The development of multicellular tissues involves the process of patterning, wherein pluripotent cells adapt distinct fates, often with specialized functions in response to spatial and temporal signaling activities. Concomitantly, in a process called morphogenesis, cell and tissue-scale shape changes lead tissues into particular forms for proper functionality. The establishment of many complex biological structures, including the early embryonic body plan, depends on the precise coordination and interplay of these processes. The goal of this thesis is to explore how mechanisms of tissue patterning influence shape changes, and shape changes -or resulting physical alterations of tissues- in turn influence patterning during early stages of vertebrate embryonic development. To this end I use two model systems: (1) the annual killifish Nothobranchius furzeri, and human embryonic stem cell (hESC)-based in vitro models of human gastrulation. In the first chapter, we investigate the initiation of gastrulation in N. furzeri, where the unusual, seemingly disorganized pre-gastrulation movements of embryonic cells challenge conventional pre-prepatterned models of axis specification. We find that the maternally deposited dorsal organizing factor huluwa is lost in this animal, necessitating alternative strategies for axis specification. Further, we determine a novel early role for Nodal signaling in initiating axis specification by coordinating cell movements during the re-aggregation morphogenesis of the embryonic cells. These results reveal a surprising divergence from classical mechanisms of axis specification, with implications for the emergence of self-organized strategies. In the second chapter, I investigate the role of tissue-scale physical properties on the mesendodermal patterning of hESC colonies iii Thesis advisor: Professor Sharad Ramanathan Deniz Cihat Aksel downstream TGF-β and Wnt signals. By culturing and differentiating hESC colonies on tunable polyacrylamide (PAAM) and polydimethylsiloxane (PDMS) substrates, and assaying spatial expression of the mesendoderm marker Brachyury, I demonstrate that substrate stiffness does not modulate the efficacy or spatial extent of mesendodermal differentiation, challenging previous reports. Instead, I identify substrate porosity to be a key parameter regulating the spatial span of TGF-β signaling and downstream differentiation. Finally, I present preliminary results implicating protein-surface interactions in regulating the efficacy of Wnt3a-driven differentiation of hESCs. These results lead to an improved understanding of the physical parameters that do and do not influence signaling in hESCs as they differentiate towards mesendoderm. In the third chapter, I present a hESC-based toolbox for the spatiotemporal measurement and manipulation of FGF activity in vitro. I generate and validate a 4-color transgenic line for simultaneous cell tracking and measurement of the 3 intracellular cascades downstream FGF: MAPK/Erk, PI3K/Akt, and PLC-γ/Ca2+. Using this line, I observe distinct intracellular activity patterns downstream different FGF ligands. Additionally, I present lines for optogenetic control of FGF signaling, demonstrating their utility in generating diverse intracellular Erk dynamics. I discuss future applications for these tools in illuminating signal processing in the FGF pathway, as well as disentangling the roles of FGF signaling in patterning and morphogenesis during human gastrulation. Collectively, these results provide new insights as well as valuable tools for understanding the interplay of patterning and morphogenesis during early vertebrate development.