Sea urchins are virtuosi of biomineralization, the process by which organisms build mineralized tissues. The embryonic animal exemplifies this with the formation of its endoskeletal spicule. The primary mesenchyme cells (PMCs) undertake spicule synthesis, which involves deposition of the initial granule, elongation of the spicule, and several choreographed changes in crystallographic direction. The final spicule is smooth and curved single crystalline magnesium calcite (Ca0.95Mg0.05CO3) that also comprises 0.1% embedded organic matrix. Although early PMC morphogenesis has been studied in detail, the precise mechanisms through which the PMCs control spiculogenic events such as granule nucleation, spicule growth, and crystallographic direction switching, are not well-understood. Remarkably, PMC construction of the skeleton is largely autonomous, so PMCs cultured in vitro still form spicules. In this dissertation, we leverage in vitro PMC culture to dissect the molecular toolkit effecting this complex process of biogenic single crystal growth. We first build upon and further develop a technique for isolation and culture of PMCs, compatibilizing it with powerful characterization methods such as confocal and electron microscopy. Using in vitro culture, pharmacological inhibitors, and fluorescence microscopy, we then examine indirect control of spicule formation through the signaling molecule VEGF and direct control through the cytoskeleton. We find that the temporal distribution of VEGF is a control lever for crystal growth. We also find that the cytoskeleton is essential to spicule formation by enabling macropinocytosis-driven uptake of ions into the mineral. In another set of experiments, we leverage immunofluorescence to examine the spatial distribution of organic matrix proteins within the spicule. We found a heterogeneous distribution of two key spicule matrix proteins, indicating they have different functions in guiding crystal growth. Finally, in a collaborative project, we use X-ray diffraction and electron microscopy to examine the shell of the gastropod species C. fornicata and find that it comprises aragonitic crossed-lamellar microstructure. Our results expand upon theories for spiculogenesis. The formation of the sea urchin spicule is a model system for studying biomineralization, and we thus expect that the results herein will elucidate analogous processes in other organisms and in materials synthesis.

Creator

• Moreno, Bradley Keck

DOI

• https://doi.org/10.21985/n2-cbw2-jx24

Subject

• Materials Science

Language

• en

Alternate Identifier

• http://dissertations.umi.com/northwestern:16210
• etdadmin_upload_926575

Keyword

• Biomineralization
• Spicule
• Sea urchins
• Calcite

Date created

• 2022-01-01

Resource type

• Dissertation

Rights statement

• In Copyright