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 different types of division. The first type is a division (proliferative division) that generates two identical stem cells thereby triggering the exponential expansion of the stem cell pool. The second type is a division (differentiative division) that generates more differentiated cells, such us neurons, thus depleting the stem cell pool.

In the developing mouse brain, we found that the switch from proliferation to neurogenesis of neural stem cells is accompanied by a lengthening of the G1 phase of their 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 suggested that the lengthening of G1 is a cause, rather than a consequence, of differentiation of neural stem cells as a short G1 may not provide the time that is necessary for a cell fate change to occur whereas a longer G1 will. Therefore, we investigated whether expansion of neural stem cells can be obtained by shortening G1, which we achieved by in utero electroporation of cell cycle regulators (Fig. 1B) during embryonic development. We found that shortening G1 i) inhibited neurogenesis (Fig. 1C), ii) increased the generation and expansion of neural progenitors, iii) induced a thickening of the germinal zones (Fig. 1D), and, finally, iv) correlated with a three-fold increase in cortical surface area of the postnatal brain being contributed by targeted progenitors (Lange et al., in press).

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 neurogenesis and manipulation of G1 can be used to increase neural stem cell expansion and, perhaps, brain size.


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 and use 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 / esiRNA into the mouse brain followed by electroporation. We perform this technique in whole-embryo culture (Calegari et al., 2002; Calegari et al., 2004) or directly in utero (Lange et al., in press), which we also used to detect micro-RNA activity in live tissues (DePietri Tonelli, et al., 2006).

• Moreover, we recently contributed to establish a method to control gene expression in whole organisms using UV light (Cambridge et al., 2009).

• Finally, we recently set up platforms to perform cell-transplantation as well as stereotaxic viral injection in the mouse brain.
  

Legend Figure 1 (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-G) Stereotaxic injection in the adult hippocampus with onco- or lenti-viruses, respectively.

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 brain. We believe this will be relevant for understanding tissue formation, brain function and, perhaps, to better use neural stem cells in regenerative therapy.

Group Members

List of group members

Selected Publications

Lange C. Huttner W.B. and Calegari F. 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.

DePietri-Tonelli D., Calegari F., Ji-Feng F., Nomura T., Osumi N., Heisenberg C.P. and Huttner W.B. (2006) Single-cell detection of microRNAs in developing vertebrate embryos after acute targeting using dual fluorescent reporter/sensor plasmid. Bio Techniques 41:727-732

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., Marzesco A.M., Kittler R., Buchholz F. and Huttner W.B. (2004) Tissue-specific RNA interference in postimplantation mouse embryos using directional electroporation and whole embryo culture. Differentiation 72:92-102.

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.