The basic helix-loop-helix transcription factor, heart and neural crest derivatives expressed transcript 2, marks hepatic stellate cells in zebrafish: analysis of stellate cell entry into the developing liver

Abstract

Hepatic stellate cells (HSCs) are liver-specific mesenchymal cells that play vital roles in liver development and injury. Our knowledge of HSC biology is limited by the paucity of in vivo data. HSCs and sinusoidal endothelial cells (SECs) reside in close proximity, and interactions between these two cell types are potentially critical for their development and function. Here, we introduce a transgenic zebrafish line, Tg(hand2:EGFP), that labels HSCs. We find that zebrafish HSCs share many similarities with their mammalian counterparts, including morphology, location, lipid storage, gene-expression profile, and increased proliferation and matrix production, in response to an acute hepatic insult. Using the Tg(hand2:EGFP) line, we conducted time-course analyses during development to reveal that HSCs invade the liver after SECs do. However, HSCs still enter the liver in mutants that lack most endothelial cells, including SECs, indicating that SECs are not required for HSC differentiation or their entry into the liver. In the absence of SECs, HSCs become abnormally associated with hepatic biliary cells, suggesting that SECs influence HSC localization during liver development. We analyzed factors that regulate HSC development and show that inhibition of vascular endothelial growth factor signaling significantly reduces the number of HSCs that enter the liver. We also performed a pilot chemical screen and identified two compounds that affect HSC numbers during development.

Conclusion: Our work provides the first comprehensive description of HSC development in zebrafish and reveals the requirement of SECs in HSC localization. The Tg(hand2:EGFP) line represents a unique tool for in vivo analysis and molecular dissection of HSC behavior.

Fig. 1

Tg(hand2:EGFP) is expressed in a novel cell population within the zebrafish liver. (A) Whole-mount in situ hybridization shows the endogenous expression of hand2 in wild-type larvae at 4 days post fertilization (dpf). hand2 is expressed in the pharyngeal arch (Ph), fin bud (Fin), liver (Li), and the mesenchyme surrounding the intestine (Int). (A’) Tg(hand2:EGFP) expression resembles the endogenous expression of hand2. (B) At 4 dpf, Tg(hand2:EGFP) is expressed in a single-celled layer (arrows) lining the liver , as well as in star-shaped cells inside the liver (asterisks). (C-E) Expression of Tg(hand2:EGFP) does not overlap with the expression of the hepatocyte marker Tg(fabp10a:dsRed) (C), the biliary cell marker Alcam (D), or the endothelial cell marker Tg(kdrl:ras-mCherry) (E). Notably, Tg(hand2:EGFP)-expressing cells appear to wrap around endothelial cells (E, arrows). (F) Tg(hand2:EGFP) expression in a vibratome section of adult zebrafish liver. Similar to what is observed in the larval liver, Tg(hand2:EGFP)-expressing cells in the adult liver reside in close proximity to endothelial cells. (A, A’) Whole-mount zebrafish larvae, dorsal views, anterior to the top. (B-F) Confocal single-plane images of zebrafish livers. (B-E) Dorsal views, anterior to the top. A, anterior; P, posterior; L, left; R, right. Scale bars: (A, A’) 100 μm; (B-F) 20 μm.

Fig. 2

Tg(hand2:EGFP) expression marks HSCs in the zebrafish liver. (A-B) Tg(hand2:EGFP) expression in the liver at 5 dpf. (A’-B’) Same views as (A-B), but showing immunostaining for GFAP and desmin. Tg(hand2:EGFP) expression largely overlaps with GFAP and desmin antibody labeling at this stage (arrowheads). (C) Vibratome section of adult zebrafish liver shows the presence of vitamin A as revealed by gold chloride staining. (D) Tg(hand2:EGFP)-expressing cells deposit lipid droplets as shown by Oil Red O staining on a vibratome section of adult liver. (A-B, A’-B’, D) Confocal single-plane images of the zebrafish liver. (A-B, A’-B’) Dorsal views, anterior to the top. Scale bars, 20 μm.

Fig. 3

Acute ethanol treatment leads to increased deposition of extracellular matrix proteins and HSC number. (A, B) Confocal single-plane images of HSCs in untreated controls (A) and larvae treated with 2% ethanol from 96 to 120 hpf (B). Animals were collected and examined immediately after treatment. (A’, B’) same views as (A, B), but showing the deposition of laminin. HSCs in ethanol-treated animals upregulated their production of laminin and exhibited changes in morphology. 30 control and 30 ethanol-treated animals from six clutches were examined and all showed an increase in laminin deposition. (C, D) Confocal projections showing HSCs in untreated controls and ethanol-treated larvae at three days post treatment (dpt). HSCs in ethanol-treated larvae are more numerous, and show more elongated cell bodies and less complex cytoplasmic processes. (E) Numbers (mean±SEM) of HSCs in control and ethanol-treated animals immediately after treatment (0 dpt), and at 1, 2, and 3 days post treatment. At each time point, 10 control and 10 ethanol-treated larvae from two clutches were examined. The differences in HSC cell number between control and treated animals at 1, 2, and 3 dpt were statistically significant (p<0.05). (F) Percentages (mean ±SEM) of HSCs that had incorporated EdU during or at one day after ethanol treatment. At both time points, 10 control and 10 ethanol-treated larvae were examined. Asterisk indicates statistical significance: *p<0.05. (A-D) Dorsal views, anterior to the top. Scale bars, 20 μm.

Fig. 4

HSC development in zebrafish. (A-F) Time course analysis of HSCs and SECs in Tg(hand2:EGFP; kdrl:ras-mCherry) larvae. Eight larvae were fixed every two hours between 62 and 76 hpf, and stained for GFP (green) and dsRed (red). Arrows and asterisks in (A-B) mark the positions of SECs and HSCs, respectively. Arrows in (C-D) point to SECs that have entered the liver without being accompanied by HSCs. Arrowheads in (D) point to HSCs that have entered the liver in association with SECs. (A-F) Confocal projections of transverse vibratome sections, dorsal to the top. (G-H) HSCs inside the liver exhibited low proliferation rates. Five Tg(hand2:EGFP) larvae were fixed every two hours between 65 and 81 hpf and stained for Phospho-histone 3 (blue) which labels proliferating cells. 100 Tg(hand2:EGFP)-expressing cells were found to be Phospho-histone 3 positive, but only eight of them were located inside the liver (arrow in G). The remaining cells were located at the periphery of the liver (arrows in H-J). Among these cells, 39 of them were located proximal to the gut (arrows in H), 25 were located posteriorly (arrow in I), and 11 were located distal to the gut (arrow in J). (G-J) Confocal single-plane images of the liver, anterior to the top. (A-J) Dashed lines outline the liver. Scale bars, 20 μm. D, dorsal; V, ventral.

Fig. 5

clo mutant livers still contain Tg(hand2:EGFP)-expressing cells . (A-B) Wild-type and clo mutant larvae were collected from an incross of clo heterozygous fish that were also homozygous for the hand2:EGFP and kdrl:ras-mCherry transgenes. By 4 dpf, whereas wild-type livers formed a clear vascular network as revealed by Tg(kdrl:ras-mCherry) expression (A), endothelial cells were completely missing in clo mutant livers (B). (A’-B’) Confocal-reconstructed transverse sections of the livers shown in (A-B). White dashed lines in (A-B’) outline the livers. Yellow dashed lines in (A-B) mark the levels where the sections were reconstructed. (C-D) Distribution of hepatic biliary cells that express Alcam (red) and HSCs that express Tg(hand2:EGFP) (green). In wild-types (C), most HSCs (asterisk) are separated from biliary cells (arrow) by hepatocytes (indicated by the bracket). In contrast, in clo mutant livers (D), HSCs are closely associated with biliary cells (arrows). (E) Numbers (mean±standard deviation) of hepatocytes and HSCs in wild-type and clo mutant larvae. Hepatocytes were detected by Prox1 staining (25). 4 wild-type and 4 clo mutant larvae were analyzed at 4 dpf. Asterisks indicate statistical significance: *p < 0.05; ***p < 0.001. (A-B, C-D) Confocal single-plane images, anterior to the top. Scale bars, 20 μm.

Fig. 6

Inhibition of VEGF signaling during development decreases HSC number. (A-C) Inhibition of VEGF signaling reduced the number of intrahepatic vascular branches and HSCs in a dose and time-dependent manner. (A) Confocal projections of the livers in Tg(hand2:EGFP; kdrl:ras-mCherry) larvae that were treated with DMSO or the VEGF receptor inhibitor SU6415 from 55 to 80 hpf, or from 72 to 96 hpf. Dorsal views, anterior to the top. (B) Numbers (mean±SEM) of intraphepatic vascular branches in animals treated with DMSO, 1 μM SU6415, or 2 μM SU6415. (C) Numbers (mean±SEM) of HSCs in the same animals. (D) Knock-down of Kdrl levels resulted in a decrease in HSC number. Left panel shows confocal projections of the livers in uninjected controls and kdrl morpholino (MO)-injected larvae at 80 hpf. Right panel shows the numbers (mean±SEM) of HSCs in uninjected controls and kdrl-knock down animals. (E) Numbers (mean±SEM) of HSCs in clo mutants treated with DMSO or SU5416. VEGF signaling inhibition decreased the number of HSCs in clo mutants. (A, D) White dashed lines outline the livers. Scale bars, 20 μm. (B-E) The numbers of animals analyzed are shown at the bottom of the graph. Asterisks indicate statistical significance: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 7

Chemical screen identifies compounds that alter HSC numbers. (A) Chemical screen set-up, with cartoon illustrating phenotypes of interest such as decreased HSC numbers (middle animal) and increased HSC numbers (right animal) compared to control (left animal). (B) Numbers (mean±SEM) of HSCs in animals treated with DMSO, AM580, or methoprene acid (MA). The numbers of animals analyzed are shown at the bottom of the graph. Asterisks indicate statistical significance: *p < 0.05; **p < 0.01. (C) Confocal projections of the livers in Tg(hand2:EGFP) larvae that were treated with DMSO, AM580, or MA from 55 to 80 hpf. Dorsal views, anterior to the top. White dashed lines outline the livers. Scale bars, 20 μm.