Lunatic and Manic Fringe Cooperatively Enhance
Marginal Zone B Cell Precursor Competition
for Delta-like 1 in Splenic Endothelial Niches
Joanne B. Tan, Keli Xu, Kira Cretegny, Ioana Visan, Julie S. Yuan, Sean E. Egan, and Cynthia J.
Figure S1: LacZ expression in Jag1+/LacZ spleen. X-GAL whole mount staining was
performed on spleens from adult Jag1+/LacZ mice. WP and central arterioles (CA) are indicated.
(a) Whole spleen viewed under dissecting scope. (b) Paraffin sections counter-stained with
Neutral Red are shown at 10X. No splenic X-GAL staining was observed in WT littermates (data
Figure S2: LacZ expression in Dll4+/LacZ spleen. X-GAL whole mount staining was performed
on spleens from adult Dll4+/LacZ mice. MS, WP, RP and CA are indicated. (a) Whole spleen
viewed under dissecting scope. (b) Paraffin sections counter-stained with Neutral Red are shown
at 10X. No splenic X-GAL staining was observed in WT littermates (data not shown).
Figure S3: Haplo-insufficiency of Notch2 and Dll1 for MZ B cell development in LacZ
reporter mice. (a) Splenocytes isolated from WT, Dll1+/LacZ, and Notch2+/LacZ mice between 8-
14 weeks of age were stained with anti-B220, anti-CD21, and anti-CD23 to define T1 B cells
(B220+ CD21int/lo CD23int), Fo B cells (B220+ CD21int CD23hi), and MZ B cells (B220+ CD21hi
CD23hi). The number of MZ B cells in Dll1+/LacZ and Notch2+/LacZ mice (triangles) compared to
their WT (Dll1+/+ or Notch2+/+) littermates (squares) were determined by the staining strategy
shown above. Horizontal bars represent the mean values of each experimental set. mean ± S.E.
of MZ B cells: Notch2+/+: 7.5x106±1.2x106, Notch2+/LacZ: 2.3x106±4.6x105, Dll1+/+:
5.1x106±9.9x105, Dll1+/LacZ: 1.3x106±2.0x105. Statistically significant differences, calculated
using one-sided Student’s T-test, are indicated. (b) Cryosections of Dll1+/+ and Dll1+/LacZ
spleens were stained with anti-IgM Cy3 (red) and anti-IgD FITC (green) to distinguish MZ B
cells inhabiting the MZ and Fo B cells found in WP splenic follicles. At the time of analyses, the
Dll1+/LacZ strain had been backcrossed to C57BL/6N more than 10 generations, and the
Notch2+/LacZ strain had been backcrossed to C57BL/6N for 4-6 generations.
Figure S4: Gating scheme for donor derived peripheral B cell subsets in Fringe chimeras.
Splenocytes were stained with anti-B220 APC, anti-IgM FITC, anti CD21-PE and biotinylated
anti-CD23 (Avidin PE-TR) to delineate peripheral B cell subsets. Chimerism was assessed using
anti-CD45.2 APC-Cy5.5 and anti-CD45.1 PE-Cy7 antibodies. Two-parameter contour plots
depicting B220 and IgM expression in gated live cells were generated to identify B cells in the
spleen (far left). B220+ cell populations were further subdivided using CD21 and IgM (left,
second column) to identify CD21hi IgMhi populations (right, third column) and CD21int IgMint
populations (far right) containing MZP (CD21hi IgMhi CD23hi), MZ (CD21hi IgMhi CD23int/lo),
and Fo B cells (CD21int IgMint CD23hi) respectively. Two-parameter contour plots depicting
CD45.1 and CD45.2 expression were used to identify the contribution of B6.CD45.2 and
B6.CD45.1; CD45.2 donor cells to the MZP, MZ, and Fo B cell subsets. The frequency of each
population within the contour plots is indicated.
Figure S5: Enumeration of MZPs versus MZ B cells.
Mixed chimeras generated in Figure 4c from Lfng+/+Mfng-/- and Lfng+/-Mfng-/- or Lfng–/–Mfng–/–
CD45.2 FL mixed with WT CD45.1; CD45.2 BM competitors were analyzed to show the
distribution of CD23hi MZPs and CD23int/lo MZ B cells among FL-derived IgMhi CD21hi
splenocytes. (A) shows an example of this distribution for Lfng+/+Mfng-/- and Lfng+/-Mfng-/-
versus Lfng–/–Mfng–/– donor cells. (B) shows the number of each subset in individual chimeras
from one experiment.