factor binding sites

• Tissue-Specific Regulation:

  • Different chromatin states in heart and skeletal muscle may explain high dhrs7c expression

  • Comparative analysis between tissues with high and low expression can reveal regulatory mechanisms

• Manipulation Strategies:

  • Morpholinos targeting specific chromatin remodelers

  • Small molecule inhibitors of chromatin modifying enzymes

  • CRISPR-based recruitment of chromatin modifiers to the dhrs7c locus

Understanding these chromatin-based regulatory mechanisms provides insights into how dhrs7c expression is precisely controlled during development and in specific tissues.

What is the evolutionary significance of DHRS7C conservation between Xenopus tropicalis and mammals?

The evolutionary conservation of DHRS7C offers important insights into its fundamental biological roles:

• Sequence Conservation:

  • DHRS7C is strongly conserved among vertebrates

  • This suggests an essential function that has been maintained throughout vertebrate evolution

• Genomic Context:

  • X. tropicalis genome shows remarkable synteny with mammalian genomes

  • Analysis of the genomic neighborhood of dhrs7c can reveal conserved regulatory elements

• Expression Pattern Conservation:

  • Similar tissue-specific expression (heart, skeletal muscle) between amphibians and mammals

  • Conservation of expression suggests conservation of function in these tissues

• Functional Conservation:

  • Down-regulation in response to adrenergic stimulation is observed in mammals

  • Testing whether this response is conserved in X. tropicalis can reveal evolutionary conservation of regulatory mechanisms

• Comparative Analysis Framework:

  • X. tropicalis as a diploid species offers advantages for comparative genomics over the pseudotetraploid X. laevis

  • Comparison with other vertebrate models can place DHRS7C function in an evolutionary context

This evolutionary perspective helps distinguish fundamental roles of DHRS7C from species-specific adaptations and provides insights into the protein's core functions.