Qiufu Ma - US grants

DESCRIPTION (provided by applicant): The trigeminal ganglia are responsible for sensory processing in the face, oral and nasal cavities. Injuries and malformations of this nerve are clinically and cosmetically devastating to humans. Two different tissues contribute to the formation of the trigeminal ganglia: the ectodermal placode and the cranial neural crest. Most mechanoceptive neurons (trkC+ and trkB+) are derived from the placode, whereas the neural crest gives rise to all of the nociceptive and thermoceptive neurons (trkA+). The broad objectives of this research are to understand how these distinct classes of sensory neurons develop and how they innervate to the locales within the face. In preliminary studies, my colleagues and I have found that a single neural determination gene, Neurogenin1 (ngnl), is required to direct formation of the entire trigeminal ganglion. Research described in this proposal is primarily based on this finding. We will address three fundamental questions: 1) how is the neural crest-derived neurogenesis regulated by extrinsic signals from the pioneer placodal neurons? 2) How are axons of crest-derived neurons projected to precise locales within the face? 3) What are the intrinsic molecular mechanisms by which sensory sublineages are specified? For the first question, we will determine if the potential of the trigeminal neural crest cells to form the trkB+ and trkC+ neurons is restricted by pioneer placodal neurons. We will also determine if specification of other neuronal features is dependent on this cell-to-cell interaction as well. To do this, we will create trigeminal crest ganglia that completely lack the pioneer placodal neurons, and then to examine how the remained crest-derived neurogenesis will be affected. For the second question, a testable hypothesis is suggested by the classic experiments of Victor Hamburger. Pioneer placode-derived neurons may play an essential role for axon pathfinding of the crest-derived neurons. This hypothesis will be tested by examining axonal projections of crest-derived neurons that develop in mice without placodal neurons. For the third question, we will use "Gene Family Differential Screening," a procedure developed by the investigator, to isolate regulatory molecules that are expressed in a subset of sensory neurons. These sublineage-specific genes will be candidate molecules that control cell subtype specification.

A small subset of genes that encode transcription factors are expressed at spatially restricted positions and times during vertebrate central nervous system (CNS) development. These "informative" transcription factors act singly and in combinations to regulate fate choice and subtype specification. The primary goal of Project by Ma is to build a comprehensive temporal/spatial map of transcription factor expression patterns in the developing mouse CNS. Our central hypothesis is that this transcription factor atlas will identify novel regulators of early cell fate choice in neural progenitors and later maturation of specialized neurons and glia. The most carefully annotated vertebrate genome - human- encodes approximately 1,600 known or putative transcription factors of all classes (homeodomain, nuclear hormone, zinc finger, etc). In preliminary work, we have i) identified murine homologues of all human transcription factor-encoding genes, ii) designed and generated PCR primer pairs for these genes, and iii) cloned approximately 75% (1200) of them into vectors suitable for in situ hybridization (ISH). Our study plan builds upon this and other preliminary groundwork. We have three specific aims: Aim 1 is to characterize the expression patterns of all transcription factor-encoding genes by ISH at key stages of forebrain, hindbrain, cerebellar and spinal cord development. Aim 2 is to catalogue the data into a searchable resource accessible to the general public via the web. The database will link ISH images to relevant annotation, accession numbers reagents, other vertebrate and invertebrate databases and human neurodevelopmental - neurodegenerative disease loci. Aim 3 is to test the central hypothesis. Towards this end, we will determine whether the atlas can be used to identify transcription factors that implement the actions of Olig and Ngn bHLH transcription factors on neuronal sub-type specification. The atlas will be scanned to identify transcription factors expressed in the Olig2 domain of spinal cord and the Ngn expression domain of the cortex. Candidate target genes will be culled by observation of aberrant expression in Olig2-/-and Ngn1/2-/- tissues, respectively and then assessed for biological function in chick neural tube assays. Aims 2 and 3 draw heavily upon the informatics and expression vector cores. The project as a whole will augment efforts in Projects by Greenberg and Stiles to identify Ngn target genes and co regulator proteins for Olig 1 and Olig2.

DESCRIPTION (provided by applicant): The mammalian nervous system is composed of thousands of distinct neuronal cell types. However, all of them are either excitatory or inhibitory. The principal excitatory and inhibitory neurotransmitters are the amino acids glutamate and GABA (gamma-aminobutyrate), respectively. During development, these two transmitters are specified in a mutually exclusive manner. The broad goal of this proposal is to understand the molecular mechanism that underlies this very important fate choice decision. In preliminary studies, we have focused upon an anatomically well-defined region of the developing nervous system - the dorsal horn of the spinal cord. We have found that the Tlx-class transcription factors have a dual function in cell fate choice: promoting glutamate and suppressing GABA neuron development. Thus, in Tlx-null mice, glutamatergic sensory cells in the dorsal horn are transformed into GABAergic neurons. Our study plan builds upon this preliminary data. We have three specific aims. Aim 1 is to define the anatomical range of Tlx function in the glutamatergic versus GABAergic fate choices. Is this Tlx function confined to the dorsal horn of the spinal cord or does it extend to other Tlx-positive regions of the central nervous system - specifically, the sensory nuclei in the hindbrain? Addressing this question will determine whether binary specification of glutamate and GABA is a common theme in the nervous system and may also provide insight into why Tlx-null mice suffer a breathing problem that resembles human congenital hypoventilation syndrome. Aim 2 is to define the roles of Tlx proteins in the fate choice process. Our preliminary studies show that Tlx proteins are necessary for glutamate neuron development. Here we will use genetic gain-of-function to determine whether Tlx proteins are sufficient to specify glutamate transmitter phenotype in various brain areas. In addition, we will determine whether Tlx proteins directly or indirectly promote glutamatergic neuron differentiation. Aim 3 is to define the structural basis of Tlx proteins in suppression of GABA neuron differentiation. Within the human CNS, a disruption of the balance between excitation and inhibition underlies neurological disorders, such as epilepsy, schizophrenia, and pain disorders. Accordingly the studies described here will have practical overtones for the management of these diseases.

DESCRIPTION (provided by applicant): The long-range goal is to identify the genetic programs controlling the formations of specific sensory pathways and to gain insight into the molecular and cellular basis of pain perception. In this renewal application, we try to address two major knowledge gaps. First, most clinically relevant pain is derived from deep tissues, such as muscle, joint, bone, and visceral organs. Despite this clinical significance, the molecular and cellular basis of deep pain is still poorly understood, which is in stark contrast to the tremendous progress made in the past decades in understanding cutaneous pain. In Aim 1 studies, we will establish a molecular map for sensory neurons innervating distinct deep tissues. We will also determine if Meis1 represents the first transcription factor that controls the development of deep tissue sensory neurons. Second, the cellular basis of mechanical allodynia (pain evoked by innocuous mechanical stimuli), a hallmark for most, if not all, chronic pain disorders, needs further clarification. In case of neuropathic pain, it was proposed that central disinhibition will allow low threshold myelinated A? mechanoreceptors to directly activate the pain pathways. However, a recent study proposed that unmyelinated low threshold c- mechanoreceptors, marked by the expression of the vesticular glutamate transporter VGLUT3, may play an essential role in the readout of the mechanical allodynia. Our preliminary genetic fate-mapping studies show that VGLUT3 lineage neurons are in fact composed of both 1) unmyelinated c-mechanoreceptors that form free nerve endings in the skin epidermis and lanceolate endings around hair follicles, and 2) myelinated m-mechanoreceptors that form the Merkel-cell neurite complex. In Aim 2, we will determine if Zfp521, a transcription factor expressed exclusively in VGLUT3 lineage c-mechanoreceptor, is necessary for the development of these c-mechnaoreceptors. We will also determine if mechanical allodynia is differentially affected in mice that will have a selective developmental defect in VGLUT3 lineage c- mechanoreceptors or a defect in the VGLUT3-expressing Merkel cell-neurite complex. Together, these studies will gain insight into 1) the genetic programs that control the formation of the deep tissue pain pathways, and 2) the identities of low threshold mechanoreceptors mediating the readout of mechanical allodynia.

DESCRIPTION (provided by applicant): The mammalian nervous system is composed of thousands of distinct neuronal cell types. However, all of them are either excitatory or inhibitory. The principal excitatory and inhibitory neurotransmitters are the amino acids glutamate and GABA (gamma-aminobutyrate), respectively. During development, these two transmitters are specified in a mutually exclusive manner. The broad goal of this proposal is to understand the molecular mechanism that underlies this very important fate choice decision. In preliminary studies, we have focused upon an anatomically well-defined region of the developing nervous system - the dorsal horn of the spinal cord. We have found that the Tlx-class transcription factors have a dual function in cell fate choice: promoting glutamate and suppressing GABA neuron development. Thus, in Tlx-null mice, glutamatergic sensory cells in the dorsal horn are transformed into GABAergic neurons. Our study plan builds upon this preliminary data. We have three specific aims. Aim 1 is to define the anatomical range of Tlx function in the glutamatergic versus GABAergic fate choices. Is this Tlx function confined to the dorsal horn of the spinal cord or does it extend to other Tlx-positive regions of the central nervous system - specifically, the sensory nuclei in the hindbrain? Addressing this question will determine whether binary specification of glutamate and GABA is a common theme in the nervous system and may also provide insight into why Tlx-null mice suffer a breathing problem that resembles human congenital hypoventilation syndrome. Aim 2 is to define the roles of Tlx proteins in the fate choice process. Our preliminary studies show that Tlx proteins are necessary for glutamate neuron development. Here we will use genetic gain-of-function to determine whether Tlx proteins are sufficient to specify glutamate transmitter phenotype in various brain areas. In addition, we will determine whether Tlx proteins directly or indirectly promote glutamatergic neuron differentiation. Aim 3 is to define the structural basis of Tlx proteins in suppression of GABA neuron differentiation. Within the human CNS, a disruption of the balance between excitation and inhibition underlies neurological disorders, such as epilepsy, schizophrenia, and pain disorders. Accordingly the studies described here will have practical overtones for the management of these diseases.

DESCRIPTION (provided by applicant): The long-range goal of this competitive renewal of a previous R01 is to investigate genetic programs that control pain relay sensory neuron phenotypes in the dorsal spinal cord. During the previous funding interval, my colleagues and I have compiled a genome-scale expression map of transcription factors in the mouse nervous system. Subsequent genetic studies demonstrate that Tlx3, a homeobox class transcription factor, is a pivotal regulator of spinal relay sensory neurons, including specification of both glutamate and peptide neurotransmitters. Furthermore, persistent Tlx3 expression in adult animals is confined to superficial laminae, where putative pain relay neurons are located. The research described here builds upon this preliminary work. The goal of our research over the next five years is to illustrate the roles of Tlx3 in regulating spinal relay nociceptor phenotypes and pain behaviors and to gain insights into the molecular and cellular basis underlying pain perception. We have four specific Aims. Aim 1 is to determine the roles of Tlx3 in controlling the development of ascending projection neurons that are critical for pain perception. Aim 2 is to determine how dynamic Tlx3 expression controls lamina organization of the dorsal spinal cord. Aim 3 is determine the roles of Tlx3 in maintaining dorsal horn excitatory neuron phenotypes, thereby determining if Tlx3-mediated core transcription program is a potential target for pain treatment. Aim 4 is to determine the roles of Tlx3-dependent differentiation programs in controlling pain behaviors. Each of these aims is built upon a set of preliminary data that lead to a testable hypothesis. A panel of genetic tools that we have already developed will test the predictions of these hypotheses. PUBLIC HEALTH RELEVANCE: Pain management remains a major medical problem in a variety of human diseases. Chronic pain, moreover, is associated with worse disease outcome and depression. In the fullness of time, the work may allow us to determine whether the Tlx3-mediated core transcriptional program is a valid and novel therapeutic target for pain management.

The long-term goal of Project 2 is to develop new targets for the treatment of chronic pain. Specifically, our research has been focusing on those core transcriptional programs that control nociceptor phenotypes and pain behaviors. In the previous funding cycle, we have compiled a genome-scale analysis of the expression of transcription factors (TFs) in the developing nervous system. From this screen, we identified a small number of TFs expressed in the pain circuitry. Subsequent genetic studies demonstrated that the runt class transcripfion factor Runxl is a key regulator of nociceptor development, and mice lacking Runxl exhibit a marked deficit in inflammatory pain and neuropathic pain. The experimental plan of this project is built on these preliminary studies, and we have three specific aims. Aim 1 is to determine the roles of Runxl in controlling two types of cancer pain: pain induced by tumor growth or by chemotherapy. This aim is built on the facts that cancer pain is composed of both inflammatory and neuropathic pain components, and Runxl is required for these two types of chronic pain. Aim 2 is to determine Runxl targets that serve as candidates critical for neuropathic pain. This aim is built on the observation that Runxl activity at embryonic stages, rather than at postnatal stages, is required for neuropathic pain, implying that early Runxl targets are later required for the development of this type of chronic pain. Aim 3 is to determine signaling pathways that modulate Runxl expression in adult nociceptors. This aim is built on the finding that persistent Runxl activity is required for inflammatory pain. Accordingly, compounds capable of extinguishing Runxl expression may serve as new targets for inflammatory pain treatment. The studies of these aims will be enabled by the availability of various Runxl mutant mice and from the Druggable Mechanisms Core (the DMC), including high throughput single molecule DNA sequencing and bioinformatics analyses.

Project Summary The broad goal of this research is to improve (i) the potency, (ii) safety, and (iii) the credibility of nerve stimulation as a treatment for sepsis by defining the functional neural circuitry. In the USA, among 1-3 million patients suffering sepsis each year, 15-30% will die and many of the survivors will suffer organ damage. Conventional therapeutic regimens are thus far inadequate. Against this backdrop, nerve stimulations such as non-invasive electroacupuncture stimulation (ES) at specific acupoints can attenuate systemic inflammation associated with sepsis and promote survival of laboratory animals. To date, one dominant view is that ES drives vagal nerve-dependent anti-inflammatory pathways, involving activation of sympathetic cells and subsequent modulation of pro-inflammatory cytokine release from immune cells. However, there are two important unresolved issues that must be addressed in order to realize the full potential of ES as a therapeutic modality for sepsis. First, the roles of sympathetic neurons are still ill-defined. Noradrenaline (NA), one of transmitters released from sympathetic cells, has long been proposed to suppress systemic inflammation, via activation of ?2 adrenergic receptors. However, NA can also promote inflammation via activation of ?2 adrenergic receptors, and this pro-inflammatory signaling can be sensitized following macrophage pre- exposure to bacteria-derived endotoxins such as the lipopolysaccharide (LPS). Accordingly, it remains unknown i) if ES could be counterindicated when sepsis has progressed to certain stages, and ii) if a strategy to maximize the anti-inflammatory over the pro-inflammatory activity is pivotal for ES to treat severe sepsis. Second, it has been long known that ES can drive vagal parasympathetic reflexes only in specific acupoints, but the underlying neural basis is entirely unknown. Because of unknown identities and anatomical distributions of somatosensory neurons driving vagal reflexes, it becomes difficult to optimize stimulation parameters to activate this anti-inflammation pathway. To address these unresolved issues, we have developed innovative genetic tools to ablate, silence or activate molecularly defined sympathetic and somatosensory neurons. Built upon strong preliminary results, we postulate i) that sensory neurons marked by the expression of the G protein-coupled receptor Prokr2 are required to for low electric intensity ES (0.5 mA) to drive vagal reflexes, and ii) that noradrenergic sympathetic neurons marked by the expression of the neuropeptide NPY, which can be activated by high intensity ES (3 mA), may suppress and promote inflammation, dependent on ES delivered before or after sepsis manifestation. A series of predictions from these hypotheses will be tested. In the fullness of time, the studies outlined in this application will enable us to illustrate distinct neuronal pathways that can dynamically modulate sepsis-associated systemic inflammation, and will help to improve sepsis management by promoting the anti-inflammatory over the pro-inflammatory pathways. !

Abstract The goal of this application is to explore therapeutic opportunities for visceral pain in a new set of cellular targets within the spinal cord that will drive affective pain, rather than reflexive-defensive reactions. Pain from deep tissues (e.g. visceral organs, joints, muscles and bones) is among the most prevalent and disruptive. Major causes include inflammation, nerve injury and tumor growth. Translation of preclinical insight into the neurobiology of pain towards new medications has been disappointing. In preliminary studies, my colleagues and I have unmasked one of potential explanations for the lack of progress. Put briefly, in a series of studies on cutaneous pain, we have identified a group of spinal ascending projection neurons, marked by Tac1-Cre, that are essential for affective pain-indicative coping behaviors, but dispensable for first-line nocifensive-defensive reactions. This disconnect at the level of cutaneous pain leads to a testable hypothesis. We propose that affective and defensive elements of the response to noxious visceral stimuli could also be segregated at the level of the spinal cord. Our study plan uses innovative genetic tools to probe testable predictions of this hypothesis. We have two specific aims: Aim 1 is to test the prediction that unique spinal substrates are associated with acute visceromotor reflexes versus affective visceral pain. This prediction is based on our preliminary results, showing that two groups of spinal excitatory neurons (one is Tac1-Cre neurons), which are activated by noxious colorectal distensions (CRD), send extensive ascending projections to distinct, though partially overlapped sets of thalamic and midbrain nuclei. Meanwhile, we will test if a group of spinal inhibitory neurons activated by CRD act to gate visceral motor reflexes and/or affective pain. Aim 2 is to test the prediction that unique spinal substrates are associated with inflammatory visceral pain. Our pilot studies have identified two groups of spinal excitatory neurons that are activated by CRD only after gastrointestinal (GI) inflammation, and one of which (marked by VGLUT3-Cre) appears to be crucial for mediating sensitized affective visceral pain. Together with three other groups of excitatory neurons that are already activated by CRD under naïve conditions, we will determine if these five groups of neurons transmit sensitized visceromotor reflexes, affective visceral pain, as well as the referred cutaneous pain induced by GI inflammation. All together, these studies will provide new insight into spinal substrates mediating different dimensions of acute and chronic visceral pain.