2. Materials and Methods
3.2. Ablation of VAMP8 causes defects in Thymus
3.2.1. Morphological defect
*Histological staining done by Dr. Afshin Fayyazi at the Department of Pathology, Georg-August Universität Göttingen.
Several organs from a small and sick VAMP8-/- Vti1b-/- mouse were examined histologically. While kidney, intestine, brain, spinal cord, spleen and heart appeared normal, the thymus showed a morphological defect. The pancreas of VAMP8-/- Vti1b-/ mice also showed a defect. The pancreas of small as well as adult VAMP8-/- Vti1b-/- mice looked enlarged and creamy. This defect was probably due to the loss of VAMP8 similar to what was previously described by Wang and co-workers in their VAMP8-/- mice. (Wang et al. 2003). However we concentrated on studying the thymus
During the initial work (Ref. section 1.4.3, 1.4.4), thymus of a few VAMP8-/- Vti1b-/- mice was studied by histochemistry and compared to the wild type mice. The normal morphology of thymus section from an adult wild type mouse is depicted in Figure 3.5A.
The cortex (C) and medulla (M) appear as distinct compartments in Giemsa staining (Figure 3.5A). On higher magnification, a clear boundary defining the thymic cortex and the medulla was observed (Figure 3.5B). Dendritic cells are strictly confined to the medulla and the cortico-medullary junction and can be seen by Fascein staining of thymus sections.
As seen in Figure 3.5C, the yellow stained dendritic cells are present only in the medulla within a well defined boundary and are excluded from the cortex. In addition, thymus from 2 months old adult VAMP8-/- Vti1b-/- mouse showed a normal morphology (analysed during master’s thesis) with a clearly defined medullary and cortical boundary (Figure 3.5D and E) and dendritic cells strictly confined to the medullary region (Figure 3.5F). However, thymus from small and sick VAMP8-/- Vti1b-/- mice showed no distinction into cortex and medullary compartments (Figure 3.5G).
Chapter 3. Results
Figure 3.5. Morphology of thymus is disrupted in VAMP8-/- small and sick mice: HE stained sections from the thymus of a wild type mouse showed a distinction into two compartments, the cortex (C) and the medulla (M) (A,B). Fascein stained thymus sections from wild type mouse showed the dendritic cells strictly confined to the medullary region (brown E). Thymus from a healthy adult VAMP8-/- mouse also showed a clear distinction into cortex and the medulla (C-D). In contrast, the thymus section from small and sick VAMP8-/- Vti1b-/- (G-H) and from a 8 day old VAMP8-/- (I,J) mice did not show the characteristic compartmentalization into cortex and medulla with medullary cells (pink) scattered through the thymic cortex (blue). Fascein stained thymus sections from a small and sick VAMP8-/- Vti1b-/- mouse showed a scattered pattern of dendritic cells (brown F). The pictures were taken by Leica-DM IRB microscope at the indicated magnifications.
*Histological staining done by Dr.
Afshin Fayyazi at the Department of Pathology, Georg-August Universität Göttingen.
On higher magnification, complete disruption of the cortico-medullary boundary was more prominent. There were few pink stained medullary cells randomly scattered between the
Chapter 3. Results
cortical cells (Figure 3.5H). Dendritic cells were also scattered in these thymii, as seen by fascein staining (Figure 3.5I).
During this work, the disruption of thymus morphology in VAMP8-/- Vti1b-/- mice was confirmed by analysis of thymii from several small and sick and adult VAMP8-/- Vti1b-/ -mice. In addition the thymus from small and sick VAMP8-/- mice were also analyzed.
Thymii from small and sick VAMP8-/- mouse showed a morphological defect similar to the VAMP8-/- Vti1b-/- mice. There was no distinct compartmentalization into cortex and medulla and a few medullary cells appeared scattered throughout the cortex (Figure 3.5J).
In addition, thymus from a 9 day old small not sick VAMP8-/- mouse was analyzed histologically. There was a no disruption of the cortico-medullary boundary in the thymus of this mouse and the two regions were clearly demarcated. Hence, it was confirmed that the disruption of thymus morphology was a characteristic feature of the small and sick stage of both VAMP8-/- and VAMP8-/- Vti1b-/- mice, while the thymus is normal at the small not sick stage.
3.2.2. Reduction in thymus size and total cell count
Total cell count of thymus was analyzed from several VAMP8-/- small and sick, small not sick and adult mice. The thymii were homogenized in 10ml of RPMI-1640 supplemented medium and the cells counted in a neubauer chamber. It was seen that the thymus cellularity in the VAMP8 -/- mice was reduced quite rapidly with the progression of phenotypic manifestation. While the small not sick mice already showed a signs of reduction in thymic cellularity, the most severe reduction was seen in the thymii from the small and sick mice.
The VAMP8-/- small not sick mice had an average of 140 million cells compared to 260 million in the VAMP8+/-controls i.e. a reduction by around 1.8 fold (Figure 3.6). The cell count in the small and sick VAMP8-/- mice rapidly reduced by upto 10 folds. There were just about 26 million cells in the thymus of the small and sick mice compared to 260 million in the controls (Figure 3.6). Thymus from the adult VAMP8-/- mice also showed a slight reduction by around 1.4 folds compared to littermate controls (Figure 3.6).
As previously observed, similar reduction was observed in the total thymus cell count of VAMP8-/- Vti1b-/- mice at the small and sick stage. On an average, the small and sick VAMP8-/- Vti1b-/- mice had around 40 million cells compared to around 510 million in the
Chapter 3. Results
littermate controls i.e. a reduction of up to 13 fold in the total cell count (Ref. section 1.4.4 and Figure 3.7A).
Cell count (Mio) 26 261 140 265 170 241
Figure 3.6. Thymus cellularity is progressively reduced in VAMP8-/-small and sick mice: The size of the thymus is highly reduced in the VAMP8-/- small and sick mice (A). The total cell count is also reduced by around 10 folds in the VAMP8-/- mice (n=7 age8-12 days) at the small and sick stage. The reduction in cell number can also be seen in the small but not so sick stage (n=5 age 8-12 days) and in the adults (n=3 age 1.5-2 months), (1.8 fold, 1.4 fold respectively) (B).
Figure 3.7. The cellularity of thymus is reduced in small and sick VAMP8-/- Vti1b-/- but not in Vti1b-/-mice: Small and sick VAMP8-/- Vti1b-/- mice (n=5,12-25 days) show up to 13 fold reduction in total cell count (A).
*Onset of phenotype was spread over first 1 month of age in VAMP8-/- Vti1b-/- mice compared to 7 days in the VAMP8-/- mice.
The data shown here is from VAMP8-/- Vti1b-/- mice between days 12-25. Hence the control VAMP8+/- Vti1b+/- have a higher number of total cell count compared to VAMP8-/+ controls.
Vti1b small mice (10-12 days, n=4) do not show any reduction in the thymus cellularity (B).
Chapter 3. Results
However in Vti1b-/- mice aged day 10-12, there was no reduction in the thymus cellularity compared to littermate Vti1b -/+ control mice (Figure 3.7B). The control mice had an average of 220 million cells compared to average 200 million cells in the Vti1b-/- mice.
Table XIII: A summary of reduction in total cell count in different studied genotypes.
Test Genotype Test
Thymocytes (Mio)
Control Thymocytes*
(Mio)
Fold decrease
VAMP8-/- small and sick 26 260 10 x
VAMP8-/- small not sick 140 266 1.8 x
VAMP8-/- adult 170 242 1.4x
**VAMP8-/- Vti1b-/- small and sick 40 510 13x
Vti1b -/- 200 220 -
*Vamp8 +/- littermates were used as controls for the Vamp8-/- mice , Vamp8+/- Vti1b+/- littermates for the Vamp8-/- Vti1b-/- and Vti1b +/- littermates for the Vti1b-/- mice. The same number of control and test mice was used. ** The window of phenotypic onset is extended over post natal days 11 to 29 in the Vamp8-/- Vti1b-/ -small and sick mice. The mice analyzed were aged 12-25 days and therefore the corresponding controls have a higher total cell count compared to the Vamp8+/- aged between 8-12 days.
Additionally, the spleen was severely reduced in the VAMP8-/- Vti1b-/- and VAMP8-/- small and sick mice.The total count of T lymphocytes in the spleen of VAMP8-/- Vti1b-/- small and sick mice was 13.8 million compared to 135 million in the controls i.e. a reduction by 10 folds. However there was no reduction in the T lymphocytes cell count in the spleen of VAMP8-/- adult mice (263 million) compared to the controls (266 million).
The above data indicates that VAMP8 is important for development of the thymus in mice and possibly for early postnatal survival. It was also confirmed that the previously observed phenotype in the VAMP8-/- Vti1b-/- mice is contributed entirely by the loss of VAMP8 while the loss of Vti1b does not affect thymus development.
3.2.3. SNARE proteins in the thymus
In order to confirm that the VAMP8tm1Lex mice do not have VAMP8 protein, protein levels in the VAMP8+/+, VAMP8+/- and VAMP8-/- mice were checked by western blot. VAMP8 was present in thymus of VAMP8+/+ and VAMP8+/- mice while VAMP8 was not detect in thymii from the VAMP8-/- mice (Figure 3.8A). From previous work in our lab it was known that syntaxin8 is degraded in the absence of Vti1b in the Vti1b-/- mice (Atlashkin et
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al. 2003). Therefore, we wanted to know if levels any of the SNARE proteins are changed due to the loss of VAMP8.
Deficiency of VAMP8 did not have a considerable effect on the amount of its complex partners syntaxin7, syntaxin8 and Vti1b nor of the early endosomal Vti1a or SNAP-29.
Western blot analysis showed a reduced amount of syntaxin 8 in the thymus from VAMP8 -/- Vti1b-/- mice (Fig. 3.8B) as expected for Vti1b-deficient tissue (Atlashkin et al. 2003).
The loss of the late endosomal SNARE complex could possibly be compensated for by the early endosomal SNARE complex. However, there was no major change in the amounts of the early endosomal SNAREs VAMP4, Vti1a or syntaxin16.
VAMP8 +/+ VAMP8 +/- VAMP8
Figure 3.8A. SNARE profile of the thymus from healthy VAMP8-/- mouse aged ≥ 1.5 months in comparison to the VAMP8-/+ and wild type mice.
There was no difference in the expression level of SNAP29, Vti1b, syntaxin 8 and Vti1a in thymii of control and test mice.
Figure 3.8B. SNARE profile of thymus from VAMP8-/- Vti1b-/- mouse(≥1.5 months) and littermate wild type mouse. There was no difference in the expression of Vti1a, VAMP4, SNAP29, syntaxin 7 and syntaxin 16 between the two samples. There was no Vti1b detected in the double knock out thymus while syntaxin 8 was reduced due to the deficiency of Vti1b in the VAMP8-/- Vti1b-/- mouse.