Alvarado-Mallart et al. (1984) - Metencephalon transplant

 Transplanting experiment
Figure 9 modified from "Development of the Nervous System" 2nd Ed, Fig. 2.13.

A signaling center at the midbrain-hindbrain boundary organizes this region of the brain. (A) Normally, in this region there is an expression of the homeodomain transcription factor engrailed which contains the progenitor cells for the cerebellum and midbrain. (B) Alvardo-Mallart et al. transplanted a small part of the telencephalon to the forebrain of another chick embryo. The cerebellum still developed from the transplanted tissue but additionally induced a new mesencephalon to develop ectopically in the forebrain.


McMahon and Bradley (1990) - wnt1 knockout

The met-mes boundary acts as an inducing center for the midbrain and cerebellum. This region contains localized mRNA of key signaling molecules such as wnt1, engrailed, and FGF8. McMahon and Bradley (1990) performed an investigation on wnt1 function by creating KO mice and found that this resulted in the absence of the midbrain and cerebellum. The exact reason for this phenotypical change and wnt1 function is not known but wnt1 -/- mice loose the expression of the transcription factor engrailed, which is usually co-expressed. This may indicate that engrailed is part of the wnt1 pathway and thus gets blocked. In Drosophila, the homologous gene for wnt1, wingless, is responsible for maintaining the expression of Drosophila engrailed gene at the segment boundaries. Deletions of engrailed and fgf8 yield similar phenotypical abnormalities as wnt1 KO.


Crossley et al (1996) - Ectopic Fgf8

FGF8 is important for the organizing signaling events occurring in the mes-met border. (A) Crossley et al (1996) placed a bead with fgf8 protein onto the telencephalon and discovered that it was sufficient to induce repatterning of this region to midbrain and hindbrain structures creating a new mes-met boundary with mirror duplicated midbrain. These results are similar to Alvarado-Mallart’s transplant experiment.


Cell cycle oscillations

 Figure 10: Birthdating
Figure 10 modified from "Development of the Nervous System" 2nd Ed, Fig. 3.2. and "Neuroscience 2nd Edition, S. Mark Williams" 2nd Ed, Box D.

Cell cycle lengths of progenitor cells can be revealed by thymidine labeling, where 3H- thymidine gets injected into a developing embryo. All the S-phase cells incorporate the label into their DNA. Once these cells continue to enter M-phase, they are recognizable as mitotically active. A graph of mitotic figures against time after thymidine injection shows how S-phase cells increase in number when reaching M-phase. Then these cells proceed from M-phase to G1 and are not mitotic anymore making their count decrease, creating oscillations of this sort where the time from one mitosis to the other represents the cell cycle length.


Retroviral lineage tracing

Retroviral tracing

A common method used for determining relationships between different cell types is called retroviral lineage tracing. Retroviruses can insert their genome into host cells only during S phase of the cell cycle. Genetically manipulating the retrovirus to express the genes for a traceable enzyme such as GFP and inserting them into the developing brain will allow to identify the only cells that will contain this foreign gene. Effectively, only the progenitor cells will be marked by this manipulation and if a small amount of viruses are added, it is possible to trace the progeny of the progenitor cells.


 Figure 11 modified from "Development of the Nervous System" 2nd Ed, Fig. 3.5.





Richard Sidman (1960) - Thymidine "birthdating"

3H-thymidine labeling in mice was used to determine when neurons and glia are formed- ie. become post-mitotic. Due to the dilution of the isotope over time it is also possible to find the newly generated post-mitotic cells which will be heavily stained. This technique was pioneered by Richard Sidman (1960). These thymidine “birthdating” studies revealed that neurogenesis is highly ordered with specific temporal and spatial gradients of neuron production as shown in fig 12 in a slice of the mouse hippocampus. In another example, the cerebral cortex gets generated in a medial-to-lateral fashion and the sequence of laminae production is conserved amongst species.

Figure 12 modified from "Development of the Nervous System" 2nd Ed, Fig. 3.7 C.

Inside-out development of the cerebral cortex

Inside-out development

Birthdating studies with the injection of a thymidine isotope reveal the inside-out pattern of the cerebral cortex. This experiment was performed on a rhesus monkey brain. Neurons that become postmitotic early in the embryo, are found close to the white matter of the subplate, while neurons developing later in gestation are found in more superficial cortical layers.

 Figure 13 modified from "Neuroscience, S. Mark Williams" 2nd Ed, Fig. 22.7.


Reeler Mice


The reeler mutant in mice disrupts cerebellar development by affecting migration. Cortical neurons develop normally but fail to take their normal positions, resulting in disorganized cortical layers and oblique radial glia. The reason is the lack of Reelin which is an extracellular matrix glycoprotein secreted by the Cajal-Retzius cells during corticogenesis. Histogenesis of the cortex has shown that the failure in migration results in the assumption of an inverted pattern. As a result, the mice start to loose their motor coordination (see video below). Mutations in the genes coding for tyrosine kinases called disabled, LDLR8 and VLDLR, ApoE and cdk5 show similar defects as the reeler mice. 

Figure 14: Image courtesy of https://upload.wikimedia.org/wikipedia/commons/8/80/Corticogenesis_in_a_wild-type_mouse_with_captions_in_english_copy.gif and https://upload.wikimedia.org/wikipedia/commons/4/4f/Corticogenesis_in_reeler_mutant_mouse_with_captions_in_english.gif. These images are in the public domain and thus free of any copyright restrictions.



Figure 15: Reelin

To test whether Reelin is a chemorepellent or chemoattractant, a transgenic mouse with Reelin expressed in wrong places was created. (A) shows the sagittal section of the area of cortical extraction for the experiment. (B) Mice with Reelin expression controlled by the nestin promoter, thus expressed in the ventricular zone, creates a Reelin “sandwich” for migrating neurons. Surprisingly, the nestin-Reelin mice had normal cortical lamination. (C) Mating nestin-Reelin mice with reeler-deficient mice only keeps Reelin in the ventricular zone with no expression in the Cajal-Retzius cells. This actually improved the lamination compared to the reeler-deficient mice, showing that the precise location of Reelin is insignificant; what counts is its expression in general.


Figure 15 modified from "Development of the Nervous System" 2nd Ed, Fig. 3.29.