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Mfsd2a + hepatocytes repopulate the liver during injury and regeneration

View Article: PubMed Central - PubMed

ABSTRACT

Hepatocytes are functionally heterogeneous and are divided into two distinct populations based on their metabolic zonation: the periportal and pericentral hepatocytes. During liver injury and regeneration, the cellular dynamics of these two distinct populations remain largely elusive. Here we show that major facilitator super family domain containing 2a (Mfsd2a), previously known to maintain blood–brain barrier function, is a periportal zonation marker. By genetic lineage tracing of Mfsd2a+ periportal hepatocytes, we show that Mfsd2a+ population decreases during liver homeostasis. Nevertheless, liver regeneration induced by partial hepatectomy significantly stimulates expansion of the Mfsd2a+ periportal hepatocytes. Similarly, during chronic liver injury, the Mfsd2a+ hepatocyte population expands and completely replaces the pericentral hepatocyte population throughout the whole liver. After injury recovery, the adult liver re-establishes the metabolic zonation by reprogramming the Mfsd2a+-derived hepatocytes into pericentral hepatocytes. The evidence of entire zonation replacement during injury increases our understanding of liver biology and disease.

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PP hepatocytes reduced during liver homeostasis.(a) Schematic figure showing experimental strategy for tamoxifen treatment (Tam) and tissue analysis at the indicated time points after Tam. (b) Whole-mount fluorescence view of Mfsd2a-CreER;Rosa26-RFP livers collected at the indicated time points. Scale bars, 1 mm. (c) Quantification of the percentage of RFP+ and RFP− area. n=4. Error bars are s.e.m. of the mean. (d) Immunostaining for RFP, CK19 and HNF4a on liver sections shows that RFP+ hepatocytes remains in the PP regions during homeostasis. Scale bars, 100 μm. (e) Quantification of the percentage of RFP+ hepatocytes in the liver. (f) In situ hybridization of PP or PC genes on liver sections from mice at 6–20 weeks old. Scale bars, 500 μm. (g) Schematic figure showing PP hepatocytes reduced during liver homeostasis, whereas expression (Expr.) of zonation genes is maintained. Each image is a representative of four individual samples.
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f3: PP hepatocytes reduced during liver homeostasis.(a) Schematic figure showing experimental strategy for tamoxifen treatment (Tam) and tissue analysis at the indicated time points after Tam. (b) Whole-mount fluorescence view of Mfsd2a-CreER;Rosa26-RFP livers collected at the indicated time points. Scale bars, 1 mm. (c) Quantification of the percentage of RFP+ and RFP− area. n=4. Error bars are s.e.m. of the mean. (d) Immunostaining for RFP, CK19 and HNF4a on liver sections shows that RFP+ hepatocytes remains in the PP regions during homeostasis. Scale bars, 100 μm. (e) Quantification of the percentage of RFP+ hepatocytes in the liver. (f) In situ hybridization of PP or PC genes on liver sections from mice at 6–20 weeks old. Scale bars, 500 μm. (g) Schematic figure showing PP hepatocytes reduced during liver homeostasis, whereas expression (Expr.) of zonation genes is maintained. Each image is a representative of four individual samples.

Mentions: Although the PP and PC hepatocyte populations are distinct in terms of their molecular profiles and functions7812, we sought to address their repopulation capacity during homeostasis. We first analysed the proliferation of PP and PC hepatocytes by 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay in the wild-type mice (Fig. 2a) by adopting similar experimental strategies as previously described1417. Immunostaining for EdU, HNF4a (hepatocyte marker) and CDH1 (PP zonation marker) showed that a subset of hepatocytes (EdU+HNF4a+) were proliferating in both CDH1+ and CDH1− regions (Fig. 2b). Quantification of proliferating hepatocytes in both PP and PC regions showed a significantly higher number of proliferating hepatocytes in PC region than that in PP regions (percentage of EdU+ hepatocytes: 1.76±0.17% versus 1.01±0.092%, n=6; Fig. 2c). Similarly, we found more proliferating hepatocytes in PC region than in PP region of Mfsd2a-CreER transgenic mice (percentage of EdU+ hepatocytes: 1.84±0.24% versus 0.99±0.12%, n=6; Fig. 2d). We next performed genetic lineage-tracing experiments using Mfsd2a-CreER;Rosa26-RFP by administering tamoxifen at 6 weeks and performing analysis at later stages (Fig. 3a). Whole-mount fluorescence views of livers collected at 6-, 12- and 20-week-old mice showed a significant regression of RFP+ area with time, indicating that those unlabelled hepatocytes expanded and replaced some of the Mfsd2a-derived PP hepatocytes (n=4; Fig. 3b,c). However, there was no significant regression of the RFP+ area from 20 to 36 weeks (Fig. 3b,c). Immunostaining for RFP and CK19 on liver sections demonstrated that prelabelled PP hepatocytes reduced from 8–9 to 3–4 cell layers from week 6 to 20, which maintained thereafter till week 36 (Fig. 3d). Quantification for the percentage of RFP+ hepatocytes among all hepatocytes showed a significant reduction in RFP+ hepatocytes at 6- to 20-week-old mice (Fig. 3e), indicating that unlabelled hepatocytes competed over the labelled PP population during homeostasis. There is no significant reduction in RFP+ hepatocytes at 20- to 36-week-old mice (Fig. 3e).


Mfsd2a + hepatocytes repopulate the liver during injury and regeneration
PP hepatocytes reduced during liver homeostasis.(a) Schematic figure showing experimental strategy for tamoxifen treatment (Tam) and tissue analysis at the indicated time points after Tam. (b) Whole-mount fluorescence view of Mfsd2a-CreER;Rosa26-RFP livers collected at the indicated time points. Scale bars, 1 mm. (c) Quantification of the percentage of RFP+ and RFP− area. n=4. Error bars are s.e.m. of the mean. (d) Immunostaining for RFP, CK19 and HNF4a on liver sections shows that RFP+ hepatocytes remains in the PP regions during homeostasis. Scale bars, 100 μm. (e) Quantification of the percentage of RFP+ hepatocytes in the liver. (f) In situ hybridization of PP or PC genes on liver sections from mice at 6–20 weeks old. Scale bars, 500 μm. (g) Schematic figure showing PP hepatocytes reduced during liver homeostasis, whereas expression (Expr.) of zonation genes is maintained. Each image is a representative of four individual samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f3: PP hepatocytes reduced during liver homeostasis.(a) Schematic figure showing experimental strategy for tamoxifen treatment (Tam) and tissue analysis at the indicated time points after Tam. (b) Whole-mount fluorescence view of Mfsd2a-CreER;Rosa26-RFP livers collected at the indicated time points. Scale bars, 1 mm. (c) Quantification of the percentage of RFP+ and RFP− area. n=4. Error bars are s.e.m. of the mean. (d) Immunostaining for RFP, CK19 and HNF4a on liver sections shows that RFP+ hepatocytes remains in the PP regions during homeostasis. Scale bars, 100 μm. (e) Quantification of the percentage of RFP+ hepatocytes in the liver. (f) In situ hybridization of PP or PC genes on liver sections from mice at 6–20 weeks old. Scale bars, 500 μm. (g) Schematic figure showing PP hepatocytes reduced during liver homeostasis, whereas expression (Expr.) of zonation genes is maintained. Each image is a representative of four individual samples.
Mentions: Although the PP and PC hepatocyte populations are distinct in terms of their molecular profiles and functions7812, we sought to address their repopulation capacity during homeostasis. We first analysed the proliferation of PP and PC hepatocytes by 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay in the wild-type mice (Fig. 2a) by adopting similar experimental strategies as previously described1417. Immunostaining for EdU, HNF4a (hepatocyte marker) and CDH1 (PP zonation marker) showed that a subset of hepatocytes (EdU+HNF4a+) were proliferating in both CDH1+ and CDH1− regions (Fig. 2b). Quantification of proliferating hepatocytes in both PP and PC regions showed a significantly higher number of proliferating hepatocytes in PC region than that in PP regions (percentage of EdU+ hepatocytes: 1.76±0.17% versus 1.01±0.092%, n=6; Fig. 2c). Similarly, we found more proliferating hepatocytes in PC region than in PP region of Mfsd2a-CreER transgenic mice (percentage of EdU+ hepatocytes: 1.84±0.24% versus 0.99±0.12%, n=6; Fig. 2d). We next performed genetic lineage-tracing experiments using Mfsd2a-CreER;Rosa26-RFP by administering tamoxifen at 6 weeks and performing analysis at later stages (Fig. 3a). Whole-mount fluorescence views of livers collected at 6-, 12- and 20-week-old mice showed a significant regression of RFP+ area with time, indicating that those unlabelled hepatocytes expanded and replaced some of the Mfsd2a-derived PP hepatocytes (n=4; Fig. 3b,c). However, there was no significant regression of the RFP+ area from 20 to 36 weeks (Fig. 3b,c). Immunostaining for RFP and CK19 on liver sections demonstrated that prelabelled PP hepatocytes reduced from 8–9 to 3–4 cell layers from week 6 to 20, which maintained thereafter till week 36 (Fig. 3d). Quantification for the percentage of RFP+ hepatocytes among all hepatocytes showed a significant reduction in RFP+ hepatocytes at 6- to 20-week-old mice (Fig. 3e), indicating that unlabelled hepatocytes competed over the labelled PP population during homeostasis. There is no significant reduction in RFP+ hepatocytes at 20- to 36-week-old mice (Fig. 3e).

View Article: PubMed Central - PubMed

ABSTRACT

Hepatocytes are functionally heterogeneous and are divided into two distinct populations based on their metabolic zonation: the periportal and pericentral hepatocytes. During liver injury and regeneration, the cellular dynamics of these two distinct populations remain largely elusive. Here we show that major facilitator super family domain containing 2a (Mfsd2a), previously known to maintain blood–brain barrier function, is a periportal zonation marker. By genetic lineage tracing of Mfsd2a+ periportal hepatocytes, we show that Mfsd2a+ population decreases during liver homeostasis. Nevertheless, liver regeneration induced by partial hepatectomy significantly stimulates expansion of the Mfsd2a+ periportal hepatocytes. Similarly, during chronic liver injury, the Mfsd2a+ hepatocyte population expands and completely replaces the pericentral hepatocyte population throughout the whole liver. After injury recovery, the adult liver re-establishes the metabolic zonation by reprogramming the Mfsd2a+-derived hepatocytes into pericentral hepatocytes. The evidence of entire zonation replacement during injury increases our understanding of liver biology and disease.

No MeSH data available.