Long-term labeling of hippocampal neural stem cells by a lentiviral vector

Using a lentivirus-mediated labeling method, we investigated whether the adult hippocampus retains long-lasting, self-renewing neural stem cells (NSCs). We first showed that a single injection of a lentiviral vector expressing a green fluorescent protein (LV PGK-GFP) into the subgranular zone (SGZ) of the adult hippocampus enabled an efficient, robust, and long-term marking of self-renewing NSCs and their progeny. Interestingly, a subset of labeled cells showed the ability to proliferate multiple times and give rise to Sox2+ cells, clearly suggesting the ability of NSCs to self-renew for an extensive period of time (up to 6 months). In addition, using GFP+ cells isolated from the SGZ of mice that received a LV PGK-GFP injection 3 months earlier, we demonstrated that some GFP+ cells displayed the essential properties of NSCs, such as self-renewal and multipotency. Furthermore, we investigated the plasticity of NSCs in a perforant path transection, which has been shown to induce astrocyte formation in the molecular layer of the hippocampus. Our lentivirus (LV)-mediated labeling study revealed that hippocampal NSCs are not responsible for the burst of astrocyte formation, suggesting that signals released from the injured perforant path did not influence NSC fate determination. Therefore, our studies showed that a gene delivery system using LVs is a unique method to be used for understanding the complex nature of NSCs and may have translational impact in gene therapy by efficiently targeting NSCs

Long-Term Labeling of Hippocampal Neural Stem Cells by a Lentiviral Vector

Using a lentivirus-mediated labeling method, we investigated whether the adult hippocampus retains long-lasting, self-renewing neural stem cells (NSCs). We first showed that a single injection of a lentiviral vector expressing a green fluorescent protein (LV PGK-GFP) into the subgranular zone (SGZ) of the adult hippocampus enabled an efficient, robust, and long-term marking of self-renewing NSCs and their progeny. Interestingly, a subset of labeled cells showed the ability to proliferate multiple times and give rise to Sox2+ cells, clearly suggesting the ability of NSCs to self-renew for an extensive period of time (up to 6 months). In addition, using GFP+ cells isolated from the SGZ of mice that received a LV PGK-GFP injection 3 months earlier, we demonstrated that some GFP+ cells displayed the essential properties of NSCs, such as self-renewal and multipotency. Furthermore, we investigated the plasticity of NSCs in a perforant path transection, which has been shown to induce astrocyte formation in the molecular layer of the hippocampus. Our lentivirus (LV)-mediated labeling study revealed that hippocampal NSCs are not responsible for the burst of astrocyte formation, suggesting that signals released from the injured perforant path did not influence NSC fate determination. Therefore, our studies showed that a gene delivery system using LVs is a unique method to be used for understanding the complex nature of NSCs and may have translational impact in gene therapy by efficiently targeting NSCs

Homeobox gene <italic>Pitx2</italic> controls pituitary development and cell specification.

The pituitary gland is a neuroendocrine organ that produces and releases peptide hormones from specialized cells in the anterior lobe. Pituitary development is controlled by the combined activity of extrinsic signaling molecules and intrinsic transcription factors. Pitx2 encodes a homeobox transcription factor that is expressed in embryonic and adult pituitary gland. Individuals heterozygous for a mutant allele of PITX2 have Rieger syndrome (RGS), a disorder that is characterized by defects in eyes, teeth and umbilicus, and occasionally heart and pituitary. Most PITX2 lesions cause loss of function suggesting that Pitx2 gene dosage is critical for normal development. This hypothesis was tested by manipulating the activity of Pitx2 genes in mice. The targeted inactivation of Pitx2 in mice revealed its essential role in development of the eyes, heart, lungs, pituitary and ventral body wall. Pitx2 is required for the expansion of Rathke's pouch and survival of the precursors of terminally differentiated hormone producing cells. The size of the pituitary gland decreases as Pitx2 expression is reduced across an allelic series. Increased apoptosis is responsible for the extreme hypopituitarism of Pitx2 -/- mice. In addition, fewer pituitary cell types undergo specification in animals with reduced Pitx2 dosage. Mechanistically this is due to the failure of several lineage transcription factors, including Gata2, Sf1, Egr1 and Pit1 , to be expressed at normal levels. Elevated levels of Pitx2 also interfere with normal pituitary gland development in transgenic mice. More luteinizing hormone producing gonadotropes are formed in response to increased Pitx2 activity, revealing a direct correlation between Pitx2 gene dosage and gonadotrope specification. Thus, pituitary gland development is dependent upon a precise level of Pitx2. These studies provided a mechanistic understanding of Pitx2 gene dosage effects on pituitary development and explain the basis for RGS phenotype in patients with gain and loss of function mutations in PITX2. The model I developed for proliferation and cell death may be applicable to understanding the hypopituitarism characteristic of several other pituitary transcription factor mutants.Ph.D.Biological SciencesNeurosciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/132939/2/3058053.pd

The bicoid -related Pitx gene family in development

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42115/1/335-10-2-197_10n2p197.pd

BACE1 Deficiency Causes Abnormal Neuronal Clustering in the Dentate Gyrus

BACE1 is validated as Alzheimer's β-secretase and a therapeutic target for Alzheimer's disease. In examining BACE1-null mice, we discovered that BACE1 deficiency develops abnormal clusters of immature neurons, forming doublecortin-positive neuroblasts, in the developing dentate gyrus, mainly in the subpial zone (SPZ). Such clusters were rarely observed in wild-type SPZ and not reported in other mouse models. To understand their origins and fates, we examined how neuroblasts in BACE1-null SPZ mature and migrate during early postnatal development. We show that such neuroblasts are destined to form Prox1-positive granule cells in the dentate granule cell layer, and mainly mature to form excitatory neurons, but not inhibitory neurons. Mechanistically, higher levels of reelin potentially contribute to abnormal neurogenesis and timely migration in BACE1-null SPZ. Altogether, we demonstrate that BACE1 is a critical regulator in forming the dentate granule cell layer through timely maturation and migration of SPZ neuroblasts

PITX genes are required for cell survival and Lhx3 activation.

The PITX family of transcription factors regulate the development of many organs. Pitx1 mutants have a mild pituitary phenotype, but Pitx2 is necessary for the development of Rathke's pouch, expression of essential transcription factors in gonadotropes, and expansion of the Pit1 lineage. We report that lack of Pitx2 causes the pouch to undergo excessive cell death, resulting in severe pituitary hypoplasia. Transgenic overexpression of PITX2 in the pituitary can increase the gonadotrope population, suggesting that the absolute concentration of PITX2 is important for normal pituitary cell lineage expansion. We show that PITX1 and PITX2 proteins are present in similar expression patterns throughout pituitary development and in the mature pituitary. Both transcription factors are preferentially expressed in adult gonadotropes and thyrotropes, suggesting the possibility of overlap in maintenance of adult pituitary functions within these cell types. Double knockouts of Pitx1 and Pitx2 exhibit severe pituitary hypoplasia and fail to express the transcription factor LHX3. This indicates that these PITX genes are upstream of Lhx3 and have compensatory roles during development. Thus, the combined dosage of these PITX family members is vital for pituitary development, and their persistent coexpression in the adult pituitary suggests a continued role in maintenance of pituitary function