The Heart of glass-Cerebral Cavernous Malformation (Heg-CCM) pathway is essential for heart development in zebrafish and mouse. In zebrafish, mutants for the Heg-CCM genes ccm1, ccm2, and heg exhibit an extreme dilation of the heart chambers and inflow tract and completely lack blood circulation. The mechanisms by which this pathway regulates heart development are incompletely understood. Two major impediments to our knowledge are the paucity of genes known to participate in the Heg-CCM pathway and a lack of information about how the Heg-CCM pathway interacts with other signaling pathways in live embryos.

Somatosensory neurons are essential for detecting diverse environmental stimuli, thus critical for survival of mammals. In order to achieve sensory modality specificity, many somatosensory subtypes emerge with various receptor and ion channel expression, as well as terminal morphologies. How the somatosensory system achieves such a high variety of neuronal subtypes is unknown. In this thesis, I used a newly discovered subtype, VGLUT3-expressing unmyelinated low-threshold mechanoreceptors (C-LTMRs), as a model to try to answer this question. C-LTMRs have been proposed to play a role in pleasant touch in humans or pain in mice. Previously, our lab has identified the Runt domain transcriptional factor Runx1 to be pivotal for the development of a cohort of sensory neurons such as pain related nociceptors, thermal receptors, as well as itch related pruriceptors. Here I found that Runx1 is also required to establish all known features associated with C-LTMRs. In search of the mechanism of how Runx1 controls C-LTMR development, I found that the zinc finger protein Zfp521 is predominantly expressed in C-LTMRs and its expression is Runx1 dependent. By generating and analyzing Zfp521 conditional knock out animals, I found Zfp521 is required for part of C-LTMR molecular identities and nerve terminal morphologies. Our studies suggest that Runx1 acts through Zfp521-dependent and Zfp521-independent pathways to specify C-LTMR identities. To study C-LTMR functions, we performed a series of behavioral analysis and found the loss of VGLUT3 and mechanosensitivities in C-LTMRs does not markedly affect acute or chronic mechanical pain measured from the hind paws, which argues against the proposed role of VGLUT3 in C-LTMRs in mediating mechanical pain in mice. In the future, we will continue to use our mutant mice to study physiological functions of C-LTMRs.

A remarkable property of developing organisms is the consistency and robustness within the formation of the body plan. In many animals, morphological pattern formation is orchestrated by conserved signaling pathways, through a process of strict spatio-temporal regulation of cell fate specification. Although morphological patterns have been the focus of both classical and recent studies, little is known about how this robust process is modified throughout evolution to accomodate different morphological adaptations.

Gene expression is critical for animal development as cells divide and differentiate into multiple cell types. Cell-type specific gene expression is controlled by enhancers, DNA sequences that can direct expression of a target gene from hundreds of kilobases away. Gene promoters contact at least two enhancers on average, and enhancers may also contact each other. A key question is therefore how enhancers operate in this complex regulatory DNA context. It has long been assumed that enhancers act as independent modules based on their ability to drive gene expression when isolated in reporter constructs. To test assumptions of enhancer modularity, I probed interactions between two developmental enhancers from the even-skipped locus in Drosophila melanogaster blastoderm embryos. My results contradict the classic definition of enhancers; I found that the arrangement of enhancers relative to one another and the promoter influences levels of gene expression while not affecting its spatial pattern within the embryo. These results are described in Chapter 2. However, these enhancers are modular in one aspect: when fused directly together, they still direct their distinct spatial expression patterns. In Chapter 3 I describe a collaboration with Md Abul Hassan Samee in Saurabh Sinha's group at the University of Illinois Urbana-Champaign to apply computational sequence-to-expression models to my data. We found that a mechanistic model describing interactions between transcription factors was unable to fit our data well; in contrast, a phenomenological model that finds active sequences fits the data much better. These results indicate that to predict gene expression from sequence we will need to learn how enhancer boundaries are defined. In summary, I present evidence that the organization of enhancers within a locus impacts expression of the target gene. This finding overturns assumptions about enhancer modularity and emphasizes the importance of considering higher level interactions across a locus. Structural variation is common in natural populations, and our results highlight a novel way in which these sequence variants may alter gene expression. To realize the long-standing goal to predict gene expression directly from sequence we must investigate how enhancers interact within a complex locus.

The fruit fly Drosophila melanogaster has been a powerful model system in which to study the immune response. When microorganisms breach the mechanical barrier of the insect, phagocytosing cells and a battery of induced antimicrobial molecules rapidly attack them. These antimicrobial peptides can reach micromolar concentrations within a few hours. This immediate response is reminiscent of the mammalian innate immune response and utilizes transcription factors of the NF-κB family. We have generated loss-of-function mutants of the NF-κB-like transcription factor Relish in order to investigate Relish's role in the Drosophila immune response to microbes. Relish mutant flies have a severely impaired immune response to Gram-negative (G-) bacteria and some Gram-positive (G+) bacteria and fungi and succumb to an otherwise harmless infection. The main reason for the high susceptibility to infection is that these mutant flies fail to induce the antimicrobial peptide genes. The cellular responses appear to be normal. Relish is retained in the cytoplasm in an inactive state. We designed a set of expression plasmids to investigate the requirements for activation of Relish in a hemocyte cell line after stimulation with bacterial lipopolysaccharide. Signal-induced phosphorylation of Relish followed by endoproteolytic processing at the caspase-like target motif in the linker region released the inhibitory ankyrin-repeat (ANK) domain from the DNA binding Rel homology domain (RHD). Separation from the ANK domain allowed the RHD to move into the nucleus and initiate transcription of target genes like those that encode the inducible antimicrobial peptides, likely by binding to κB-like sites in the promoter region. By studying the immune response of the Relish mutant flies in combination with mutants for another NF-κB-like protein, Dorsal-related immunity factor (Dif), we found that the Drosophila immune system can distinguish between various microbes and generate a differential response by activating the Toll/Dif and Imd/Relish pathways. The recognition of foreign microorganisms is believed to occur through pattern recognition receptors (PRRs) that have affinity for selective pathogen-associated molecular patterns (PAMPs). We found that the Drosophila PRRs can recognize G- bacteria as a group. Interestingly, the PRRs are specific enough to distinguish between peptidoglycans from G+ bacteria such as Micrococcus luteus and Bacillus megaterium and fungal PAMPs from Beauveria bassiana and Geotrichum candidum. This thesis also investigates the expression of the antimicrobial peptide genes, Diptericin B and Attacin C, and the putative intracellular antimicrobial peptide gene Attacin D, and explores a potential evolutionary link between them.

In this project I have tried to obtain stable transformants of Drosophila melanogaster flies using electroporation. I have completed approximately 200 tests using different DNA concentrations, voltages and cuvettes, including a novel Petri dish cuvette which I developed and manufactured myself. I also developed new and more efficient procedures of egg collection and egg dechorionation. Although I was not  successful in obtaining true stable transformants, control experiments indicate that electroporation of DNA into embryos could be accomplished under the conditions used. The lack of stable transformants was probably due to failure of the electroporated DNA to integrate into the host genome. The reasons for why the DNA did not integrate was not further investigated in this study.

The renal capsule is a flattened layer of cells which surround the kidney. Expression of the transcription factor Foxd1 is required for normal development of the capsule. Furthermore, current evidence suggests that during development the capsule progenitors are in a state of active hedgehog signaling. We hypothesize that hedgehog plays a role in modulating capsule morphogenesis in the embryonic kidney. To test the hypothesis hedgehog signaling was inhibited in the capsule via Foxd1Cre mediated deletion of Smoothened (Smo), the activator of the pathway. Mutant kidneys were approximately 48% smaller in volume and had a 42% decrease in nephron number. Furthermore, mutants displayed abnormal patterning of the capsule where regions on the surface of the kidney had no capsule cells. The discontinuous capsule phenotype was observed only after E13.5. Additionally, capsule cells progressively lost expression of known markers Foxd1 and Raldh2 and their proliferative capacity was decreased by 54% at E13.5.

The renal capsule is a flattened layer of cells which surround the kidney. Expression of the transcription factor Foxd1 is required for normal development of the capsule. Furthermore, current evidence suggests that during development the capsule progenitors are in a state of active hedgehog signaling. We hypothesize that hedgehog plays a role in modulating capsule morphogenesis in the embryonic kidney. To test the hypothesis hedgehog signaling was inhibited in the capsule via Foxd1Cre mediated deletion of Smoothened (Smo), the activator of the pathway. Mutant kidneys were approximately 48% smaller in volume and had a 42% decrease in nephron number. Furthermore, mutants displayed abnormal patterning of the capsule where regions on the surface of the kidney had no capsule cells. The discontinuous capsule phenotype was observed only after E13.5. Additionally, capsule cells progressively lost expression of known markers Foxd1 and Raldh2 and their proliferative capacity was decreased by 54% at E13.5.

<p> Learning to provide emergency care alone and with others in the clinical environment imposes unexplored stresses on novice caregivers. It is unclear whether this stress inhibits or promotes performance and learning. Many academic health professions programs incorporate simulation as a method for teaching patient care emergencies. This study employed a modified switching replications design to explore the relationships and differences between psychological, physiological, and performance measures in health professions students who participated in acutely stressful health care simulation scenarios. Twenty-seven volunteer participants recruited from nursing, medicine, pharmacy, physical therapy, occupational therapy and speech therapy were assigned to teams in either a simulation treatment or a control group. Teams participated in two simulations scenarios where a fallen patient required assistance. Subjects in the simulation treatment groups received a standardized training module called the First Five Minutes^&reg; between simulation experiences. Mean heart rate, maximal mean heart rate, salivary alpha amylase levels, and salivary cortisol levels were compared at intervals before, during, and after each simulation scenario. Psychological stress was evaluated using the Stressor Appraisal Scale (SAS). Team performance during scenarios was scored by independent evaluators using an skills checklist adapted from a standardized commercially available training module, The First Five Minutes&trade;. Performance scores improved in both groups during the second simulation. Mean performance scores of the simulation intervention teams (<i>M</i> = 14.1, <i> SD</i> = 1.43) were significantly higher (<i>t</i> = 4.54, <i> p</i> &lt; .01) than the performance scores of the control teams (<i> M</i> = 10.6, <i>SD</i> = .96). Psychological and physiological measures did not significantly predict performance. Psychological and physiological indicators were reactive to the simulations across time, but did not differ significantly between the control and simulation intervention groups. This investigation explored the multi-dimensional nature of stress (psychological and physiological) that health professions students experience while learning. Simulation intervention did significantly improve group performance, but did not mitigate individual participant stress. Future research should include study with teams of working professionals to determine whether performance and stress measures differ with experience and expertise.</p>

<p>Sonic hedgehog (Shh) is a critical secreted signaling molecule that regulates many aspects of organogenesis. In the absence of Shh, many organs, including the foregut, larynx, palate, cerebellum and heart do not form properly. However, the cellular details of the roles of Shh, including the relevant domains of Shh expression and reception, have not been elucidated for many of these processes. </p><p>The single embryonic foregut tube must divide into the trachea and esophagus, which does not occur in the Shh-null mutant. In Chapter 5, I use Cre-Lox technology to determine that the ventral foregut endoderm is the relevant source of Shh for this process and the mesoderm must directly receive that Shh signal. Surprisingly, this signaling event appears to occur two days before the foregut begins to divide, indicating an early essential role for Shh in foregut division. </p><p>Shh is also expressed at later stages in the maturing trachea and esophagus. In Chapter 6, I demonstrate that these domains serve to establish differentiated mesoderm. In the trachea, Shh from the endoderm signals directly to the mesoderm to form the tracheal cartilage rings. In the esophagus, the roles of Shh are more complex. Shh regulates the size of the esophagus and controls patterning of the concentric rings of esophageal mesoderm, however this process seems to be indirect, requiring autocrine Shh signaling within the esophageal endoderm. </p><p>The laryngeal apparatus is entirely absent in the Shh-null mouse. I n Chapter 3, I dissect the domains of Shh expression and reception required for laryngeal development and demonstrate that loss of endodermal Shh expression causes laryngotracheoesophageal clefts and malformed laryngeal cartilages. As much of laryngeal morphogenesis poorly understood, I also utilize dual mesodermal and neural crest fate maps to determine the embryonic origins of various laryngeal tissues. Finally, as Shh signaling often occurs in concert with Bone Morphogenic Protein (BMP) signaling, I investigate the roles of BMP signaling in laryngeal development. </p><p>Much of Shh signaling occurs at the primary cilium, to which Smoothened, a critical pathway member, must translocate upon Shh signal transduction. This process requires a Smo-Kif3a-&#946;arretin complex in mammalian cell culture. However, the roles of &#946;arrestins in mouse development, and their relationship to Shh signaling have not been investigated in vivo. To do so, in Chapter 4, I analyze the phenotypes of the &#946;arr1/&#946;arr2 double knockout embryos and demonstrate that they have palatal, cerebellar, cardiovascular and renal defects consistent with a specific impairment of mitogenic Shh signaling. </p><p>Altogether, my work dissects the cellular details of Shh signaling during multiple organ systems in the mouse embryo. I further analyze the consequences of absent or misregulated Shh signaling across multiple developmental contexts and determine that Shh plays critical and diverse roles in organogenesis.</p> / Dissertation