Browsing by Author "Klingensmith, John"

The means by which cells, tissues, and organisms undergo morphogenesis are variable and highly regulated, and the mechanisms that govern cellular changes in response to signaling cues are poorly understood. This study seeks to address the role of a newly characterized protein in zebrafish in translating signaling cues into physical changes within a cell.

The Cordon–bleu (Cobl) gene is widely conserved in vertebrates, with developmentally regulated axial and epithelial expression in mouse and chick embryos. In vitro, Cobl can bind monomeric actin and nucleate formation of unbranched actin filaments, while in cultured cells it can modulate the actin cytoskeleton. However, an essential role for Cobl in vivo has yet to be determined. We have identified the zebrafish cobl ortholog and have used zebrafish as a model to assess the requirements for Cobl in embryogenesis. We find that cobl shows enriched expression in ciliated epithelial tissues during zebrafish organogenesis. The utilization of antibodies developed against Cobl shows that the protein is concentrated along the apical domain of ciliated cells, in close proximity to the apical actin cap.

Reduction of cobl by antisense morpholinos reveals an essential role in embryonic morphogenesis and organ development. A requirement for Cobl was shown for the proper function of various and ciliated epithelial organs. Cobl appears to direct the elongation of motile cilia in organs such as Kupffer’s vesicle and the pronephros. In Kupffer’s vesicle, the reduction in Cobl coincides with a reduction in the amount of apical F-actin. Additionally, Cobl may play a role during gastrulation cell movements and convergence and extension morphogenesis during early embryonic development. Thus, Cobl may represent a molecular activity that couples developmental patterning signals with local intracellular cytoskeletal dynamics to support elongation of motile cilia and tissue morphogenesis.

The actin cytoskeleton is a fundamental component of the cell and is involved in many processes, including cell division, cell migration, vesicle trafficking and cell polarity. The actin cytoskeleton has a very important role in embryogenesis as the cells within developing tissues proliferate, migrate, interpret extracellular cues, and shape complex tissues. The molecules that help the cell to interpret their environment and turn those cues into morphological changes are of great interest. One protein which may be involved in this manner is Cordon-bleu (Cobl).

In mouse embryos, Cobl's expression pattern resembles that of important developmental genes, is restricted to distinct domains, and changes dynamically throughout development as tissues are formed. While it is known that Cobl expression is regulated by developmental signaling pathways such as Shh and BMP, its molecular function at the cellular level remains elusive. In this study, we have identified molecular functions of Cobl. Cobl has C-terminal Wasp Homology-2 (WH2) domains which bind actin and nucleate new actin filaments in in vitro polymerization assays. Using cultured cells, we have determined that Cobl is involved in cytoskeletal remodeling during neurite branching and epithelial cell migration. We also demonstrate that Cobl interacts with the Syndapin family of adaptor proteins that link endocytosis and vesicle trafficking. Cobl colocalizes with Sdp2 in cultured epithelial cells and similarly localizes with Sdp1 and Sdp2 in developing mouse embryos. The localization of Cobl or Sdp2 in cultured epithelial cells is dependent on the other, as demonstrated using shRNA knockdown.

Previous studies demonstrated that a hypomorphic allele of Cobl interacts genetically with Looptail, in midbrain neurulation. Looptail mutants are deficient in the gene Vangl2, a member of the planar cell polarity pathway that coordinates the morphogenesis of a sheet of cells. To discover other roles for Cobl in the developing mouse, we have generated a conditionally null allele of Cobl. We find that outbred Cobl homozygous mutants are viable, but that they have inner ear defects. Together, our studies demonstrate that Cobl is a tissue-specific actin nucleator whose localization is regulated by its interaction with Syndapins and which functions in the development of sensory epithelia.

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.

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.

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.

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.

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-βarretin complex in mammalian cell culture. However, the roles of βarrestins 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 βarr1/βarr2 double knockout embryos and demonstrate that they have palatal, cerebellar, cardiovascular and renal defects consistent with a specific impairment of mitogenic Shh signaling.

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.

In the past two decades there has been a significant interest in the ethical and legal concerns surrounding the development of Lethal Autonomous Weapon Systems (LAWS). These arguments are principally based on a killer robot conception of future autonomy - that is that futuristic LAWS are capable of near-human like moral reasoning and judgement. My argument is twofold – first, I argue that this conception of future weapon autonomy might be flawed, and future LAWS technology might be better understood as existing on a continuum of weapons autonomy. Machine guns, precision guided missiles, remote piloted drones, and the Aegis Combat System, among others, represent this continuum. While successive technological development might introduce greater levels of autonomy to the battlefield, critical human functions of moral reasoning and judgement are still a necessary and critical component of their design. In this framework, LAWS can be reduced to just another weapon ultimately put to use by human soldiers and commanders. Understood in this way it becomes difficult for critical arguments to hold much force as they must define at what point on this continuum that autonomy meaningfully represents a paradigm shift in warfare to the point that it violates just war norms and international law. Second, I will argue that even if we do assume a killer robot level of autonomy, the utilitarian arguments against LAWS might still be flawed. The arguments rely on the notion that a LAWS could potentially make a mistake and cause unnecessary harm on the battlefield. This notion is flawed because it fails to articulate how that is meaningfully different than a battlefield characterized by human warfare. Humans are also subject to mistakes and error in judgement that could result in unnecessary harm. With that in mind, I will specifically address four of the most prominent utilitarian arguments against LAWS – responsibility gap, proportionality, distinction, and the idea of ‘riskless warfare’.

Abstract

During embryonic development many structures crucial for breathing and eating arise from the pharyngeal and anterior foregut epithelium (FGE), which contains the oral ectoderm and the foregut endoderm. Proper differentiation and signaling within and from this epithelial tissue is necessary for the development of the mandible, the esophagus, and the trachea. Many birth defects occur in these structures that greatly disrupt the ability of affected infants to breathe and eat. This dissertation investigates the importance of the pharyngeal and anterior FGE in mandible and foregut development.

The most rostral portion of the pharyngeal epithelium contributes to the development of the mandible. At embryonic day 10.5 the mandible is a bud structure, composed of neural crest-derived mesenchyme and core mesoderm surrounded by pharyngeal epithelium. The mesenchyme needs to receive Hedgehog signaling for mandible development, but the epithelial tissue that signals to the mesenchyme has not been identified in mammals. Data presented in Chapter 2 show that Sonic Hedgehog is necessary at two distinct stages of mandible development by using a tissue specific genetic ablation to remove Sonic Hedgehog from the pharyngeal endoderm. First, we show that Sonic Hedgehog promotes cell survival prior to cartilage differentiation through immunostaining for Caspase-9, an apoptosis marker. Second, a rescue of early cell death with the p53 inhibitor pifithrin-α shows that Sonic Hedgehog is necessary for cartilage condensation and differentiation later in development. Without cartilage differentiation the mandible is unable to elongate properly and hypoplasia occurs.

Caudal to the pharyngeal epithelium is the anterior FGE, which develops into the larynx, esophagus and trachea. The anterior FGE is a single endodermal tube at E9.5 and by E11.5 compartmentalizes into two distinct tubes: the esophagus and trachea. While the signaling pathways involved in proper compartmentalization of the foregut are well studied, nothing is known about the cellular behaviors that drive this complex event. One important event during foregut compartmentalization is the establishment of dorso-ventral patterning, which is necessary for separation to occur. To elucidate the importance of dorso-ventral patterning, we take advantage of two genetic mouse models with disrupted patterning, an activation of and a removal of β-catenin in the ventral foregut endoderm. Data presented in Chapter 3 show that β-catenin is important for epithelial pseudostratification and the establishment of a region of double-positive cells at the dorso-ventral midline through close examination of epithelial morphogenesis at E10.5 prior to compartmentalization. This data has established two mouse models for studying changes in epithelial morphology during foregut compartmentalization. In total, this body of work details how signals originating in the pharyngeal and anterior foregut epithelium regulate both mesenchymal and epithelial behaviors during mandible and foregut development.

Proper morphogenesis is essential for both form and function of mammalian craniofacial and neural tube development. Craniofacial deformities and neural tube defects are highly prevalent human birth defects. Although studies concerning craniofacial and neural tube development have revealed important genetic and/or environmental factors, understanding the mechanisms underlying proper development and the defects remain incomplete.

Among many genes that were cloned as the gastrula organizer genes in 1990s, Nog, a secreted BMP antagonist, is expressed in the relevant domains during craniofacial and neural tube development. Previous studies show that Nog null embryos exhibit fully penetrant spina bifida (open spine) and to the lesser extent exencephaly (open brain). Moreover, Nog null mice display deformities in skeletal structures including defects in craniofacial skeleton. As such, Nog is essential for proper neural tube and craniofacial development. However, it is still not clear that which domain(s) of Nog are responsible for proper craniofacial development or neural tube closure. In addition, it is also an important question when, and in what capacity Nog is necessary during development of craniofacial and neural tube.