Elsevier

Gene Expression Patterns

Volume 32, June 2019, Pages 53-66
Gene Expression Patterns

Identification of regulatory elements recapitulating early expression of L-plastin in the zebrafish enveloping layer and embryonic periderm

https://doi.org/10.1016/j.gep.2019.03.001Get rights and content

Highlights

  • Zebrafish L-plastin (lcp1) is expressed in the enveloping layer (EVL) of the gastrula.

  • A 300 bp non-coding element is sufficient to recapitulate EVL and periderm expression.

  • Our new transgenic line expresses nuclear Cre and membrane EGFP in all EVL cells.

  • Live imaging provides enhanced views of EVL migration and membrane extensions.

Abstract

We have cloned and characterized an intronic fragment of zebrafish lymphocyte cytosolic protein 1 (lcp1, also called L-plastin) that drives expression to the zebrafish enveloping layer (EVL). L-plastin is a calcium-dependent actin-bundling protein belonging to the plastin/fimbrin family of proteins, and is necessary for the proper migration and attachment of several adult cell types, including leukocytes and osteoclasts. However, in zebrafish lcp1 is abundantly expressed much earlier, during differentiation of the EVL. The cells of this epithelial layer migrate collectively, spreading vegetally over the yolk. L-plastin expression persists into the larval periderm, a transient epithelial tissue that forms the first larval skin. This finding establishes that L-plastin is activated in two different embryonic waves, with a distinct regulatory switch between the early EVL and the later leukocyte. To better study L-plastin expressing cells we attempted CRISPR/Cas9 homology-driven recombination (HDR) to insert a self-cleaving peptide (Cre-P2A-EGFP-CAAX) downstream of the native lcp1 promoter. This produced a stable zebrafish line expressing Cre recombinase in EVL nuclei and green fluorescence in EVL cell membranes. In vivo tracking of these labeled cells provided enhanced views of EVL migration behavior, membrane extensions, and mitotic events. Finally, we experimentally dissected key elements of the targeted lcp1 locus, discovering a ∼300 bp intronic sequence sufficient to drive EVL expression. The lcp1: Cre-P2A-EGFP-CAAX zebrafish should be useful for studying enveloping layer specification, gastrulation movements and periderm development in this widely used vertebrate model. In addition, the conserved regulatory sequences we have isolated predict that L-plastin orthologs may have a similar early expression pattern in other vertebrate embryos.

Introduction

The major cytoskeletal protein of most cells is actin, which exists in both monomer and polymer form. Once polymerized, the resulting actin filaments are organized into parallel bundles and branching networks by numerous accessory proteins. Most actin-organizing proteins are ubiquitous, appearing in all eukaryotic cells. However some are remarkably lineage-specific, which prompts the question of what unique functions these proteins provide and how their expression is controlled. In combination, both generalist and specialist actin organizers increase the capabilities of the cytoskeleton, expanding the cellular repertoire.

In this paper, we present our recent studies on a cell-specific actin-bundling protein called lymphocyte cytosolic protein 1 (lcp1). This is a highly-conserved, calcium-dependent phosphoprotein within the fimbrin family of proteins (Galkin et al., 2008; Zhang et al., 2016a, Zhang et al., 2016b). Originally isolated from neoplastic human fibroblasts, it was then termed ‘p65’ for its molecular weight of 65 kDa (Goldstein et al., 1985; Lin et al., 1988). Later, the same protein was identified as highly expressed in activated mouse macrophages, leading to the name of ‘leukocyte-specific plastin’ or ‘L-plastin’ (Shinomiya et al., 1994). Current research on L-plastin follows two well-traveled roads: its role in normal cells, and its role in tumor cells. In the normal context, L-plastin is characteristic of B-cells, T-cells, macrophages, monocytes, natural killer cells, and neutrophils (reviews by Delanote et al., 2005; Morley, 2012). In these cells, L-plastin is required for several critical functions including maturation, migration and immune synapse formation (Wang et al., 2010; Todd et al. 2011, 2016; Deady et al., 2014; Zhou et al., 2016). L-plastin is also expressed in osteoclasts, where it is necessary for cell-substrate contact and thus bone resorption (Ma et al., 2010; Chellaiah et al., 2018). Immune cells and osteoclasts are similar in that they are wandering cells that must make high-affinity contacts with specific extracellular targets. Thus, they likely require enhanced actin-bundling to stabilize cell membrane projections used in directed migration or substrate adhesion.

Enhanced migration is the hallmark of cancer, and most cancer deaths result from cellular spread from the primary tumor. L-plastin is thus additionally relevant due to its widespread expression in cancer cells of multiple origins. In animal models, overexpression of L-plastin contributes to apoptotic resistance, matrix invasion and metastatic spread (Janji et al., 2010), whereas siRNA suppression has the opposite effect (Chaijan et al., 2014; Inaguma et al., 2015). In humans, L-plastin has been suggested as diagnostic or prognostic marker for colon, kidney, nasopharyngeal, and oral cancers (Klemke et al., 2007; Samstag and Klemke, 2007; Li et al., 2010; Li and Zhao, 2011), and the significance of such expression is the subject of periodic reviews (Samstag and Klemke, 2007; Shinomiya, 2012). The question still unsolved from L-plastin's discovery 30 years ago is how the same gene is regulated to be constitutive in selected lineages where it is useful, yet simultaneously repressed in all other cells. Therefore the regulatory control of L-plastin is also of interest, and will enhance the study of both normal and cancerous cells.

Here we show that L-plastin is not restricted to zebrafish leukocytes, but has an additional major expression domain during early embryonic development. Consistent with its reported mRNA expression, L-plastin protein is first detected at late blastula. Spatially, expression is restricted to the enveloping layer (EVL), an extraembryonic tissue with epithelial characteristics. Temporally, expression coincides with EVL differentiation and migration, when cells migrate rapidly over the yolk. Weak periderm expression persists to the tailbud stage, but quickly fades; by 24 h, expression is restricted to individual, wandering myeloid cells. These observations establish that zebrafish L-plastin is activated in two embryonic waves, with a distinct regulatory switch between the early, EVL and the later leukocyte.

To better study L-plastin expressing cells, we generated a novel stable transgenic zebrafish– Tg (lcp1:Cre-P2A-EGFP-CAAX)– containing a multifunction protein cassette downstream of a 990 bp lcp1 intronic fragment. This cassette drives nuclear-localized Cre recombinase and membrane-enriched EGFP in all L-plastin expressing cells. F2 embryos show Mendelian inheritance of the transgene, as well as triple expression of Cre, EGFP and L-plastin in the EVL. The fluorescent membrane label provides excellent detail of EVL migratory activity, membrane protrusions and mitotic events in vivo, as demonstrated by time-lapse confocal microscopy. To isolate the sequences that drive early embryonic expression in zebrafish, we dissected key elements of the targeted lcp1 locus and tested a series of promoter constructs in vivo. This yielded a ∼300 bp intronic sequence sufficient to drive EVL and periderm expression, but unable to activate leukocyte expression. Our in silico analysis identifies this sequence as an alternative transcriptional start site (TSS) within the lcp1 first intron, which is active only during zebrafish gastrulation and segmentation (an 18-h interval). Overall, our novel, stable zebrafish line should be useful for studying enveloping layer specification, gastrulation movements and periderm development in this widely used vertebrate model. In addition, the regulatory sequences we have identified will enhance the study of how L-plastin expression is controlled in specific lineages such as skin, bone and the immune system.

Section snippets

L-plastin protein is expressed in the enveloping layer and the embryonic periderm

When our study began, we knew that L-plastin mRNA was expressed in the EVL from late gastrulation to the early somite stage, and in wandering leukocytes at later stages (Thisse et al., 2001). To identify the earliest cells in which L-plastin protein is expressed, we performed whole-mount immunohistochemistry on wild type embryos. Our survey began at the 1–2 cell stage – a time at which L-plastin localization has not been well described – and ended at 2 dpf, when leukocyte expression is clearly

Characterization of L-plastin expression in early zebrafish development

L-plastin's best known role is as a leukocyte marker (Morley, 2012). Less well understood is its expression in other contexts. Here, we extend previous work on this gene's mRNA localization and show that L-plastin is a highly specific marker for the EVL and its derivative, the embryonic periderm. Using a zebrafish-specific L-plastin antibody, we find that protein expression in these cells is punctate, with clusters or dots spread throughout the cytoplasm. This spatial pattern is consistent with

Fish strains and breeding

The DePaul University Institutional Animal Care and Use Committee (IACUC) approved all animal protocols. Adult fish were kept in a heated, recirculating rack system at 28 °C on a 14:10 light-dark cycle with a maximum stocking density of 1 fish/200 mL. The adult diet was a 50:50 mix of finely ground flake food (TetraMin) and decapsulated brine shrimp eggs (American Brine Shrimp) given once or twice a day.

Fertilized eggs were obtained by placing adult pairs in a 500 mL crossing tank filled with

Declarations of interest

None.

Acknowledgements

Financial support was provided by grants from the National Institute of General Medical Sciences (R15-GM120664, to E. E. LeClair) and the DePaul University Research Council (URC). Confocal analysis was made possible in part by FluoRender software funded by the National Institutes of Health (NIH R01-GM098151 and P41-GM103545). We thank Janine Kirin, J.D. Davis, Jesse Hacker and Jordan Johnson for their contributions to fish care, and Mason Posner (Ashland University, OH) for valuable

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