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 any other somatic stem cell, can divide to generate either two identical stem cells (proliferative division) or more differentiated cells, such us neurons (differentiative division). We found that the length of the G1 phase of the cell cycle acts as a crucial switch determining whether a NSC will undergo a proliferative vs. differentiative division (Fig. 1A). In short, stem cells need Time in order to differentiate and this is provided during G1. The cool part is that we can change G1 length as we want… well, sort of.

Neurogenesis in brain development, evolution, adulthood, aging, disease and protecting Earth from Aliens’ invasion

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 the lengthening of G1 is a cause, rather than a consequence, of differentiation (Calegari and Huttner, 2003). Thus, all we need to do to expand NSC is to force them to cycle fast & furious. Christian did exactly that by overexpressing Cdk4/cyclin D1 (4D) (Fig. 1B and C) and, luckily for his PhD and my contract, that worked quite well leading to inhibition of neurogenesis and progenitor expansion (Fig. 1D) (Lange et al., 2009). As such, a short G1 increases the number of NSC in the developing brain (Salomoni and Calegari, 2010) and since we scientists like to over speculate beyond our own expertise we concluded that this must work in any other stem cell system ever to have existed in the Universe (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 mice with folded brains like the human brain??? That was a project we could not miss, the only problem was to find a student crazy enough to buy all that crap. We achieved the latter by recruiting Miki and making her an offer she could not refuse. The rest was downhill. Miki generated a dual-transgenic, tissue-specific, inducible and reversible 4D line in no time, which resulted in a brainy mouse that, needless to say, was called the Miki Mouse (Nonaka et al. 2013). Yet, as big as these brains were, they remained boringly smooth and we couldn’t get any gyrus out of them (Fig. 1F). So how can gyri have emerged during evolution? Alternatively, is the Miki Mouse the final prove of Intelligent Design? 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 absent in mouse to get new folds and gyri (Nonaka et al. 2013) (Fig. 1G).

That for the easy part. The real problem was to find editors, and creationists alike, able to understand that not all brains are created equal and get the story published. We tried many Journals but they kept saying that our study was not novel since “viruses that increase NSC were already shown to increase brain size and cognitive function in primates”. 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 change our story by claiming the importance of extending this to other species and criticizing Wyatt’s report for the admittedly poor description of their methods… By the way, the scary part was that in contrast to our well-behaving Miki Mouse both Victor’s ferrets and Wyatt’s apes became very aggressive with the former attacking the students and the latter trying to conquer the world. In turn, this suggests that gyrification correlates with megalomaniac world-domination ambitions as fully supported by two extreme examples of hyper-aggressive, super-gyrified species: humans on this planet and Martians on our neighbor planet… I mean, look at those brains (Fig 1H), no wonder they tried to destroy us over again (reviewed by Burton et al., Mars Attacks, 1996). Victor and I could not allow those green midgets to invade us again so we published a paper describing the Achilles’ heel of the Martians’ brain and so we saved the World (Borrell and Calegari, 2014).

 

(A) Progenitors with different G1 length do different things. (B-D) We manipulate G1 and guess what? We get more progenitors (Lange et al., 2009). (E) So we get fame and glory (Salomoni and Calegari, 2010; Lange and Calegari, 2010). (F-G) The Miki mouse gets more cortical surface (F) while the Miki ferret gets more gyri (G; red lines). (H) Victor and I fight to the bitter end and repel a Mars attack. (I) Using ubb-GFPloxNLS-T2A-cdk4-T2A-ccnd1lox HIV infection in nestin-CreERT2 mice gave us more adult neurogenesis. (J) Manipulating the switch of neurogenesis will guide us toward solving all mysteries in science.

Development is interesting and fun but adult somatic stem cells are interesting, fun, and useful (so everybody keep saying here at the CRTD, so it must be true…right?). Therefore it was obvious to investigate if 4D could give us more NSC and then more neurons pretty much as it did in development. Benedetta tried that by inventing an overly complicated VSVG-pseudotyped, HIV-lentiviral system based on stereotaxic injection of tissue-specific, temporally-controlled, tamoxifen-dependent, ubb::GFPloxNLS-T2A-cdk4-T2A-ccnd1lox in C57BL/6J nestin::CreERT2 mice (whatever, if you could understand all that, including the abbreviations, you already deserved your PhD in my lab!!!). To cut it short, it worked (Artegiani et al., 2011) (Fig. 1I) giving us the opportunity to investigate the effects of increased neurogenesis in cognitive function, aging, neurodegenerative disease and understanding why humans like iPhones and mice blackberries (Fig. 1J).

Our tools

We have a lot of fun trying to develop new tools and techniques. You might be surprised to know that from time to time some also work. These are some of those we have contributed in the past (omitted are those that “did not work” as the students keep telling me without giving any detail).
•    a whole-embryo culture system to grow mouse embryos without their mothers (Calegari and Huttner., 2003).
•    a system to overexpress/knock-down genes in NSC by injecting plasmids and delivering electric shocks (among others: Calegari et al., 2002; Lange et al., 2009).
•    Viral-infections in all flavors and colors (Artegiani et al., 2011; Artegiani and Calegari, 2013).
•    Methods to control gene expression in whole organisms by UV light (Cambridge et al., 2009), monitor miRNA activitly live (DePietri et al., 2006) and check for miRNA targets (Ghosh et al., 2014).
•    Any sort of transgenic mouse, can’t even remember them all I only know that the bill from the animal house is horrendous. The latest one is a cool red and green mouse made by Silvia and used by Julieta to sequence all non-coding transcripts of NSC (Aprea et al., 2013).
We also made a movie for a couple of those techniques (PG-17: Requires Accompanying 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 thus far, and could understand more than the average editor does, then you must also be able to have a fair idea of our future ambitions. Figure 1J depicts them all; except for iPhones and blackberries for trademark issues.

 

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

Borrell V, Calegari F (2014) Mechanisms of brain evolution: regulation of neural progenitor cell diversity and cell cycle length. Neurosci Res. DOI: 10.1016/j.neures.2014.04.004

Aprea J, Prenninger S, Dori M, Ghosh T, Monasor LS, Wessendorf E, Zocher S, Massalini S, Alexopoulou D, Lesche M, Dahl A, Groszer M, Hiller M, Calegari F (2013) Transcriptome sequencing during mouse brain development identifies long non-coding RNAs functionally involved in neurogenic commitment. EMBO J. 32:3145-60.

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, 32(13):1817-28

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

Calegari F, Haubensak W, Haffner C and Huttner WB (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 WB (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 WB 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: 23/09/2014