Growing Up POMC: Pro-opiomelanocortin in the Developing Brain Stephanie Louise Padilla Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy under the Executive Committee of the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2011 © 2011 Stephanie Louise Padilla All Rights Reserved ABSTRACT Growing Up POMC: Pro-opiomelanocortin in the Developing Brain Stephanie Louise Padilla Neurons in the arcuate nucleus of the hypothalamus (ARH) play a central role in the regulation of body weight and energy homeostasis. ARH neurons directly sense nutrient and hormonal signals of energy availability from the periphery and relay this information to secondary nuclei targets, where signals of energy status are integrated to regulate behaviors related to food intake and energy expenditure. Transduction of signals related to energy status by Pro-opiomelanocortin (POMC) and neuropeptide-Y/agouti-related protein (NPY/AgRP) neurons in the ARH exert opposing influences on secondary neurons in central circuits regulating energy balance. My thesis research focused on the developmental events regulating the differentiation and specification of cell fates in the ARH. My first project was designed to characterize the ontogeny of Pomc- and Npy-expressing neurons in the developing mediobasal hypothalamus (Chapter 2). These experiments led to the unexpected finding that during mid-gestation, Pomc is broadly expressed in the majority of newly-born ARH neurons, but is subsequently down-regulated during later stages of development as cells acquire a terminal cell identity. Moreover, these studies demonstrated that most immature Pomc-expressing progenitors subsequently differentiate into non-POMC neurons, including a subset of functionally distinct NPY/AgRP neurons. The second aspect of my work focused on characterizing Pomc-expressing progenitors throughout the brain (Chapter 3). Similar to findings in Chapter 2, Pomc was broadly expressed in many aspects of the developing brain and subsequently down-regulated as cells further differentiated into non-Pomc terminal identities. In the CNS, the percentage of POMC neurons derived from Pomc-expressing progenitors are marginal. These findings are of general importance to the field of energy homeostasis research, because many genetically targeted functional manipulations of POMC cells have been induced during early development, a time in which Pomc is expressed in many cells that will not persist into a POMC terminal identity. The off target consequences of these manipulations have yet to be considered. Our work will provide the foundational evidence for potential confounds of targeted genetic POMC manipulations and may help to explain some of the unexpected phenotypes that have arisin using a BAC transgenic Pomc-Cre reagent. My current research efforts are focused on elucidating the molecular mechanism regulating differentiation and cell fate specification in the ARH during gestation and the early postnatal period (Chapter 4). Consistent with the identification of Pomc transcriptional activity during embryogenesis, functional POMC-processing products (β-endorphin and α-melanocyte- stimulating hormone) are also detected in embryonic hypothalamic extracts (at embryonic day (E) 13.5 and E15.5, respectively). The presence of POMC-derived peptides in the embryo, prior to the establishment of circuits regulating food intake, may be involved in the local differentiation events. Preliminary loss-of-function studies are underway to determine the role of POMC- derived peptides in differentiation of hypothalamic terminal fates. Early results from this work indicate that both β-endorphin and α-melanocyte-stimulating hormone are critical to the differentiation of the Pomc lineage. The goal of this work is to define the developmental origins of critical components of neuronal circuits that determine body weight. Evidence in the literature supports that maternal signals influence these events, our studies may provide a means to the design of effective strategies to reduce transmission of signals that increase susceptibility to obesity in offspring. TABLE OF CONTENTS CHAPTER 1: BACKGROUND OVERVIEW ............................................................................... 1 Body Weight Homeostasis and the Sensation of Hunger .................................................. 1 The Hypothalamic-Adipose Axis ....................................................................................... 4 Hypothalamic Determinants of Body Weight: Melanocortin System .................................. 5 Hypothalamic Determinants of Body Weight: NPY/AgRP .................................................11 POMC and NPY Neurons: Response to the Environment ................................................15 Development of the ARH .................................................................................................18 Programming of Body Weight Outcomes .........................................................................20 Hypothalamic Projections Related to Feeding ..................................................................22 Concluding Remarks .......................................................................................................25 References ......................................................................................................................26 Figure 1.1 ............................................................................................................................. 9 Figure 1.2 ...........................................................................................................................25 CHAPTER 2 “Pomc-expressing Progenitors Give Rise to Antagonistic Neuronal Populations in Hypothalamic Feeding Circuits.” ...............................................................................................38 Introduction ......................................................................................................................38 Manuscript .......................................................................................................................39 Methods ...........................................................................................................................47 References ......................................................................................................................58 Figure 2.1 ............................................................................................................................45 Figure 2.2 ............................................................................................................................46 Supplementary Figure 2.1 ...................................................................................................51 i Supplementary Figure 2.2 ...................................................................................................52 Supplementary Figure 2.3 ...................................................................................................53 Supplementary Figure 2.4 ...................................................................................................54 Supplementary Figure 2.5 ...................................................................................................55 Supplementary Figure 2.6 ...................................................................................................56 CHAPTER 3 “Defining POMC neurons utilizing transgenic reagents: Impact of transient Pomc expression in diverse immature neuronal populations.” .............................................................60 Introduction ......................................................................................................................60 Manuscript .......................................................................................................................62 Abstract ...........................................................................................................................62 Introduction ......................................................................................................................63 Results ............................................................................................................................65 Discussion .......................................................................................................................72 Methods ...........................................................................................................................78 Acknowledgements ..........................................................................................................81 References ......................................................................................................................92 Table 3.1 .............................................................................................................................82 Figure 3.1 ............................................................................................................................83 Figure 3.2 ............................................................................................................................84 Figure 3.3 ............................................................................................................................85 Figure 3.4 ............................................................................................................................86 Figure