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.

我们克隆并表征了斑马鱼淋巴细胞胞质蛋白 1（ lcp1 ，也称为 L-plastin）的内含子片段，该片段驱动斑马鱼包膜层 (EVL) 的表达。 L-塑性蛋白是一种钙依赖性肌动蛋白成束蛋白，属于塑性蛋白/纤毛蛋白家族，对于包括白细胞和破骨细胞在内的几种成体细胞类型的正确迁移和附着是必需的。然而，在斑马鱼中， lcp1在 EVL 分化过程中更早地大量表达。该上皮层的细胞集体迁移，在卵黄上生长。 L-塑蛋白表达持续到幼虫周皮中，这是一种形成幼虫第一层皮肤的短暂上皮组织。这一发现证实，L-塑蛋白在两个不同的胚胎波中被激活，早期 EVL 和晚期白细胞之间存在明显的调节开关。为了更好地研究 L-plastin 表达细胞，我们尝试使用 CRISPR/Cas9 同源驱动重组 (HDR) 在天然lcp1启动子下游插入自切割肽 (Cre-P2A-EGFP-CAAX)。这产生了在 EVL 细胞核中表达 Cre 重组酶并在 EVL 细胞膜中表达绿色荧光的稳定斑马鱼系。这些标记细胞的体内追踪提供了 EVL 迁移行为、膜延伸和有丝分裂事件的增强视图。最后，我们通过实验解剖了目标lcp1位点的关键元件，发现了足以驱动 EVL 表达的~300 bp 内含子序列。 lcp1 ：Cre-P2A-EGFP-CAAX 斑马鱼应可用于研究这种广泛使用的脊椎动物模型中的包络层规格、原肠胚形成运动和周皮发育。 此外，我们分离出的保守调控序列预测L-塑蛋白直向同源物在其他脊椎动物胚胎中可能具有类似的早期表达模式。