Additional lines created; use of a high-throughput embryo sorter allowed screening of very large numbers of animals for GFP-expressing protein trap insertions.
A set of transgenic insertion stocks derived by TE mobilization using the protein-trap constructs P{PTT-GA}, P{PTT-GB}, and P{PTT-GC}. The constructs carry a w+mC mini-white visible marker and an Avic\GFP vital fluorescent protein-trap marker. In each of the three constructs, the splice acceptor and splice donor consensus sequences are in a different reading frame relative to the Avic\GFP sequence. For a successful GFP-positive insertion into an intron of a protein-coding gene, translation results in a fusion of the GFP to both the amino- and carboxyl-terminal parts of the trapped protein.
trol reporter expression becomes visible around cardioblasts at embryonic stage 14. By stage 16, expression also surrounds pericardial nephrocytes and the surface of the lymph gland. Shortly after larval hatching, expression is observed covering each individual hematopoietic progenitor of the lymph gland. Expression is particularly strong around cells of the posterior signalling center. By the second instar stage, trol expression forms a solid layer around the surface of the primary and secondary lobes of the lymph gland. Within the lobes, expression is seen strongly around clusters of cells. The trol reporter pattern outlines a system of chambers that each contain small clusters of hematopoietic progenitors or differentiated hemocytes. Basement membrane components collagen and laminin are co-exexpressed with trol in the lymph gland. The structures containing trol reporter are revealed as basement membrane by electron microscopy.
GFP-tagged trol protein encoded by AvicGFPtrol-ZCL1973 is first detected in the basement membrane surrounding cardioblasts at embryonic stage 14. By embryonic stage 16, tagged trol protein is associated with the basement membranes of pericardial nephrocytes, and at the surface of the embryonic/larval lymph gland. In newly hatched larvae, tagged trol protein surrounds hematopoietic progenitors of the lymph gland; expression is pronounced in cells of the posterior signalling center of the lymph gland primary lobe. In second and third instar larvae, tagged trol protein is concentrated in the medullary zone and expression is decreased in the cortical zone of lymph glands.
