Rolf O. Karlstrom - US grants

neurogenetics; neuroanatomy; neuronal guidance; gene mutation; brain; zebrafish; genetic mapping;

DESCRIPTION (Applicant's abstract reproduced verbatim): The hedgehog (Hh) family of secreted proteins plays a fundamental role in cell differentiation within the brain, spinal cord, limbs, somites and circulatory system. Defects in Hh signaling during embryonic development are associated with human congenital malformations, including holoprosencephaly. Mis-regulation of Hh signaling later in life is associated with basal cell carcinoma, the most common form of cancer affecting fair-skinned adults. The study of Hh signaling during development is thus important for understanding human congenital malformations as well as the formation of certain tumors. The major goal of this project is to understand how Hh signaling contributes to cell differentiation during normal development in the zebrafish embryo. We have used a genetic approach to study the role of Hh signaling in the formation of a defined region of the nervous system, the post optic area of the ventral forebrain. We have recently shown that two zebrafish forebrain mutations encode Hh-responsive transcription factors of the gli family. Cell differentiation defects in the zebrafish forebrain mutants appear to be caused by mis-regulation of Hh signaling. Consistently, it was also recently demonstrated that another zebrafish forebrain mutation encodes sonic hedgehog. The fact that three of the zebrafish forebrain mutants encode members of the Hh pathway underscores the importance of this signaling cascade in development. This application focuses on a fourth zebrafish forebrain mutant called umleitung (uml). Like the known Hh pathway mutations, uml interferes with Hh signaling and cell differentiation in the ventral forebrain. We now have evidence that uml may encode another zebrafish gli gene. While gli genes appear to be central to the regulation of Hh signaling, little is known about how gli genes function in vertebrates. There is evidence that some gli genes activate the transcription of Hh target genes, while others act to repress Hh targets. We propose to analyze the role of gli mediated hedgehog signaling in zebrafish forebrain development by A) identifying the gene encoded by uml, determining the genetic lesion in uml, and isolating null alleles of uml, B) determining how gli genes contribute to cell differentiation in the ventral forebrain, C) determining how gli genes regulate, and in turn are regulated by, Hh signaling in vivo, and D) identifying and characterizing genes that are transcriptionally regulated by gli genes in the zebrafish embryo. The zebrafish embryo provides a powerful model for the study of Hh regulation in vertebrates as it allows a genetic approach to be combined with ectopic expression studies. The combination of these two approaches is well suited to the study of molecular signaling pathways that regulate cell fate decisions and promises to provide insights not readily attainable in other vertebrate systems.

[unreadable] DESCRIPTION (provided by applicant): The pituitary gland is known as the master endocrine gland and controls multiple hormonal responses including those regulating reproduction, homeostasis, and responses to stress. The hormone-secreting lobe of the pituitary gland, or adenohypophysis, forms at the anterior end of the developing embryo through inductive interactions between neurally and epidermally derived tissues. Hedgehog (Hh) signaling molecules help mediate these inductive events, a role that has been conserved across vertebrate species from fish to mammals. Human mutations in Hh signaling lead to a variety of syndromes that affect pituitary development, including Holoprosencephaly and Pallister-Hall syndrome. In fact, human congenital pituitary defects are quite common and range from the loss of all endocrine function (panhypopituitarism) to the loss of single hormone function. The loss of GH is the most common single endocrine deficiency in humans, occurring in 1 in 4000 embryos. We previously showed that this range of pituitary defects also occurs in zebrafish Hh pathway mutants, providing a unique resource for the study of pituitary development. Among these, the uncharacterized uml mutation eliminates some cell types (e.g. GH) and uniquely affects cell fate decisions in the pituitary. We also showed that Gli mediated Hh signaling is needed for pituitary induction and endocrine cell differentiation. Here we propose to continue our use of the zebrafish as a model system to investigate the molecular and cellular mechanisms of Hh regulated cell differentiation in the vertebrate pituitary gland. We will first test whether Hh acts as a morphogen or mitogen in pituitary development and determine which Gli transcription factors mediate the pituitary Hh response. Using newly developed techniques to temporally and cell-autonomously disrupt Hh signaling, we will then test the direct requirement for Hh signaling in pituitary precursor cells and endocrine cell lineages and determine when direct Hh signaling is needed for these cell differentiation events. Finally, we will determine the molecular basis of the zebrafish umleitung (uml) mutation as part of a genetic investigation of Hh involvement in endocrine cell lineage determination. This work will provide fundamental knowledge about the role of Hh signaling in guiding cell specification in the vertebrate pituitary. Our research plan takes advantage of zebrafish to combine genetic, cellular, and molecular analyses at a level not possible in other vertebrates. Our new genetic tools will be useful to researchers investigating Hh in any embryonic tissue. The characterization of a potentially novel regulator of Hh signaling (the uml locus) is also likely to provide new insights into the regulation of Hh signaling throughout the developing embryo. Because the Hh signaling pathway has been highly conserved through evolution, this work will apply directly to our understanding of Hh signaling in higher vertebrates and will impact on our ability to direct stem cell differentiation for therapeutic purposes. Ultimately, this zebrafish research promises to contribute to our understanding of human birth defects affecting the pituitary and may shed light on tumorigenesis caused by mis-regulation of Hh signaling postnatally. [unreadable] [unreadable]

Pituitary adenomas are among the most prevalent of human tumors, affecting over 15% of the population in the United States. However, it is not yet known what regulates the division of endocrine cells in adults. Hedgehog (Hh)/Gli and Fgf-mediated cell-cell signaling play critical roles in pituitary development in the embryo, a requirement that has been conserved across vertebrate species from fish to humans. We have shown that Hh/Gli signaling plays a role in controlling the number and position of endocrine cell types in the embryo. Little is known about the cellular and molecular mechanisms by which Hh acts in the pituitary, or how these signaling systems continue to influence endocrine cell differentiation and proliferation post-embryonically. Our goal is to understand the mechanisms by which Hh induces and functionally patterns the vertebrate pituitary in the embryo and to determine how Hh/Gli signaling influences pituitary growth post- embryonically. In Aim #1 we seek to determine the embryonic origin of the two sub-domains of the adenohypophysis and determine whether Hh/Gli signaling is directly required in placodal cells, a crucial step toward understanding the cellular and molecular mechanisms that induce and pattern the pituitary gland. The goal of Aim #2 is to characterize and quantify endocrine cell proliferation in the larval pituitary and determine how Hh/Gli signaling regulates endocrine cell numbers post-embryonically. These studies are designed to provide insight into how the ordered complexity of this tissue arises, how different endocrine cell types are generated, and how pituitary growth and differentiation are regulated. Answering these questions is important, not least because many diseases have their origins in embryonic development. Errors in Hh signaling at this time, if not corrected, can develop into medical conditions such as holoprosencephaly or endocrine dysfunction, including dwarfism and hypopituitarism. Because Hh inhibitors are being used now to treat several other human tumors, this work may help provide the molecular background needed to bring similar treatments to patients with pituitary adenomas. Comment [MB1]: I assume that stat is in the US? I would insert US here, given that you are applying for PHS money.

All living organisms must regulate their internal salt and water content to function routinely and survive. In more complex animals, such as vertebrates, hormones and the receptors to which they bind are critical signaling molecules involved in regulation of salt and water transport. Fish are in direct contact with water, and they must actively take up salts from freshwater and actively secrete them in seawater to be able to regulate internal salt concentrations. To date research on the hormonal control of salt regulation has been limited to studies on advanced fish species. This project will examine the hormonal control of salt regulation in two basal vertebrates, lamprey eel and sturgeon. The researchers will examine how growth hormone and prolactin, two hormones secreted by the pituitary gland, and their receptors are altered by external salinity. This research will determine the effect of these hormones on the ability of lamprey and sturgeon to tolerate changes in external salinity. The results of these studies will yield insights about the functional evolution of these hormones, which in turn could be applied to enhancing agricultural production of fish and management of invasive lamprey. The results will be rapidly transferred to textbooks in comparative endocrinology and physiology, published in peer-reviewed journals, and shared at scientific meetings.

Studies supported by this award will investigate the functional evolution of osmoregulatory hormones in aquatic vertebrates. Our current understanding of the hormonal control of osmoregulation in aquatic vertebrates comes almost entirely from advanced bony fish (teleosts), and there is no information on how pituitary hormones control osmoregulation in basal vertebrates. It is now well-established that prolactin (PRL) is critical for survival of most teleosts in freshwater (FW) and that growth hormone (GH), at least in some lineages, promotes osmoregulation in seawater (SW). Lamprey, as basal vertebrates, and chondrosteans (including sturgeon), as basal bony fish, have the same osmoregulatory strategy as teleosts (namely, maintaining nearly constant levels of plasma ions irrespective of external salinity), yet there is currently no information on whether PRL or GH play a role in osmoregulation of either lamprey or chondrosteans. There is strong evidence that lamprey possess only one member of the PRL/GH family of pituitary hormones (GH), making them especially critical for these studies. Our specific aims are: 1) to determine the response of GH and GH receptor to salinity change in lamprey; 2) to determine the osmoregulatory actions of GH in lamprey; and 3) to determine the osmoregulatory roles of GH and PRL in sturgeon. For each species we will examine the time course of physiological and endocrine responses to reciprocal transfers between FW and SW. We will utilize recombinant technology to make lamprey GH in order to develop homologous radioimmuno- and radioreceptor- assays, and determine its osmoregulatory actions in vivo and in vitro.

How biodiversity arises and is maintained over time remains an important area of study. Adaptive radiations represent extreme instances of morphological evolution in response to discrete ecological shifts, and are thought to have made significant contributions to biodiversityon this planet. This research seeks to identify and characterize the genetic and cellular mechanisms that promote adaptive radiations, and thus to provide novel insights into the origins of biodiversity. This highly interdisciplinary project provides a rich intellectual landscape to train students in various methods and theories. It also offers an explicit framework to develop pedagogical tools for conveying evolutionary principals to the public. This will be part of on-going efforts to develop a series of animated and interactive evolutionary "origin stories" that detail the genetic mechanisms that underlie the development and evolution of notable traits, including turtle shells, bat wings, and limb loss in whales. Here the team proposes a new chapter-- Making faces: How the fish changes its skull.

A focal point of this project is phenotypic plasticity, which refers to the ability of an organism to change its appearance in response to a change in the environment. The ability of an individual to change its phenotype in different environments may increase its fitness in changing and/or fluctuating environments, which suggests that plasticity may be adaptive and therefore subject to selection itself. It is predicted that species that live in fluctuating environments will maintain a high degree of plasticity. Conversely, species that live in more stable environments are predicted to loose the ability to mount a plastic response, and exhibit fixed phenotypes. Unfortunately, a strict genetic basis for phenotypic plasticity has remained elusive, and thus the evolutionary potential for this trait remains largely unknown. The goal of this research is to characterize the molecular basis for phenotypic plasticity of the teleost jaw. An emphasis on the jaws has direct ecological consequences, as different jaw shapes will determine where a fish lives and what it feeds on. Mutant and transgenic zebrafish will be used to assess the degree to which key molecules involved in bone formation, including those that underlie the primary cilia and Hedgehog signaling, are necessary to promote phenotypic plasticity in the skull and jaws. The molecular mechanisms that underlie plasticity will then be compared to patterns of genetic evolution in a textbook adaptive radiation, East African cichlid fishes, which exhibit extensive diversity in the shape of their jaws. It is predicted that the genes that underlie plasticity of the feeding apparatus, also underlie evolution of the jaws. This is because genetic variation that leads to plasticity is predicted to become "fixed" as populations adapt to new environments over time. In all, research under this award will facilitate a better understand of how the genome and environment interact to promote biodiversity.