Research
Below are selected highlights of my work.
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These studies relied heavily upon mammalian (murine) whole-embryo culture. This culture method utilizes a specialized strategy that adapts to the needs of the growing organism. Gas balance, nutrient uptake, and waste removal must be carefully managed to ensure morphogenesis that mirrors in vivo development.
Molecular differences between the two liver mesenchymes
NGS Sequencing and validation
The liver is patterned by surrounding mesenchyme to form distinct lobes. Previous studies have shown that the liver is divided into rostral and caudal lobes, originating form two distinct progenitor populations, induced by different molecular signals, emanating from seemingly unique mesenchymes. These mesenchymes, Septum Transversum Mesenchyme (STM) and here named Caudal Lobe Mesenchyme (CLM), are derived from different portions of the embryonic mesenchyme and remain distinct throughout early development. To test the hypothesis that the STM and CLM are molecularly distinct, I performed RNA sequencing on dissected livers from early stages of development. The STM and CLM show distinct molecular signatures, which could indicate parallel developmental programs: two ways to make a liver.
A) Livers were isolated from embryonic day (E) 10.0, 10.5, and 11.5, and separated into rostral (blue) and caudal (yellow) lobes, and RNA extracted for sequencing. B) Experimental design: from sequencing data, genes from nearby vascular and cardiac structures were removed, leaving 49 candidate genes to be validated. C) Sequencing data shows separation of rostral vs. caudal populations at E10.0 and 11.5. D) Validation shows distinct molecular markers of STM (blue outline) and CLM (yellow outline).
Mesenchyme migration and differentiation in the embryo
DiI fate mapping
The pancreas is formed from two organ buds: the dorsal and ventral. These buds are induced in the naïve endoderm by surrounding mesenchyme. Using a strategy of DiI labeling, whole-embryo culture, and section analysis, I performed fate-mapping of the early mesenchyme from early formation to differentiation. The dorsal and ventral pancreas buds are surrounded by distinct populations of mesenchymes from different origins that infiltrate shortly after induction. This proximity is maintained from early budding through the first stages of pancreatic differentiation, poising this mesenchyme to be the source of inductive signals.
A) A representative 8 somite embryo, labeled lateral to the to 6th somite with DiI (red) and cultured to the 26 somite stage (B). After culture the embryo was sectioned, immunofluorescence performed for (Isl-1 white, DAPI blue, DiI red). Labeled cells were Isl-1 positive mesenchyme adjacent to and surrounding the dorsal pancreas bud (dp). D -E) Representative 8 somite embryo labeled lateral to the 4th somite and cultures to the 25 somite stage. F-G) Section analysis shows DII (red) in mesenchyme anterior to the dorsal pancreas bud, an in the pancreas tissue demonstrating separation of tissues during development.
Development of a technique
Laser-mediated cell ablation
Excision studies in mammalian development are hampered by the strict physical needs of the viviparous embryo. Not only must strict culture conditions be met, but the outer tissues of the murine embryo are under considerable physical strain from gastrulation to gut-closure stages. A small tear is fatal during this time. To overcome these barriers, I devised a laser-mediated ablation method. This technique can be used to specifically ablate different sized areas of cells with minimal disruption to adjacent cells or the underlying cell matrix. Culture after ablation yields normal gross development through gut closure stages. This targeted approach was used to excise a portion of the notochord, an essential patterning structure, and examine the effects on dorsal pancreas development.
A) An illustration of a transverse section of an embryonic day (E) 8.5 embryo. Neural tube (blue), notochord (green), mesenchyme (orange) and endoderm (red) are drawn to scale. The predicted area of laser penetration (black ellipse) lies within the focal plane of the 20X objective (grey). The maximum temperature is generated at the center of the focal plane and decreases away from the focal plane as indicated (red-orange scale to the right). B) Similar sized areas were pulsed on the endoderm/visceral endoderm border of an E8.5 embryo with durations ranging from 25 to 200 μs as indicated. After treatment the embryo was stained with both propidium iodide (PI): red fluorescence in the nucleus of cells with ruptured membranes, and the nuclear stain Hoechst. C-D’) Whole mount views of an embryo treated with 150 ms pulses targeted to the exposed endoderm before (C-C’) and after (D-D’) addition of trypan blue (TB). E-F) Transverse sections through an embryo treated as above (C-D) then sectioned and subject to immunofluorescence, demonstrates that TB staining (E-F arrowheads) is limited to FOXA1 expressing cells (green, F) on the embryonic surface.