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High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function
Published on: October 19, 2013
Zebrafish (Danio rerio) matrilins: shared and divergent characteristics with their mammalian counterparts
Ya-Ping Ko1, Birgit Kobbe, Mats Paulsson
1Centre for Biochemistry, Medical Faculty, University of Cologne, Joseph-Stelzmann-Str. 52, D-50931 Cologne, Germany.
Abstract:
We have cloned the cDNAs of the zebrafish (Danio rerio) members of the matrilin family of extracellular adaptor proteins. In contrast to mammals, no orthologue of matrilin-2 was found in zebrafish, either by RT (reverse-transcriptase) PCR using degenerated primers or by screening the databases (Ensembl and NCBI); however, two forms of matrilin-3, matrilin-3a and -3b, were present. The identity with the mammalian matrilins is from more than 70% for the VWA (von Willebrand factor A)-like domains to only 28% for the coiled-coil domains of matrilin-3a and -3b. In all zebrafish matrilins we found a greater variety of splice variants than in mammals, with splicing mainly affecting the number of EGF (epidermal growth factor)-like repeats. The exon-intron organization is nearly identical with that of mammals, and also the characteristic AT-AC intron interrupting the exons coding for the coiled-coil domain is conserved. In the matrilin-3b gene a unique exon codes for a proline- and serine/threonine-rich domain, possibly having mucin-like properties. The matrilin-1 and -3a genes were mapped to chromosome 19 and 20 respectively by the radiation hybrid method. The temporal and spatial expression of zebrafish matrilins is similar to that seen in the mouse. Zebrafish matrilin-4 is highly expressed as early as 24 hpf (h post fertilization), whereas the other matrilins show peak expression at 72 hpf. By immunostaining of whole mounts and sections, we found that matrilin-1 and -3a show predominantly skeletal staining, whereas matrilin-4 is more widespread, with the protein also being present in loose connective tissues and epithelia.
Insights
Zebrafish possess matrilin-1, -3a, -3b, and -4, but lack matrilin-2 found in mammals. These extracellular proteins exhibit diverse splice variants and conserved gene structures, with distinct expression patterns and tissue localization.
Area of Science:
- Extracellular matrix biology
- Comparative genomics
- Developmental biology
Background:
- Matrilins are extracellular adaptor proteins crucial for tissue development and homeostasis.
- Understanding matrilin family evolution and function in zebrafish provides insights into vertebrate development.
Purpose of the Study:
- To identify and characterize zebrafish matrilin family members.
- To compare their gene structure, splice variants, and expression patterns with mammalian counterparts.
Main Methods:
- cDNA cloning and database screening (Ensembl, NCBI).
- Reverse-transcriptase PCR (RT-PCR) with degenerate primers.
- Radiation hybrid mapping.
- Whole mount immunostaining and section analysis.
Main Results:
- Zebrafish have matrilin-1, -3a, -3b, and -4; matrilin-2 orthologue is absent.
- Matrilins exhibit high sequence identity in VWA domains but lower in coiled-coil domains.
- Greater splice variant diversity observed in zebrafish matrilins, particularly affecting EGF-like repeats.
- Conserved exon-intron organization and AT-AC introns.
- Matrilin-3b gene contains a unique proline- and serine/threonine-rich domain.
- Matrilin-1 and -3a mapped to chromosomes 19 and 20.
- Temporal expression peaks differ, with matrilin-4 early (24 hpf) and others later (72 hpf).
- Matrilin-1 and -3a localize to skeletal tissues; matrilin-4 is more widespread.
Conclusions:
- Zebrafish matrilin gene repertoire differs from mammals, notably lacking matrilin-2.
- Conserved structural elements suggest functional importance of matrilin domains.
- Differential expression and localization indicate specialized roles for zebrafish matrilins in development.
