University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations Summer 2011 The Role of Sonic Hedgehog in the Hypothalamus Solsire E. Zevallos University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Congenital, Hereditary, and Neonatal Diseases and Abnormalities Commons, Developmental Biology Commons, Developmental Neuroscience Commons, Disease Modeling Commons, Embryonic Structures Commons, Endocrine System Commons, Endocrine System Diseases Commons, Eye Diseases Commons, Genetic Phenomena Commons, Genetic Processes Commons, Genetics Commons, Medical Genetics Commons, Medical Neurobiology Commons, Medical Pathology Commons, Molecular Genetics Commons, Nervous System Commons, Nervous System Diseases Commons, and the Neurosciences Commons Recommended Citation Zevallos, Solsire E., "The Role of Sonic Hedgehog in the Hypothalamus" (2011). Publicly Accessible Penn Dissertations. 357. https://repository.upenn.edu/edissertations/357 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/357 For more information, please contact [email protected]. The Role of Sonic Hedgehog in the Hypothalamus Abstract ABSTRACT THE REQUIREMENT OF SONIC HEDGEHOG IN THE HYPOTHALAMUS Solsire E. Zevallos Douglas J. Epstein Sonic hedgehog (Shh) is a morphogen secreted during early development that is required for the formation of the ventral neural tube, including the ventral forebrain. The prechordal source of Shh underlying the forebrain is required early to bifurcate the cerebral hemispheres and eye vesicles as well as to specify a rostroventral forebrain region, the hypothalamus. We hypothesized that Shh expressed later in the hypothalamus may be required for other known functions of Shh, such as patterning within a region, cell proliferation, cell specification, and axon guidance. To evaluate this hypothesis, Shh was genetically excised from the hypothalamus using a cre/loxp strategy: Shh brain enhancer 2 (SBE2), with specific activity in the hypothalamus, drove cre recombination of the Shh conditional allele. SBE2-cre;Shh-/loxp mice (or ShhDhyp) exhibited patterning abnormalities that resulted in morphologically abnormal hypothalamic ventral midline, pituitary, and optic nerve. The constellation of patterning defects and phenotypic abnormalities reflect the human disorder known as septo-optic dysplasia (SOD). These findings contribute ot our understanding of the developmental mechanisms involved in proper formation of the hypothalamic-pituitary axis and the optic nerve, the two main aspects of the etiology of SOD. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Neuroscience First Advisor Douglas Epstein Keywords Sonic hedgehog, hypothalamus, pituitary, optic nerve, optic disc, sept-optic dysplasia Subject Categories Congenital, Hereditary, and Neonatal Diseases and Abnormalities | Developmental Biology | Developmental Neuroscience | Disease Modeling | Embryonic Structures | Endocrine System | Endocrine System Diseases | Eye Diseases | Genetic Phenomena | Genetic Processes | Genetics | Medical Genetics | Medical Neurobiology | Medical Pathology | Molecular Genetics | Nervous System | Nervous System Diseases | Neurosciences This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/357 THE REQUIREMENT OF SONIC HEDGEHOG IN THE HYPOTHALAMUS Solsire E. Zevallos A DISSERTATION in Neuroscience Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2011 Supervisor of Dissertation ___________________________________ Douglas J. Epstein, Assoc. Prof. of Genetics Graduate Group Chairperson ___________________________________ Rita Balice-Gordon, Prof. of Neuroscience Dissertation Committee Jonathan Raper, Prof. of Neuroscience and Thesis Committee Chair Jeffrey Golden, Prof. of Pathology and Laboratory Medicine Loretta Flanagan-Cato, Assoc. Prof of Psychology Greg Bashaw, Assoc. Prof. of Neuroscience ABSTRACT THE REQUIREMENT OF SONIC HEDGEHOG IN THE HYPOTHALAMUS Solsire E. Zevallos Douglas J. Epstein Sonic hedgehog (Shh) is a morphogen secreted during early development that is required for the formation of the ventral neural tube, including the ventral forebrain. The prechordal source of Shh underlying the forebrain is required early to bifurcate the cerebral hemispheres and eye vesicles as well as to specify a rostroventral forebrain region, the hypothalamus. We hypothesized that Shh expressed later in the hypothalamus may be required for other known functions of Shh, such as patterning within a region, cell proliferation, cell specification, and axon guidance. To evaluate this hypothesis, Shh was genetically excised from the hypothalamus using a cre/loxp strategy: Shh brain enhancer 2 (SBE2), with specific activity in the hypothalamus, drove cre recombination of the Shh conditional allele. SBE2-cre;Shh-/loxp mice (or Shhhyp) exhibited patterning abnormalities that resulted in morphologically abnormal hypothalamic ventral midline, pituitary, and optic nerve. The constellation of patterning defects and phenotypic abnormalities reflect the human disorder known as septo-optic dysplasia (SOD). These findings contribute to our understanding of the developmental mechanisms involved in proper formation of the hypothalamic-pituitary axis and the optic nerve, the two main aspects of the etiology of SOD. ii TABLE OF CONTENTS PAGE ABSTRACT.…………………………………………………………..….………... ii LIST OF ILLUSTRATIONS………………………………………..…………...... iv CHAPTER 1: Introduction…………………………………………..…………..... 1 CHAPTER 2: Disruption of the SoxB1 dependent regulation of Sonic hedgehog transcription in the hypothalamus results in septo-optic dysplasia……………………………………………………………………….….. 38 CHAPTER 3: Shh restricts expansion of the non-proliferative ventral midline and specifies hypothalamic progenitor domains……….…............... 70 CHAPTER 4: Summary, Discussion and Future Directions……….....…….100 iii LIST OF ILLUSTRATIONS FIGURE PAGE CHAPTER 2: Disruption of the SoxB1 dependent regulation of Sonic hedgehog transcription in the hypothalamus results in Septo-Optic Dysplasia 1. The hypothalamic source of Shh is required for proper pituitary morphology at E12.5…………………………………………………………………………………... 58 2. Shh is required for proper anteroposterior patterning in the hypothalamus… 60 3. The optic disc fails to form in Shhhyp embryos ………………………………... 61 4. SoxB1 proteins are dose dependent regulators of Shh expression in the anterior hypothalamus ………......................................................................................... 63 5. Sox2 binds directly to SBE2 and is necessary for its activation ….……….… 64 6. SBE2 activity in the hypothalamus is dependent on a highly conserved SoxB1-binding .........………………………………………….………...…….…….. 66 Figure S1. Shh expression in the hypothalamus is absent in SBE2-cre;Shh-/loxp (Shhhyp) embryos…………………………………………….… 67 Figure S2. Shhhyp embryos exhibit pituitary dysmorphology, ectopic positioning, and reduction in hormone-producing cell types at E18.5..………………………. 68 iv FIGURE PAGE CHAPTER 3: Shh restricts expansion of the non-proliferative ventral midline and specifies hypothalamic progenitor domains 1. Shh in the hypothalamus is required for ventral midline morphology and proper dorsoventral patterning ……..………................................................................. 92 2. Shh in the hypothalamus restricts lateral expansion of the ventral midline…. 94 3. Shh in the hypothalamus is required to restrict anterior expansion of Bmp4 and Tbx3……..……………………………………………………………….……….. 96 4. Shh in the hypothalamus is required for specifying anteroventral and tuberal nuclei identity, but not the identity of the dorsal supra-optoparaventricular and caudal mamillary areas ………………………………………………...…….…….. 97 5. Model: Antagonism between Shh and Bmps mediates ventral midline patterning .……………………………………………………………………….……. 99 CHAPTER 4: Summary, Discussion and Future Directions 1. Proposed model of ventral midline patterning ………………………………... 112 v CHAPTER 1 INTRODUCTION Sonic hedgehog (Shh) is required during vertebrate embryonic development to mediate the formation of the brain and spinal cord (Chiang et al., 1996). The Shh mutant and a portion of patients with holoprosencephaly (HPE) have deficiencies in Shh that result in the severe malformations of the brain and face that are salient and characteristic (Chiang et al., 1996; Solomon et al., 2010). The cerebral hemispheres and optic vesicles fail to separate into bilateral structures and instead appear fused. Ventral subcortical structures such as the hypothalamus are particularly affected, appearing fused across the midline in many HPE patients or even absent in Shh-/- mice (Hahn and Barnes, 2010). Shh is not only required to attain the overall end result of proper morphology, but more importantly, to establish the comprising pattern of distinct neural regions and cell fates within the CNS (Ribes and Briscoe, 2009). Shh has a critical role as a morphogen, and in this capacity, Shh patterns the ventral neural tube, including the hypothalamus and adjacent optic vesicles and pituitary (Szabo et al., 2009; Shimogori et al., 2010; Wang et al., 2010; Zhao et al., 2010). Sonic hedgehog: A secreted signal activates a transcriptional cascade Shh is a secreted protein that activates a transcriptional cascade in receptive cells. Transcription factor activity