Previous and current research

The goal
Our goal is to understand and manipulate the mechanisms controlling the proliferation vs. differentiation of neural stem cells in vivo.

The length of G1 as a crucial switch of neurogenesis
Neural stem cells, like any other somatic stem cell, can undergo essentially two types of division. The first is a division (proliferative division) that generates two identical stem cells thereby expanding the stem cell pool. The second is a division (differentiative division) that generates more differentiated cells, such us neurons, and depletes the stem cell pool.

We found that in the developing mouse brain the switch from proliferation to differentiation of neural stem cells is accompanied by a lengthening of the G1 phase of the cell cycle (Calegari et al., 2005) (Fig. 1A) and that an artificial lengthening of G1 alone is sufficient to induce premature neurogenesis (Calegari and Huttner, 2003). These findings indicated that the lengthening of G1 is a cause, rather than a consequence, of differentiation of neural stem cells. In essence, a short G1 may not provide the time that is necessary for a cell fate change to occur whereas a longer G1 will. Importantly, this suggested that expansion of neural stem cells can be achieved by “simply” shortening G1. Indeed, by using in utero electroporation, we overexpressed the positive regulators of G1 (Fig. 1B) during embryonic development and found that shortening G1 i) inhibited neurogenesis (Fig. 1C), ii) increased the expansion of neural progenitors, which resulted in iii) a thicker germinal zone (Fig. 1D), and, finally, iv) a three-fold increase in cortical surface area of the postnatal brain (Lange et al., 2009).

Legend Figure 1 (A) progenitors undergoing different division have different G1 length. (B) plasmids used to manipulate G1 length and (C) their effect on neurogenesis and (D) on thickness of germinal zones.

Therefore, G1 lengthening is both necessary and sufficient to induce the switch of neural stem cells from proliferation to differentiation (Salomoni and Calegari, 2010). We believe that our finding may not be limited to neural stem cells but may also hold true, in principle, for any other paradigm of stem cell differentiation (Lange and Calegari, 2010).

Manipulating the expansion of neural stem cells in the adult brain
We recently investigated whether a manipulation of G1 may allow the expansion of neural stem cells also in the adult mouse brain. To this end, we developed a system to acutely control the expression of cell cycle regulators, which allowed us to expand the NSC pool for the desired amount of time followed by their switch to physiological differentiation. As a result, we could double the number of newborn neurons generated in the adult hippocampus (Artegiani et al., 2011). To the best of our knowledge, this is the first temporally controlled expansion of any somatic stem cell ever performed acutely in an adult mammal and we believe that this achievement has a huge potential for better understanding i) stem cell contribution to tissue formation and homeostasis, ii) the role of brain size and adult neurogenesis in cognitive function, and iii) using neural, and perhaps also other somatic, stem cells for therapy; all lines of research that we are currently investigating.

Our tools
One of the strengths of our laboratory is the use, establishment and development of state-of-the-art technologies for the study of mammalian neurogenesis. The most important tools we contributed to develop are:

• a whole-embryo culture system that allows to reproduce mouse development ex utero (Calegari and Huttner., 2003).

• A platform to acutely overexpress / knock-down genes in neural stem cells by injection of DNA / siRNA into the mouse brain followed by electroporation. We perform this technique in whole-embryo culture (Calegari et al., 2002) or directly in utero (Lange et al., 2009).

• Moreover, we contributed to establish a method to control gene expression in whole organisms using UV light (Cambridge et al., 2009) and set up platforms to conditionaly and temporaly control the expression of multiple genes by injection of viral particles in the adult mouse brain (Artegiani et al., 2011).

Legend Figure 2 (A) Picture of mouse embryos developing in Whole Embryo Culture. (B-B’) neural progenitors transplanted in utero from a GFP donnor to a WT mouse. (C) Schematic representation of in utero electroporation and (D-E) its effect on the expression of GFP in the brain. (F-H) Adult mouse hippocampus after stereotaxic injection with onco- (F) or lenti- (G) viral particles and 3D reconstruction of the whole hippocampus shwoing GFP+ infected cells (H).

Future prospects and goals

In our future research we want to understand how cell cycle progression of neural stem cells is controlled in vivo and we want to investigate the consequences of manipulating the expansion of neural, and perhaps also other somatic, stem cells in the embryonic or adult mouse. We believe that this will be relevant for understanding tissue formation, brain function and, perhaps, to better use stem cells in regenerative therapy.

Group Members

List of group members

Selected Publications

Artegiani B, Lindemann D and Calegari F (2011) Overexpression of cdk4 and cyclinD1 triggers a greater expansion of neural stem cells in the adult mouse brain. J Exp Med 208:937-948.

Lange C and Calegari F (2010) Cdks and cyclins link G(1) length and differentiation of embryonic, neural and hematopoietic stem cells. Cell Cycle 9:1893-1900.

Salomoni P and Calegari F (2010) Cell cycle control of mammalian neural stem cells: putting a speed limit on G1. Trends Cell Biol 5:332-342.

Lange C. Huttner W.B. and Calegari F. (2009) Cdk4/cyclinD1 overexpression in neural stem cells shortens G1, delays neurogenesis, and promotes the generation and expansion of basal progenitors. Cell Stem Cell 5:320-31

Cambridge SB, Geissler E, Calegari F, Anastassiadis K, Hasan MT, Stewart AF, Huttner WB, Hagen V and Bonhoeffer T (2009) Cellular resolution, doxycycline-dependent photoactivated gene expression in eukaryotic systems. Nat Methods 6:527-31.

Calegari F., Haubensak W., Haffner C. and Huttner W.B. (2005) Selective lengthening of the cell cycle in the neurogenic subpopulation of neural progenitor cells during mouse brain development. J Neurosci 25:6533-6538.

Calegari F. and Huttner W.B. (2003) An inhibition of cyclin-dependent kinases that lengthens, but does not arrest, neuroepithelial cell cycle induces premature neurogenesis. J Cell Sci 116:4947-4955.

Calegari F., Haubensak W., Yung D., Huttner W.B. and Buchholz F. (2002) Tissue-specific RNA interference in postimplantation mouse embryo using endoribonuclease-prepared short interfering RNA. Proc Nat Acad Sci USA 99:14236-14239.