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MATERIALS AND METHODS

Fertile chicken eggs (Gallus gallus domesticus) were obtained from a commercial supplier and incubated in a rotating egg incubator (PROFI-I; Lyon Technologies) at 38° and 50% to 60% humidity. On ED4, 0.5 to 1 μL of human recombinant bFGF (100 ng/μL, dissolved in 0.1 M PBS solution and dyed with methylene blue; R&D Systems) was injected into the lateral or tectal ventricles. The injected FGF2 rapidly diffused throughout the ventricles, regardless of injection site. Control chicks were injected with 0.5 to 1 μL of dyed 0.1 M PBS solution. After injection, the eggs were resealed and transferred to the incubator until ED7, ED10, or ED12. The embryos were then immersion-fixed overnight in methacarn (by volume 60% methanol, 30% chloroform, 10% glacial acetic acid), dehydrated, embedded in paraffin, and sectioned at 18 μm. Approximately 40 to 70 evenly spaced sections from each brain were mounted onto Superfrost Plus slides (Fisher Scientific).

For morphometric measurements, sections were stained with Giemsa stain (Sigma-Aldrich) and coverslipped. Brain regions were delineated and volumes estimated using the Cavalieri method, as described previously (Striedter and Charvet, 2008). As defined here, the telencephalon includes the evaginated hemispheres and midline telencephalic structures; the tectum corresponds to what others have called the optic or dorsal tectum (Delgado et al., 2005). Telencephalon and tectum volumes for the ED12 embryos were normalized by comparing them to the rest of the brain, including diencephalon, pretectum, tegmentum, torus semicircularis, and hindbrain (but excluding tectum or telencephalon, respectively). The hindbrain was excluded from the normalization factor for the ED7 embryos, because it had not been sectioned completely in all the embryos. Ventricular surface area was estimated by summing ventricular surface lengths from a series of regularly spaced sections and multiplying the sum by the section spacing. The tectum’s radial thickness was quantified by dividing tectum volume by the tectum’s ventricular surface area.

To examine whether FGF2 injections delay tectal neurogenesis, we measured the proliferative and postproliferative zones in FGF2-treated and control chickens at ED7. As development proceeds, cells exit the proliferative ventricular zone and form a postproliferative mantle zone. As the mantle zone expands, the ventricular zone wanes. Therefore, a region’s PZF is a good measure of how far neurogenesis has progressed; the higher the PZF, the more neurogenesis has been delayed (Striedter and Charvet, 2008). To estimate the tectum’s PZF, we stained sections with antibodies against PCNA. Measurements were made on four equally spaced sections through each tectum and then averaged. Mounted sections were incubated with anti-PCNA (clone PC10; mouse; 1:500; Zymed), followed by a secondary antibody (anti-mouse IgG; 1:200; Vector Labs). They were then



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