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Published on: May 17, 2017
Insight into Nephrocan Function in Mouse Endoderm Patterning
Martina Addeo1,2, Silvia Buonaiuto2, Ilaria Guerriero1
1Istituto di Ricerche Genetiche "G. Salvatore", Biogem s.c.ar.l, Ariano Irpino, 83031 Avellino, Italy.
Abstract:
Endoderm-derived organs as liver and pancreas are potential targets for regenerative therapies, and thus, there is great interest in understanding the pathways that regulate the induction and specification of this germ layer. Currently, the knowledge of molecular mechanisms that guide the in vivo endoderm specification is restricted by the lack of early endoderm specific markers. Nephrocan (Nepn) is a gene whose expression characterizes the early stages of murine endoderm specification (E7.5-11.5) and encodes a secreted N-glycosylated protein. In the present study, we report the identification of a new transcript variant that is generated through alternative splicing. The new variant was found to have differential and tissue specific expression in the adult mouse. In order to better understand Nepn role during endoderm specification, we generated Nepn knock-out (KO) mice. Nepn-/- mice were born at Mendelian ratios and displayed no evident phenotype compared to WT mice. In addition, we produced nullizygous mouse embryonic stem cell (mESC) line lacking Nepn by applying (CRISPR)/CRISPR-associated systems 9 (Cas9) and employed a differentiation protocol toward endoderm lineage. Our in vitro results revealed that Nepn loss affects the endoderm differentiation impairing the expression of posterior foregut-associated markers.
Insights
Nephrocan (Nepn) is crucial for early endoderm development. Its absence impairs in vitro endoderm differentiation, highlighting its role in specifying posterior foregut markers.
Area of Science:
- Developmental Biology
- Regenerative Medicine
- Molecular Genetics
Background:
- Endoderm-derived organs like the liver and pancreas are key targets for regenerative therapies.
- Understanding endoderm specification pathways is vital, but limited by a lack of early endoderm-specific markers.
- Nephrocan (Nepn) is a secreted protein expressed during early murine endoderm specification (E7.5-11.5).
Purpose of the Study:
- To investigate the role of Nephrocan (Nepn) in endoderm specification.
- To identify and characterize new transcript variants of Nepn.
- To analyze the in vivo and in vitro effects of Nepn deficiency on endoderm development.
Main Methods:
- Identification of a novel Nepn transcript variant via alternative splicing.
- Generation of Nepn knock-out (KO) mice (Nepn-/-).
- Creation of a nullizygous mouse embryonic stem cell (mESC) line using CRISPR/Cas9 technology.
- In vitro differentiation of mESCs towards an endoderm lineage.
Main Results:
- A new Nepn transcript variant with differential, tissue-specific adult expression was identified.
- Nepn-/- mice exhibited no observable phenotype and were born at Mendelian ratios.
- In vitro, Nepn-deficient mESCs showed impaired endoderm differentiation.
- Loss of Nepn affected the expression of posterior foregut-associated markers during differentiation.
Conclusions:
- Nephrocan plays a significant role in the in vitro specification of endoderm, particularly the posterior foregut.
- While Nepn is not essential for overall embryonic development in vivo, it is critical for proper endoderm differentiation in vitro.
- Further research into Nepn's function could advance regenerative therapies for endoderm-derived organs.
