The vertebrate brain develops embryonically in such a way that parallels the evolutionary development of the brain. In the embryo there is a tubular dorsal nerve cord and it is from this cord that the vertebrate central nervous system (including the brain and spinal cord) arises. Early in the differentiation process, the embryonic nerve cord develops three distinct anterior bulges, which are called the forebrain, midbrain, and hindbrain. These same structures comprise the earliest form of the brain thought to have evolved. Later in the progression of both embryonic and evolutionary development, additional divisions of the brain appear. A five-week-old human embryo, for example, has a forebrain that has differentiated into two regions, termed the telencephalon and the diencephalon, and a hindbrain that has differentiated into the metencephalon and the myelencephalon. Modifications of the midbrain have also taken place by this point in embryonic development, when it is known as the mesencephalon.

Neurofilaments, which are found specifically in neurons and are especially prominent in the axons of the cells, were targeted in a rat brain horizontal section (shown above) with mouse anti-NF-P antibodies followed by goat anti-mouse secondary antibodies conjugated to Alexa Fluor 488. The glial fibrillary acidic protein was simultaneously targeted in the specimen with mouse anti-GFAP antibodies visualized with goat anti-rabbit secondary antibodies conjugated to Alexa Fluor 568. Nuclear DNA was subsequently labeled with Hoechst 33342. Images were recorded in grayscale with a 12-bit digital camera coupled to a Nikon Eclipse 80i microscope equipped with bandpass emission fluorescence filter optical blocks. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles.