Hoonkyo Suh

In Vivo Fate Analysis Reveals the Multipotent and Self-Renewal Capacities of Sox2+ Neural Stem Cells in the Adult Hippocampus

To characterize the properties of adult neural stem cells (NSCs), we generated and analyzed Sox2-GFP transgenic mice. Sox2-GFP cells in the subgranular zone (SGZ) express markers specific for progenitors, but they represent two morphologically distinct populations that differ in proliferation levels. Lentivirus- and retrovirus-mediated fate-tracing studies showed that Sox2+ cells in the SGZ have potential to give rise to neurons and astrocytes, revealing their multipotency at the population as well as at a single-cell level. A subpopulation of Sox2+ cells gives rise to cells that retain Sox2, highlighting Sox2+ cells as a primary source for adult NSCs. In response to mitotic signals, increased proliferation of Sox2+ cells is coupled with the generation of Sox2+ NSCs as well as neuronal precursors. An asymmetric contribution of Sox2+ NSCs may play an important role in maintaining the constant size of the NSC pool and producing newly born neurons during adult neurogenesis.

Signaling in Adult Neurogenesis

The identification of neural stem cells (NSCs) and their contribution to continuous neurogenesis has shown that the hippocampus and olfactory bulb are plastic. Brain plasticity, achieved at the level of cell genesis, has an essential role in the maintenance of brain homeostasis. Via combinatorial functions of extrinsic signals and intrinsic programs, adult neurogenesis is tightly regulated in a specialized microenvironment, a niche. Misregulated neurogenesis is detrimental to normal brain functions and, in extreme cases, pathogenic. Hence, understanding signaling in adult neurogenesis is not only important to understand the physiological roles of neurogenesis, but also to provide knowledge that is essential for developing therapeutic applications using NSCs to intervene in the progression of brain diseases.

BRCA1 tumour suppression occurs via heterochromatin-mediated silencing

Mutations in the tumour suppressor gene BRCA1 lead to breast and/or ovarian cancer. Here we show that loss of Brca1 in mice results in transcriptional de-repression of the tandemly repeated satellite DNA. Brca1 deficiency is accompanied by a reduction of condensed DNA regions in the genome and loss of ubiquitylation of histone H2A at satellite repeats. BRCA1 binds to satellite DNA regions and ubiquitylates H2A in vivo. Ectopic expression of H2A fused to ubiquitin reverses the effects of BRCA1 loss, indicating that BRCA1 maintains heterochromatin structure via ubiquitylation of histone H2A. Satellite DNA de-repression was also observed in mouse and human BRCA1-deficient breast cancers. Ectopic expression of satellite DNA can phenocopy BRCA1 loss in centrosome amplification, cell-cycle checkpoint defects, DNA damage and genomic instability. We propose that the role of BRCA1 in maintaining global heterochromatin integrity accounts for many of its tumour suppressor functions.

The bicoid -related Pitx gene family in development

The important roles of homeobox genes in development of the hindbrain and axial body are well established. More recently, it has become clear that certain subfamilies of homeobox genes play particularly important roles in the development of more anterior structures. These have included the paired gene family in the eye (Gehring, 1996; Hanson and Van Heyningen, 1995; Macdonald and Wilson, 1996; Wehr and Gruss, 1996), the orthodenticle and distalless gene families in the foreand midbrains (Acampora et al., 1996; Acampora et al., 1995; Price et al., 1991; Simeone et al., 1994; Williams, 1998), and the Lhx gene family in the pituitary gland (Sheng et al., 1997; Sheng et al., 1996). This review summarizes the newly identified Pitx gene family and its role in development. This family includes three vertebrate paralogues that have been cloned in multiple organisms, and a fly cognate. Mutations in two members of this gene family lead to human disease or birth defects affecting anterior structures. The nomenclature for this gene family has been complicated by the fact that members have been cloned and uniquely named by more than one laboratory (Table 1). The first member of this family, mouse Ptx1 (pituitary homeobox 1) was isolated as a transcription factor involved in pro-opiomelanocortin gene transcription in anterior pituitary corticotropes (Lamonerie et al., 1996). However, since some pentaxin genes in mouse and human had previously been assigned the Ptx gene symbol, the gene symbols for the three mouse paralogues for this new homeobox gene family are Pitx1, Pitx2, and Pitx3 (Mouse Genome Database). In this review, we have adopted the official nomenclature of the MGD and propose that, for clarity, this nomenclature be adopted for other organisms. Three vertebrate paralogues, Pitx1, Pitx2, and Pitx3, have all been cloned from mouse and human (Table 1 and references therein). Some paralogues have also been cloned from chicken (Pitx1 and Pitx2), xenopus and zebrafish (Pitx2), and rat (Pitx3) (Table 1 and references therein). In two reports, mouse Pitx1 was cloned in functional assays: in a two-hybrid screen using Pit-1 as bait (Szeto et al., 1996) and as noted above. Human PITX2 was identified by positional cloning of the Rieger Syndrome gene (Semina et al., 1996). In the other reports, cloning was the result of using degenerate PCR or low stringency hybridization to detect expressed homeobox sequences in a variety of embryonic and adult tissues. The difficulty in cloning Pitx1 from xenopus and zebrafish has suggested that this orthologue may not be as widely distributed in nature as Pitx2 (Kitamura et al., 1997). However, the recent identification of a fly Pitx gene during a chromosome walk demonstrates that this gene family arose prior to the divergence of vertebrates and invertebrates (Vorbruggen et al., 1997). Each vertebrate paralogue has been mapped genetically in mouse and human (Table 1). The Pitx proteins all belong to the bicoid-related subclass of homeodomain proteins because they encode the defining lysine at residue 50 within the homeodomain. This residue, at residue 9 within the recognition helix of the homeodomain, is the major determinant of DNA binding specificity (Gehring et al., 1994; Hanes and Brent, 1989). Several members of this small subfamily are essential for axis and pattern formation (Ang et al., 1996). Pitx2 expresses multiple protein isoforms as a result of alternative splicing (Gage and Camper, 1997; Kitamura et al., 1997) and the use of different promoters (P. Gage and E. Semina, unpublished results) (Fig. 1). The three vertebrate paralogues are all highly conserved at the amino acid level (Fig. 1). For example, in mouse the Pitx2 and Pitx3 homeodomains are identical while Pitx1 differs by only two amino acids. The paralogues are also conserved Cterminal to the homeodomain (55–70%). In contrast, the N-termini of these proteins are essentially unrelated. The vertebrate orthologues are even more highly conserved. For example, there the mouse and chicken Pitx2a proteins are 96% identical with only ten amino acid substitutions between them. The Drosophila Pitx protein shows high conservation to the vertebrate proteins within the homeodomain (90–93%) and a short region near C-terminus that has been termed the OAR sequence (Furukawa et al., 1997) or the C-peptide (Kitamura et al., 1997). This sequence is present in several homeobox genes. In Pitx2, this domain appears to function as an intrinsic inhibitor of DNA binding activity whose function can be modulated by protein-protein interactions (Amendt et al., 1998). The vertebrate Pitx genes each have unique developmental and tissue-specific expression patterns (Fig. 2 and Table 2). However, there are several significant overlaps in expression pattern (Fig. 2). The most significant may be in the eye, where both Pitx2 and Pitx3 are expressed in the mesenchyme and its derivatives (Semina et al., 1998; Semina et al., 1996; Smidt et al., 1997). Demonstration in humans that mutations to Pitx2 result in Rieger’s Syndrome (Semina et al., 1996) and mutations to Pitx3 result in anterior segment mesenchymal dysgenesis and dominant cataracts (Semina et al., 1998) confirmed the importance of these genes in eye development. These autosomal-dominant conditions each affect the development or maintenance of anterior structures of the eye. Interestingly, mouse Pitx3 maps near aphakia, a recessive mutation resulting in small eyes that lack lenses and fail to develop beyond 11 days of gestation (Semina et al., 1997). Rieger’s Syndrome patients frequently show defects in dental and umbilical development in addition to their ocular defects (Feingold et al., 1969; Rieger, 1935), and subsets of patients also present with isolated growth insufficiency (Feingold et al., 1969). Several observations suggest that Pitx genes are also important for the development and function of other organs. The stomodeum is an ectoderm-derived layer of epithelium that derives from the anterior neural ridge and forms the earliest mouth structures (Couly and Le Douarin, 1985). Pitx1 expression defines the stomodeum and continues within stomodial derivatives, including the nasal pit and Rathke’s pouch (Lanctot et al., 1997). Pitx1 is also expressed more caudally in the posterior lateral plate and extraCorrespondence to: P.J. Gage Mammalian Genome 10, 197–200 (1999).

Monosynaptic inputs to new neurons in the dentate gyrus

Adult hippocampal neurogenesis is considered important for cognition. The integration of newborn dentate gyrus granule cells into the existing network is regulated by afferent neuronal activity of unspecified origin. Here we combine rabies virus-mediated retrograde tracing with retroviral labelling of new granule cells (21, 30, 60, 90 days after injection) to selectively identify and quantify their monosynaptic inputs in vivo. Our results show that newborn granule cells receive afferents from intra-hippocampal cells (interneurons, mossy cells, area CA3 and transiently, mature granule cells) and septal cholinergic cells. Input from distal cortex (perirhinal (PRH) and lateral entorhinal cortex (LEC)) is sparse 21 days after injection and increases over time. Patch-clamp recordings support innervation by the LEC rather than from the medial entorhinal cortex. Mice with excitotoxic PRH/LEC lesions exhibit deficits in pattern separation but not in water maze learning. Thus, PRH/LEC input is an important functional component of new dentate gyrus neuron circuitry.

Gene Expression Profiling of Neural Stem Cells and Their Neuronal Progeny Reveals IGF2 as a Regulator of Adult Hippocampal Neurogenesis

Neural stem cells (NSCs) generate neurons throughout life in the hippocampal dentate gyrus (DG). How gene expression signatures differ among NSCs and immature neurons remains largely unknown. We isolated NSCs and their progeny in the adult DG using transgenic mice expressing a GFP reporter under the control of the Sox2 promoter (labeling NSCs) and transgenic mice expressing a DsRed reporter under the control of the doublecortin (DCX) promoter (labeling immature neurons). Transcriptome analyses revealed distinct gene expression profiles between NSCs and immature neurons. Among the genes that were expressed at significantly higher levels in DG NSCs than in immature neurons was the growth factor insulin-like growth factor 2 (IGF2). We show that IGF2 selectively controls proliferation of DG NSCs in vitro and in vivo through AKT-dependent signaling. Thus, by gene expression profiling of NSCs and their progeny, we have identified IGF2 as a novel regulator of adult neurogenesis.

SRY-box-containing Gene 2 Regulation of Nuclear Receptor Tailless (Tlx) Transcription in Adult Neural Stem Cells

Background: Sox2 and TLX are essential for the self-renewal of adult neural stem cells (NSCs). Results: Sox2 positively regulates TLX expression and antagonizes the negative feedback system of TLX by a physical interaction between Sox2 and TLX. Conclusion: Sox2 and TLX form a molecular network regulating adult NSCs. Significance: This molecular network is a target to discover new ways regulate endogenous neurogenesis. Adult neurogenesis is maintained by self-renewable neural stem cells (NSCs). Their activity is regulated by multiple signaling pathways and key transcription factors. However, it has been unclear whether these factors interplay with each other at the molecular level. Here we show that SRY-box-containing gene 2 (Sox2) and nuclear receptor tailless (TLX) form a molecular network in adult NSCs. We observed that both Sox2 and TLX proteins bind to the upstream region of Tlx gene. Sox2 positively regulates Tlx expression, whereas the binding of TLX to its own promoter suppresses its transcriptional activity in luciferase reporter assays. Such TLX-mediated suppression can be antagonized by overexpressing wild-type Sox2 but not a mutant lacking the transcriptional activation domain. Furthermore, through regions involved in DNA-binding activity, Sox2 and TLX physically interact to form a complex on DNAs that contain a consensus binding site for TLX. Finally, depletion of Sox2 revealed the potential negative feedback loop of TLX expression that is antagonized by Sox2 in adult NSCs. These data suggest that Sox2 plays an important role in Tlx transcription in cultured adult NSCs.

Role of BRCA1 in brain development

Significance The developing brain is highly sensitive to ionizing radiation and DNA damage. Here we report that tumor suppressor breast cancer susceptibility gene 1 (BRCA1) plays a novel role in regulating the embryonic brain development and postnatal brain size. We found that loss of BRCA1 induces p53-dependent proapoptotic pathways in the CNS. BRCA1 possibly functions as a centrosomal factor in establishing the cellular polarity of the neural progenitors through the DNA damage sensor kinase ATM. Our data provide new insight in understanding the control of DNA damage sensitivity and brain size during development and evolution. Breast cancer susceptibility gene 1 (BRCA1) is a breast and ovarian cancer tumor suppressor whose loss leads to DNA damage and defective centrosome functions. Despite its tumor suppression functions, BRCA1 is most highly expressed in the embryonic neuroepithelium when the neural progenitors are highly proliferative. To determine its functional significance, we deleted BRCA1 in the developing brain using a neural progenitor–specific driver. The phenotype is characterized by severe agenesis of multiple laminated cerebral structures affecting most notably the neocortex, hippocampus, cerebellum, and olfactory bulbs. Major phenotypes are caused by excess apoptosis, as these could be significantly suppressed by the concomitant deletion of p53. Certain phenotypes attributable to centrosomal and cell polarity functions could not be rescued by p53 deletion. A double KO with the DNA damage sensor kinase ATM was able to rescue BRCA1 loss to a greater extent than p53. Our results suggest distinct apoptotic and centrosomal functions of BRCA1 in neural progenitors, with important implications to understand the sensitivity of the embryonic brain to DNA damage, as well as the developmental regulation of brain size.

20 – Pituitary Gland Development

Pituitary gland is a small endocrine gland located at the base of the brain under the optic chiasm and the arachnoid membrane, in a bony indentation called the sella turcica. Its name comes from the Greek hypophysis, which means undergrowth. The role of the pituitary gland is the regulated synthesis and secretion of polypeptide hormones that are essential for the development and function of many other organs in the body. Releasing hormones and inhibiting factors reach the anterior pituitary via hypothalamic neurons that terminate in the capillary beds of the median eminence, just dorsal to the pituitary gland. These capillary beds are connected to the hypophyseal portal vessels that nourish the anterior pituitary. In response to these stimulatory stimulatory factors, pituitary hormones are released into hypophyseal portal blood vessels and carried through the blood stream to their target organs. An intriguing feature of pituitary development is the initial organization of the cell types into discrete patches and the loss of this spatial organization as the organ expands. The rodent pituitary is composed of three lobes. The posterior pituitary (neurohypophysis or pars nervosa) is derived from neural ectoderm and contains the nerve terminals that secrete oxytocin and vasopressin. The intermediate lobe (pars intermedia) and anterior lobe are both derived from oral ectoderm. The bulk of the anterior lobe (pars distaIis) is at the same level as the intermediate lobe, but a portion of it known as the pars tuberalis extends dorsally along the pituitary stalk.

Notch keeps ependymal cells in line.

The ependymal cells lining the lateral ventricles are not stem cells, but a study now shows that they can be activated to generate neuroblasts in a stroke model, and mature olfactory bulb neurons when Notch signaling is disrupted.