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. 2021 Jun 23;11:13099. doi: 10.1038/s41598-021-92473-9

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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The expression of marker genes indicates a sequential development from HPT cells to SGCs and finally to GCs. The differentiation of GCs from HPT cells was induced in vitro by the addition of crayfish muscle extract. (A) Images of uninduced HPT cells and cells induced for 21 days. Bar, 20 μm. (B) RNA was extracted from the cells at different times post-induction, and the expression of marker genes was analyzed by RT-PCR. Purified SGCs and GCs were used as the controls. There was a sequential change in the gene expression pattern from undifferentiated HPT cells to SGCs and finally to GCs. The genes analyzed were PCNA (proliferating cell nuclear antigen, HPT cell marker); hemolectin (expressed in HPT cells and SGCs); CHF (crustacean hematopoietic factor, expressed in HPT cells and SGCs); KPI (SGC specific Kazal proteinase inhibitor, SGC marker); peptidase (SGC marker); peroxinectin (GC marker); and MBP (mannose-binding protein, GC marker). β-actin was used as an internal control.