), expressing Cre in the BHPC lineage, was used to delete RBP-J, the common DNA-binding partner required for gene transcription downstream from all Notch receptors, giving Alb-Cre;RBP-Jflox/flox
(RBP KO) mice, or to activate Notch1 by conditional expression of the NICD (Alb-Cre; ROSA26Notch1
; NICD mice). In a CD1 out-bred mouse strain, Alb-Cre
-mediated recombination of the ROSA26 locus is complete at embryonic day 16.5 (E16.5), as indicated by reporter activity (Sparks et al., 2010
). Immunofluorescent detection of GFP expressed via the internal ribosomal entry site within the NICD allele was observed at E16.5 (supplementary material Fig. S1A-D). The RBP-J
locus begins to recombine as early as E14, as assessed by quantitative PCR analysis of genomic DNA isolated from whole liver. However, analysis of whole liver extract does not allow us to rule out the possibility that there are cells that escape recombination by Alb-Cre
(supplementary material Fig. S2A). Analysis of ROSA26-YFP reporter (R26R-YFP) expression in Alb-Cre;RBP-Jflox/flox;R26R-YFP
livers at P60 and P120 show very few cells that do not express reporter activity. These cells are only observed in hilar ducts and not in peripheral ducts, thus suggesting that the majority of cells have expressed Alb-Cre
during early hepatoblast cell fate decisions (supplementary material Fig. S2B-E). Thus, Alb-Cre
allows us to address the chronic requirement of Notch signaling within liver epithelial cell lineages derived from the BHPC, and thus infers the post-natal consequence of chronic alterations in Notch signaling within the BHPC lineage in AGS patients.
To visualize the macro-structural effect of gain or loss of Notch signaling on 3D IHBD architecture, resin casts were obtained from mice of each genotype (control, RBP KO and NICD) at P60, P90 and P120 ( and data not shown). Prior to P60, control mice are still rapidly growing (P30–P60 slope=8.43 vs P60–P120 slope=3.98; supplementary material Fig. S3B); therefore, we selected time points that allow us to specifically assay the maintenance of IHBDs subsequent to the post-natal branching and elongation period. Stereoscopic images of left lobe resin casts from control mice showed no noticeable change with age (), whereas casts from RBP KO mice appeared to lose peripheral IHBDs (). Casts from NICD mice appeared to have increased peripheral branches early in development, which are maintained with age ().
Fig. 1. Resin casts of adult mice reveal IHBD changes upon altered Notch signaling. Resin casting was performed by manual, retrograde injection of a modified acrylic into the common bile duct, followed by tissue maceration. (A–F) Representative stereoscopic (more ...)
To quantify changes in the structure of the IHBD system with age, we quantified the volume, thickness and number of branches in left lobe resin casts using microCT (). Analysis of microCT scans was achieved by adapting trabecular bone analysis software from Scanco (Perrien et al., 2007
). Left lobe casts of control, RBP KO and NICD mice were microCT scanned and analyzed at P60 (n
=4, 5 and 3, respectively), P90 (n
=4, 6 and 3, respectively) and P120 (n
=4, 4 and 3, respectively). The same casts were analyzed throughout this manuscript. The total volume of the IHBD casts did not change significantly with age in the control mice. Interestingly, the total volume of IHBD casts from RBP KO mice was reduced at P120, as compared with age-matched control mice (P≤
0.01) and P60 RBP KO mice (P≤
0.05). Conversely, NICD IHBD casts showed consistent, but not significant, increases in cast volume with age compared with control ().
Fig. 2. MicroCT analysis reveals that IHBD average volume is modulated with age upon deletion of RBP-J or activation of Notch1. Reconstruction of microCT scans provides a high-resolution global representation of resin casts. (A) Representative image from Scanco (more ...)
To further define the structural changes that are visually apparent in the resin casts, we calculated the distribution of branch thickness throughout the IHBD casts (, ; supplementary material Fig. S4). The frequency of branch diameter distribution of the resin casts is displayed in . Owing to variations in maximum thickness resulting from the imprecise method of individual lobe separation (i.e. separated manually), the analysis included only diameters less than 520 μ
m, which consistently incorporates the main branches regardless of genotype. Small branches, as defined by a diameter of <15 μ
m (Glaser et al., 2009
), are not resolved by this method due to detection limitations imposed by cast size; however, we define intermediate (20–220 μ
m) and main (240–520 μ
m) branches for our analysis. As shown in , branches with a diameter between 240 and 520 μ
m highlight main branches, whereas branches with a diameter between 20 and 220 μ
m compose intermediate branches. Control IHBD casts demonstrate a high frequency of intermediate branches, and this frequency changed little with age. By contrast, RBP KO IHBD casts showed a significant decrease in intermediate branch frequency at P120 compared with control and RBP KO casts at P60 and P90. Intermediate branch diameter frequency in NICD IHBD casts did not significantly change with time ().
Fig. 3. Alterations in Notch signaling affect the frequency of intermediate and main IHBD branches. (A,B) The frequency of intermediate branches (20–220 μm; A) and main branches (240–520 μm; B) for each genotype are shown. The (more ...)
Fig. 4. Notch signaling affects the frequency of different branch diameters in proportion to the original contribution. Individual diameter frequencies (A–C) represented as a percent contribution to the total structure (D–F). (A,D) In control (more ...)
For all genotypes, the frequency of branches with a diameter of 240–520 μm (main branches) was reduced compared with the frequency of intermediate branches. Main branch frequency remained consistent in control and NICD IHBD casts with age, but was reduced in RBP KO IHBD casts at P90 and P120 compared with control (P≤0.05 and P≤0.01, respectively), and at P120 compared with P60 RBP KO (P≤0.001) (). Further dissection of the frequency of individual diameters revealed that 80–100 μm diameters were the most frequent regardless of genotype or age (; supplementary material Fig. S4). To visualize the relative contribution of each intermediate diameter branch to the total cast, frequency was represented as a percentage of all diameters (). Consistent with the raw data, the percent contribution of intermediate branches in control IHBD casts was the greatest of all of the branch diameters and remained consistent with age (). The percent contribution of each diameter in RBP KO IHBD casts did not change with age, indicating that RBP KO IHBD casts are losing intermediate and main branch diameters at the same rate (). NICD IHBD casts demonstrated a shift in contribution towards main branches at P120, which could be attributed to the inability to resolve individual intermediate branches with increased cast density at later time points with the current scanning resolution (branch separation <20 μm; ).
For RBP KO IHBD casts, a reduction in the frequency of a specific diameter could be due to (1) branch shortening and/or (2) branch loss (). In order to ascertain which possibility is more likely, we determined the total number of segments within the IHBD casts. A segment is defined as the portion between two branch points. The total number of segments in control IHBD casts with age was not significantly changed. In RBP KO IHBD casts, there was a significant reduction of segment number at P90 (P≤0.05) that was further reduced by P120 (P≤0.05). NICD IHBD casts had a consistently, but not statistically significant, increased number of segments compared with control (). These data suggest that changes in branch diameter frequency in RBP KO are due, at least in part, to cast branch loss. However, we cannot rule out that branch loss is due to excessive branch shortening. Resin casting and microCT analysis demonstrated that intact intermediate and main branches were not maintained with age in RBP KO mice.
Fig. 5. Loss of RBP-J results in a reduction of segments due to total branch loss with age. (A) Schematic illustrating a model of branch reduction in RBP KO IHBD casts. Upper panel represents a portion of the IHBD at a younger time point. This portion contains (more ...)
We hypothesize that Notch signaling might modulate maintenance of intact IHBD structure by (1) IHBD loss or gain, (2) functional blockage of the duct due to obstruction, or (3) physical collapse of the IHBD structure. To address the underlying physiology leading to structural loss, we assessed the number of bile ducts per portal vein in control and RBP KO mice at P60 and P120. For counting purposes, bile ducts were defined as cytokeratin19 (CK19)-positive structures containing a lumen. As expected, there was a reduction in the number of bile ducts per portal vein in P60 and P120 RBP KO mice as compared with control (P≤0.0001 and P≤0.05, respectively). However, the number of bile ducts per portal vein did not change between P60 and P120 in RBP KO mice (). It remains a possibility that entire portal tracts are lost, reducing the overall number of ducts without affecting the number of bile ducts per portal vein. However, consistent with the bile ducts per portal vein analysis, there is an initially reduced number of PV/mm in P60 RBP KO compared with control, but no further reduction at P120 (). Together, 3D and 2D analyses indicate that intact branches are not maintained with age in RBP KO mice, but 2D ductal structures are maintained. Conversely, NICD mice maintain an excess of intermediate branches with age.
Fig. 6. RBP KO mice have a reduction in bile ducts per portal vein and portal vein per area (mm) ratios at P60, which are not further reduced at P120. (A,B) Regions from the left or medial lobe were counted for peripheral bile ducts per portal vein and PV/mm. (more ...)