The Journal of Neuroscience, December 8, 2004, ():
Defective Postnatal Neurogenesis and Disorganization of the Rostral Migratory Stream in Absence of the Vax1 Homeobox Gene
J. Neurosci. Soria et al.
24: 11171
Supplemental data
Files in this Data Supplement:
- supplemental material
-
Supplementary Figure 1. Long term survival of precursor cells born at E14.
Birthdating experiment, extension of what is shown in Fig.5 A,B. In this case, BrdU was injected intraperitoneally in pregnant dams at E14 and subsequently revealed by double immunohistochemistry at P14 together with anti-GABA antibody(A’, B’) and and anti Tyrosine Hydroxylase (A’’, B’’). Row A refers to wt animals, while row B to Vax1 mutants. A,B: Low magnification of sagittal sections stained with DAPI. White squares indicate the areas analyzed.
In wt mice, proliferating cells born at E14 are homogeneously distributed along the RMS (A’), while in absence of Vax1 (B’) a large fraction of long term survival proliferating cells remain in the internal zone (IZ) close to the ventricle at P14.
A’’: WT P14 OB show well organized glomeruli as revealed by TH; these glomeruli contain double stained cells (white arrows) born at E14. B’’: TH staining of Vax1-/- OB shows the complete absence of glomeruli and the presence of very few periglomerular cells (white arrows in B’’), suggesting a failure in the differentiation and migration from the mutant SVZ. EZ: external zone. IZ: internal zone. g: glomeruli, RMS: rostral migratory stream. OB: olfactory bulb. Scale bars: A, B, 500mm, A’, A’’, B’, B’’, 200 mm
- supplemental material
-
Supplemetary Figure 2. Matrigel migration assays
A: Schematic representation of the experimental procedure for culturing internal (iEZ) and external (eEZ) expand zones of the mutant SVZ. B,C: Cells cultured from the external EZ (white arrows in B) are capable of long distance migration. Inset in B shows the typical morphology of migrating cells. C: No sign of migration is instead present when the internal EZ is cultured. eEZ: external expanded zone. iEZ: internal expanded zone. OB:Olfactory bulb. Scale bars: C-F 500 mm, H,I 100mm
The aim of this experiment was to understand whether some of the cells in the SVZ were capable of migration, given the fact that at P0 some glomeruli are present in the highly hypoplastic OB. We hypothesized that the peripheral area of the expanded SVZ (Er81 positive) could be capable of some sort of migration. To assess this, we have carefully dissected two areas of the expanded SVZ from P0 brains (see schema in Suppl Fig.2A): a deeper one, in close contact with the ventricles, and a more superficial one, as indicated by the squares in Suppl Fig 2A. We have then cultured these pieces of tissues in matrigel, allowing cells to migrate away from the explant. As shown in Suppl Fig. 2, while cells from the peripheral SVZ can migrate away from the original explant to disperse into the matrigel (2B), cells from the internal expanded zone are completely unable to do so (2C), indicating most likely that cells located in the periphery of the SVZ are more differentiated and that they express the correct molecules required for migration, like for example the ApoER receptor and the tyrosine kinase receptor EphA4 (data not shown).
Materials and methods
Dissected brains obtained from newborn wild type and Vax1 mutant animals (n=4) were embedded in 3% low-melting point agarose in 0.6% Glucose 0.1M PBS and subsequently sectioned using the vibratome (Leica). A base of 25 ml of matrigel matrix solution (BD Bioscence cat n° 356230) was applied by pipetting at the bottom of a 12-well dish, allowing it to solidify for 30 min in a sterile incubator (37°C; 5% CO2/ 95% O2). The internal and external areas of knockout expanded SVZ (EZ) were carefully dissected from sagittal sections and placed on a matrigel base, which was then covered with additional 25 ml of matrigel. Explants were incubated in Neurobasal medium for 48 hrs in a sterile incubator. Cells migrating from the cultured explants were observed and photographed in bright field microscopy, as above.
