Faculty Bibliography

Sonic hedgehog (Shh) has been implicated in the ongoing neurogenesis in postnatal rodent brains. Here we adopted an in vivo genetic fate-mapping strategy, using Gli1 (GLI-Kruppel family member) as a sensitive readout of Shh activity, to systematically mark and follow the fate of Shh-responding cells in the adult mouse forebrain. We show that initially, only a small population of cells (including both quiescent neural stem cells and transit-amplifying cells) responds to Shh in regions undergoing neurogenesis. This population subsequently expands markedly to continuously provide new neurons in the forebrain. Our study of the behaviour of quiescent neural stem cells provides in vivo evidence that they can self-renew for over a year and generate multiple cell types. Furthermore, we show that the neural stem cell niches in the subventricular zone and dentate gyrus are established sequentially and not until late embryonic stages

Tumor cell adaptation to hypoxic stress is an important determinant of malignant progression. While much emphasis has been placed on the role of HIF-1 in this context, the role of additional mechanisms has not been adequately explored. Here we demonstrate that cells cultured under hypoxic/anoxic conditions and transformed cells in hypoxic areas of tumors activate a translational control program known as the integrated stress response (ISR), which adapts cells to endoplasmic reticulum (ER) stress. Inactivation of ISR signaling by mutations in the ER kinase PERK and the translation initiation factor eIF2alpha or by a dominant-negative PERK impairs cell survival under extreme hypoxia. Tumors derived from these mutant cell lines are smaller and exhibit higher levels of apoptosis in hypoxic areas compared to tumors with an intact ISR. Moreover, expression of the ISR targets ATF4 and CHOP was noted in hypoxic areas of human tumor biopsy samples. Collectively, these findings demonstrate that activation of the ISR is required for tumor cell adaptation to hypoxia, and suggest that this pathway is an attractive target for antitumor modalities

Embryo implantation in the uterus is a critical step in mammalian reproduction, requiring preparation of the uterus receptive to blastocyst implantation. Uterine receptivity, also known as the window of implantation, lasts for a limited period, and it is during this period blastocysts normally implant. Ovarian steroid hormones estrogen and progesterone (P(4)) are the primary regulators of this process. The immunophilin FKBP52 serves as a cochaperone for steroid hormone nuclear receptors to govern appropriate hormone action in target tissues. Here we show a critical role for FKBP52 in mouse implantation. This immunophilin has unique spatiotemporal expression in the uterus during implantation, and females missing the Fkbp52 gene have complete implantation failure due to lack of attainment of uterine receptivity. The overlapping uterine expression of FKBP52 with nuclear progesterone receptor (PR) in wild-type mice together with reduced P(4) binding to PR, attenuated PR transcriptional activity and down-regulation of several P(4)-regulated genes in uteri of Fkbp52(-/-) mice, establishes this cochaperone as a critical regulator of uterine P(4) function. Interestingly, ovulation, another P(4)-mediated event, remains normal. Collectively, the present investigation provides evidence for an in vivo role for this cochaperone in regulating tissue-specific hormone action and its critical role in uterine receptivity for implantation.

Ras GTPases regulate cellular growth and differentiation and are modulated by myriad stimuli including growth factors, cytokines, antigens, and UV irradiation. Ras GTPases are molecular switches that are active when GTP-bound and inactive when GDP-bound. The ability of these GTPases to signal requires that the GTP-bound form engage downstream effectors, interactions that occur only on the cytosolic surface of cellular membranes. Ras family proteins include H-Ras, N-Ras, K-Ras, and Rap1. Insight into the regulation and signaling properties of these molecules has come largely from in vitro studies relying on cellular extracts prepared following cellular stimulation. Since Ras GTPases are expressed on multiple cellular compartments that include the plasma membrane, vesicles derived from the plasma membrane, and other internal membranes such as the ER and Golgi complex, analysis of how their spatial distribution modulates signaling has remained unknown. We have developed fluorescent, GFP-based probes capable of selectively binding GTP-bound Ras or Rap1 in living cells. We have used these reporters to examine sites of cellular activation of Ras and Rap1 during growth factor stimulation. These studies have revealed new insights into the platforms from which these GTPases signal and have led to the hypothesis that GTPase signaling is modulated in a compartmentalized fashion. Here, we describe the design and implementation of fluorescent probes for Ras and Rap1

Numerous lines of evidence suggest that Notch signaling plays a pivotal role in controlling the production of neurons from progenitor cells. However, most experiments have relied on gain-of-function approaches because perturbation of Notch signaling results in death prior to the onset of neurogenesis. Here, we examine the requirement for Notch signaling in the development of the striatum through the analysis of different single and compound Notch1 conditional and Notch3 null mutants. We find that normal development of the striatum depends on the presence of appropriate Notch signals in progenitors during a critical window of embryonic development. Early removal of Notch1 prior to neurogenesis alters early-born patch neurons but not late-born matrix neurons in the striatum. We further show that the late-born striatal neurons in these mutants are spared as a result of functional compensation by Notch3. Notably, however, the removal of Notch signaling subsequent to cells leaving the germinal zone has no obvious effect on striatal organization and patterning. These results indicate that Notch signaling is required in neural progenitor cells to control cell fate in the striatum, but is dispensable during subsequent phases of neuronal migration and differentiation

Recent studies show that cadherins and catenins are hormonally regulated and carry out physiological roles during mammary development but have pathological effects when deregulated. E-cadherin expression is irreversibly lost in invasive lobular breast cancer (ILC). Animal models of ILC provide mechanistic insight, confirming that E-cadherin serves as both a tumor suppressor and an invasion suppressor in ILC. Ductal breast cancer involves complex, reversible, epigenetic modulation of multiple cadherins. Transcriptional regulators of E-cadherin have been identified that induce epithelial-to-mesenchymal transitions. Catenins are lost or mislocalized in tumors lacking cadherins. However, beta-catenin signaling is upregulated by numerous pathways in >50% of breast tumors and animal models suggest its oncogenic function in breast relates to its role in mammary progenitor cell expansion

This article reports the production of an EP-element insertion library with more than 3,700 unique target sites within the Drosophila melanogaster genome and its use to systematically identify genes that affect embryonic muscle pattern formation. We designed a UAS/GAL4 system to drive GAL4-responsive expression of the EP-targeted genes in developing apodeme cells to which migrating myotubes finally attach and in an intrasegmental pattern of cells that serve myotubes as a migration substrate on their way towards the apodemes. The results suggest that misexpression of more than 1.5% of the Drosophila genes can interfere with proper myotube guidance and/or muscle attachment. In addition to factors already known to participate in these processes, we identified a number of enzymes that participate in the synthesis or modification of protein carbohydrate side chains and in Ubiquitin modifications and/or the Ubiquitin-dependent degradation of proteins, suggesting that these processes are relevant for muscle pattern formation.

Varus or valgus malalignment of the knee may be either a cause or a consequence of unicompartmental knee arthritis in young, active adults. Proximal tibial osteotomy for the varus knee and distal femoral osteotomy for the valgus knee have been used for decades to manage this condition; however, their use has decreased significantly in recent years as the popularity of unicompartmental and total knee arthroplasty has grown. With the advent of biologic resurfacing techniques for focal full-thickness articular cartilage injury, combined or staged high tibial osteotomy is becoming increasingly popular. In addition, in the face of cruciate ligamentous instability with or without posterolateral corner instability coupled with varus malalignment, high tibial osteotomy with and without ligament reconstruction provides a solution to complex orthopedic problems. Recent long-term follow-up studies have concluded osteotomy allows for improved function and pain relief in properly selected young patients