TY - JOUR
T1 - Identification of regulatory elements recapitulating early expression of L-plastin in the zebrafish enveloping layer and embryonic periderm
AU - Baumgartner, Emily A.
AU - Compton, Zachary J.
AU - Evans, Spencer
AU - Topczewski, Jacek
AU - LeClair, Elizabeth E.
N1 - Funding Information:
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 pre-submission review.
Funding Information:
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 pre-submission review.
Publisher Copyright:
© 2019
PY - 2019/6
Y1 - 2019/6
N2 - 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.
AB - 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.
KW - Actin
KW - Cre recombinase
KW - Cytoskeleton
KW - Enhancer
KW - Epiboly
KW - Epidermis
KW - Epithelium
KW - Filopodia
KW - Gastrulation
KW - Lamellipodia
KW - Microridges
KW - Periderm
KW - Promoter
UR - http://www.scopus.com/inward/record.url?scp=85064413023&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85064413023&partnerID=8YFLogxK
U2 - 10.1016/j.gep.2019.03.001
DO - 10.1016/j.gep.2019.03.001
M3 - Article
C2 - 30940554
AN - SCOPUS:85064413023
SN - 1567-133X
VL - 32
SP - 53
EP - 66
JO - Brain research. Gene expression patterns
JF - Brain research. Gene expression patterns
ER -