Federico Calegari - Proliferation and Differentiation of Neural Stem Cells


1998  Master’s Degree,
University of Milano Italy

2000 Visiting scientist,
University of Heidelberg

2000 Ph.D., University of Milano Italy

2001-2004 Postdoctoral fellow, MPI-CBG, Dresden

2004-2006 Staff scientist,
MPI-CBG, Dresden

since January 2007 Group leader, CRTD

Previous and current research

The goal

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

It’s just a matter of Time

NSC, like all somatic stem cells, can undergo two types of division. The first is a division (proliferative) that generates two identical stem cells thereby expanding the stem cell pool. The second is a division (differentiative) that generates more differentiated cells, such us neurons, depleting the stem cell pool. We found that the length of the G1 phase of the cell cycle acts as a crucial switch determining whether a stem cell will undergo a proliferative vs. differentiative division (Fig. 1A). In short, cells need Time in order to change and this is provided during G1. The cool part is that we can change G1 as we want…well, sort of.

Neurogenesis in brain development, evolution, adulthood, disease and choosing iPhones

In the developing brain, the switch from proliferation to differentiation of NSC correlates with a lengthening of G1 (Calegari et al., 2005) (Fig. 1A) and an artificial lengthening of G1 alone was sufficient to induce neurogenesis (Calegari and Huttner, 2003). This suggested that lengthening of G1 is a cause, rather than a consequence, of differentiation (Calegari and Huttner, 2003). Thus, to expand NSC all we needed to do was to force them to keep a short G1. Christian did exactly that by overexpressing Cdk4/cyclin D1 (4D) by in utero electroporation (Fig. 1B and C) and, luckily for his PhD and my renewal, that worked quite well leading to inhibition of neurogenesis and progenitor expansion (Fig. 1D) (Lange et al., 2009). As such, manipulating G1 allowed us to control stem cell expansion in the brain (Salomoni and Calegari, 2010) and since we scientists like to over speculate beyond our own field we also concluded that this must work in any other stem cell system ever created (Lange and Calegari, 2010) (Fig. 1E).

So far so good, we can force NSC to expand during development… but can we get more neurons out of them? If so, can we generate mice with bigger brains, perhaps, with folds like human??? That was a project we could not miss, the only problem was to find a student crazy enough to buy all this. We achieved the latter by recruiting Miki, and the rest was downhill. Miki generated a Tet-On/4D line and created little brainy mice (for obvious reasons called the Miki’s mouse) (Nonaka et al. 2013). Yet, we couldn’t get any gyrus there (Fig. 1F). So how can gyri have emerged during evolution??? You can bet we were not the only ones asking that trivial question… another team was in Alicante (Victor’s group) and they were the ones to finally expand ferret progenitors that are virtually absent in mouse to get new folds and gyri (Nonaka et al. 2013) (Fig. 1G).

The next problem was to get the story published. Editors kept saying that this was not novel because “viruses that increase NSC activity were already shown to increase brain size and cognitive function in apes”. That was unfortunately true as reported by Wyatt et al. (The Rise of the Planet of the Apes. Century Fox’s, 2011). So we had to reinvent our story by claiming the importance of extending this to other species and criticizing Wyatt’s report for the admittedly poor description of material and methods. Importantly, and in contrast to Wyatt et al., our mice never showed any ambition to conquer the world, while ferrets did, suggesting that gyrification causes megalomaniac world-domination ambitions as fully supported in the human species.

 

Figure 1 (A) progenitors undergoing different division have different G1 length. (B-D) plasmids used to manipulate G1 length (B) by in utero electroporation (C) resulting in increased progenitors expansion and thicker germinal zones (D) (Lange et al., 2009). These experiments corroborated the cell cycle length hypothesis (Calegari and Huttner, 2003; Salomoni and Calegari, 2010; Lange and Calegari, 2010) (E). (F-G) Manipulation of G1 by 4D was used to increase cortical surface area in mouse (F) and gyrification in ferret (G; red lines). (G-H) 4D and control viral constructs (if you understand what are all those abbreviations you already deserve a PhD!) (G) delivered in the adult hippocampus (H). (I) This allowed us to switch neurogenesis On and Off. (J) We plan to use that to solve all mysteries in science (iPhone not depicted for copyright issues).

Development is interesting and fun but adult somatic stem cells are interesting, fun, and useful (so everybody keep saying). Therefore it was obvious to investigate if 4D could be effective in the adult brain. To investigate this, Benedetta designed 4D-lentiviral vectors by which the cell cycle regulators were overexpressed for any desired period of time followed by their Cre-mediated inactivation (Fig. 1G). Pretty much similar to developing embryos, lentiviral injection in the adult hippocampus (Fig. 1H) inhibited neurogenesis while triggering NSC expansion. Importantly, following tamoxifen-dependent recombination of the viral cassette, expanded NSC could resume neurogenesis resulting in a doubling of the number of neurons (Artegiani et al., 2011) (Fig. 1I). Being the first to achieve acute, tissue-specific, and temporally-controlled expansion of somatic stem cells in adult mammals made us very proud and gave us the opportunity to investigate the effects of increased neurogenesis in cognitive function, aging, neurodegenerative disease and understanding why people favor iPhones (Fig. 1J).

Our tools

We have a lot of fun optimizing and developing new tools and techniques. You might be surprised to know that from time to time one might also work. These are some of those we have contributed in the past (ommitted are those that “did not work” as the students keep telling me without further detail).

•    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 in the brain followed by electroporation. We perform this technique in whole-embryo culture or in utero (among others: Calegari et al., 2002; Lange et al., 2009).
•    Viral-injection systems to achieve tissue-specific and temporaly-controlled manipulation of multiple genes in the adult (Artegiani et al., 2011) and developing (Artegiani and Calegari, 2013) brain.
•    Methods to control gene expression in whole organisms by UV light (Cambridge et al., 2009) and monitor miRNA activitly live (DePietri et al., 2006).
•    Any sort of transgenic mouse, can’t even remember them all I only know that the bill from the animal house is horrendous.

We also have a movie for a couple of those techniques (PG-17: RequiresAccompanying Parent or Adult Guardian) at www.jove.com/video/4093/expansion-embryonic-adult-neural-stem-cells-utero-electroporation-or

 

Future prospects and goals

If you were able to read (and understand) thus far, you must also be able to have a fair idea of our future ambitions. Figure 1J depicts them all; except for the iPhones since mice very much favor Blackberries.

 

Group Members

List of group members

 

Alumni

•    Christian Lange (mixed hairstyle between Kerry King and Bon Jovi but apparently he uses the Gilson better than the bass)

•    Miki Nonaka (mother to the Miki mouse. She escaped back to Japan 24 hours after defending her thesis; I assume she never appreciated the fish in Germany)

•    Silvia Prenninger (very “proliferative” student: 2 kids while getting 1 PhD even without any G1 manipulation from our side)

Lab nationalities: We like to collect flags.

 

Selected Publications

1) Nonaka-Kinoshita M, Reillo I, Artegiani B, Martinez M, Nelson M, Borrell V and Calegari F (2013) Regulation of cerebral cortex size and folding by expansion of basal progenitors. EMBO J, DOI:10.1038/emboj.2013.96

2) Artegiani B and Calegari F (2013) Lentiviruses allow widespread and conditional manipulation of gene expression in the developing mouse brain. Development, DOI: 10.1242/dev.093823

3) 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.

4) 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.

5) 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.

6) 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

7) 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.

8) 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.

9) 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.

10) 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. PNAS 99:14236-14239.

Complete list of Publications

Last modified: 17/04/2014