Neurogenesis in Tα-1 tubulin transgenic mice during development and after injury

doi:10.1016/j.expneurol.2005.10.030

Experimental Neurology

Volume 197, Issue 2, February 2006, Pages 475-485

https://doi.org/10.1016/j.expneurol.2005.10.030 Get rights and content

Abstract

Tα-1 tubulin promoter-driven EYFP expression is seen in murine neurons born as early as E9.5. Double labeling with markers for stem cells (Sox 1, Sox 2, nestin), glial progenitors (S100β, NG2, Olig2), and neuronal progenitors (doublecortin, βIII-tubulin, PSA-NCAM) show that Tα-1 tubulin expression is limited to early born neurons. BrdU uptake and double labeling with neuronal progenitor markers in vivo and in vitro show that EYFP-expressing cells are postmitotic and Tα-1 tubulin EYFP precedes the expression of MAP-2 and NeuN, and follows the expression of PSA-NCAM, doublecortin (Dcx), and βIII-tubulin.

Tα-1 tubulin promoter-driven EYFP expression is transient and disappears in most neurons by P0. Persistent EYFP expression is mainly limited to scattered cells in the subventricular zone (SVZ), rostral migratory stream, and hippocampus. However, there are some areas that continue to express Tα-1 tubulin in the adult without apparent neurogenesis. The number of EYFP-expressing cells declines with age indicating that Tα-1 tubulin accurately identifies early born postmitotic neurons throughout development but less clearly in the adult.

Assessment of neurogenesis after stab wound injuries in the cortex, cerebellum and spinal cord of adult animals shows no neurogenesis in most areas with an increase in BrdU incorporation in glial and other non neuronal populations. An up-regulation of Tα-1 tubulin can be seen in certain areas unaccompanied by new neurogenesis. Our results suggest that even if stem cells proliferate their ability to generate neurons is limited and caution is warranted in attributing increased BrdU incorporation to stem cells or cells fated to be neurons even in neurogenic areas.

Introduction

The process of neurogenesis in the developing embryo follows a characteristic pattern. Cells within the neural plate undergo a series of morphogenetic movements to form a neural tube that undergoes dorsoventral and rostrocaudal patterning to generate specific regions of the brain. The initially formed neural tube is comprised of a homogenous population of dividing neuroepithelial cells that are the precursors of all differentiated cells in the nervous system. Neuroepithelial cells generate neurons via a process of differentiation that involves migration away from the zone of genesis and the sequential acquisition of markers of neuronal maturation with a concomitant exit from cell cycle. The process of neural differentiation has been recapitulated in vitro using neural stem cells and progenitor cells, while in vivo differentiation has been monitored by transplantation into fetal or adult brain (Lim et al., 1997, Wang et al., 1998, Yang et al., 2000, Vitry et al., 2001).

In general, multipotent stem cells do not express neuronal markers, but acquire them as soon as they begin to migrate. Migrating neuroblasts express doublecortin (Dcx), lissencephaly gene product, and the poly-sialyated form of neural cell adhesion molecule (PSA-NCAM). Migrating neuroblasts retain the capacity to divide, which has been shown in vitro with cells harvested from the ventricular zone (Luskin et al., 1997, Coskun et al., 2001, Soares and Sotelo, 2004), spinal cord (Kalyani et al., 1997, Kalyani et al., 1998, Rao et al., 1998, Mujtaba et al., 1999), cortex (Chang et al., 2004), striatum (unpublished results), as well as with cerebellar neuroblasts (Hatten, 1999). While it is clear that neuroblasts are lineally related to and arise from multipotent stem cells (Rao et al., 1998, Mayer-Proschel et al., 1997), it is unclear how this process is regulated and whether it persists in adulthood.

Tα-1 tubulin is expressed as cells begin to differentiate along the neuronal lineage. Antibody staining, promoter reporter constructs, as well as transgenic mice where the promoter drives the expression of β-gal or EYFP/EYFP have confirmed that Tα-1 tubulin is one of the earliest neuronal markers. The expression of Tα-1 tubulin is downregulated soon after maturation (Miller et al., 1987). However, it is detectable in sympathetic neurons of certain brain regions such as the olfactory bulb, hippocampus, hypothalamus, and thalamus during adult life and it is up-regulated in response to brain or axonal injuries (Pearson et al., 1988, Wu et al., 1993, Gloster et al., 1994, Paden et al., 1995, Wu et al., 1997, Kobayashi et al., 1997, Fournier and McKerracher, 1997, Fournier et al., 1997, Sawamoto et al., 2001). The transient and neuronal specific expression of Tα-1 tubulin may allow one to monitor neurogenesis in mice.

The vast majority of studies investigating adult neurogenesis report that ongoing neurogenesis is usually limited to the SVZ and subgranular zone of the hippocampal dentate gyrus with occasional reports of additional zones of neurogenesis in regions such as the thalamus, hypothalamus, and amygdala (Fowler et al., 2003, Gould et al., 1999, Pencea et al., 2001). Newborn neurons have also been observed in various injury models (Arvidsson et al., 2002, Magavi et al., 2000), although the origin of the neurons and the exact processes by which these newborn neurons differentiate are not yet known. These observations have raised the possibility that a well functioning endogenous neuronal repair mechanism may exist and migration from the SVZ to persistence of cortical stem cells and transdifferentiation (Emsley et al., 2004) have all been proposed as possible mechanisms. Therefore, a clear-cut marker of neurogenesis would permit the resolution of such controversies.

To assess neurogenesis during development and following injury in the adult brain, we have utilized the Tα-1 tubulin/EYFP mice where the Tα-1 tubulin promoter drives the expression of EYFP. We show that Tα-1 tubulin is an early post-mitotic neuronal marker and can be used to study neurogenesis during development and after brain injury. We find that in adult mice neurogenesis after brain injury is rather limited and restricted to known areas of neurogenesis.

Section snippets

NEP cell culture

Tα-1 tubulin-EYFP embryos were removed from timed-pregnant mice at 9 gestation days (E9) as described (Wu et al., 1993, Wu et al., 2002). The trunk segments of the E9 embryos were dissected and incubated in an enzyme solution containing Collagenase type I (1 mg/ml; Worthington Biochemical) and Dispase II (2 mg/ml; Roche) in Hank’s balanced salt solution (Gibco/BRL), at room temperature for approximately 10 min. The enzyme solution was then replaced by NEP basal medium (Kalyani et al., 1997)

EYFP/Tα-1 tubulin expression in fetal tissue

To assess the expression pattern of Tα-1 tubulin-driven EYFP expression during the early development of the nervous system, we obtained embryos at different gestational ages and processed them for histochemistry. The earliest expression of EYFP was observed in the E9.5 brain (Fig. 1A) and caudal neural tube, where isolated neurons could be identified along the entire rostrocaudal axis. Neurons appeared mature and were present outside the known proliferation zones. The number of Tα-1 tubulin

Discussion

Our results show that Tα-1 promoter-driven expression of EYFP can serve as a reliable method of detecting the birth of new neurons throughout development and in in vitro culture assays. Especially after injury Tα-1 EYFP should be used with caution as a sole marker for adult neurogenesis. Examining the expression pattern in the adult Tα-1 EYFP mice and their injury response, we find that neurogenesis is limited to known neurogenic regions and is absent in most brain regions examined.

The observed

Acknowledgments

This work was supported by NIH, NIA and the CNS foundation. We thank all members of our laboratories for constant stimulating discussions. We acknowledge the substantial improvement of our manuscript through suggestions of reviewer 2. MSR acknowledges the contributions of Dr. S. Rao that made undertaking this project possible.

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Regular ArticleNeurogenesis in Tα-1 tubulin transgenic mice during development and after injury

Abstract

Introduction

Section snippets

NEP cell culture

EYFP/Tα-1 tubulin expression in fetal tissue

Discussion

Acknowledgments

Dev. Biol.

Brain Res. Dev. Brain Res.

Int. J. Dev. Neurosci.

NeuroRx.

Neuron

Cell

Dev. Biol.

Brain Res.

Mol. Cell. Neurosci.

Dev. Biol.

Neuron

Dev. Biol.

Cell

Neurosci. Lett.

Brain Res.

Neuroscience

Mol. Cell. Neurosci.

Dev. Biol.

Neuronal replacement from endogenous precursors in the adult brain after stroke

Nat. Med.

Comparison of the expression of a T alpha 1:nlacZ transgene and T alpha 1 alpha-tubulin mRNA in the mature central nervous system

J. Comp. Neurol.

Target contact regulates GAP-43 and alpha-tubulin mRNA levels in regenerating retinal ganglion cells

J. Neurosci. Res.

Sox2 expression defines a heterogeneous population of neurosphere-forming cells in the adult murine brain

Aging Cell

Adult mammalian forebrain ependymal and subependymal cells demonstrate proliferative potential, but only subependymal cells have neural stem cell characteristics

J. Neurosci.

Doublecortin expression levels in adult brain reflect neurogenesis

Eur. J. Neurosci.

Cellular composition and three-dimensional organization of the subventricular germinal zone in the adult mammalian brain

J. Neurosci.

Expression of specific tubulin isotypes increases during regeneration of injured CNS neurons, but not after the application of brain-derived neurotrophic factor (BDNF)

J. Neurosci.

Regular Article
Neurogenesis in Tα-1 tubulin transgenic mice during development and after injury