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Sixteen D elements were characterized from the chicken genome, 15 of which are extremely homologous. Early expression of this D repertoire was studied for both DJ and VDJ alleles. No N diversification occurs at either DJ or VD junctions. Only P additions were observed, the length of which does not appear restricted to a dinucleotide. A selection for the almost exclusive usage of the first reading frame of the D elements takes place during B cell expansion in the bursa, in parallel with the selection of productive rearrangements. All three reading frames were observed for the DJ allele at each developmental stage, although some bias for the first reading frame occurs already at the junctional stage. The high incidence of D-D junctions observed (25% among DJ sequences) might represent the major functional contribution of this multigene cluster in a system in which diversity will be generated later on by successive superimposed gene conversions. Other possible functions are discussed. The onset of D diversification through gene conversion between day 15 and day 18 of embryonic development is further documented.

The formation of B lymphoid restricted progenitors was followed during chicken embryonic development by monitoring the appearance of the various Ig gene rearrangements (DJH, VHDJH, VAJX), at a sensitivity that allows the detection of a single rearranged cell. By quantifying the DJH committed progenitor populations, we describe their evolution in different compartments at different developmental stages. The yolk sac is the first site where DJH-positive cells are observed (at days 5-6 of development); via the general circulation, they then seed the various organs while undergoing VHDJH and VXJX rearrangements, which occur simultaneously but lag behind DJH by one to several days. These progenitor populations decline with time in most lymphoid sites and only expand in the bursa. RAG-I expression is observed in the bursa in the absence of ongoing rearrangement activity and thus appears to be an improper marker of rearrangement in the chicken. Commitment to the B cell lineage seems to result from an intrinsic cell program, but the survival and expansion of the committed B progenitors require the specific microenvironment of the bursa.

The thymus harbors an organ-typical dense network of branching and anastomosing blood vessels. To address the molecular basis for morphogenesis of this thymus-specific vascular pattern, we have inactivated a key vascular growth factor, VEGF-A, in thymus epithelial cells (TECs). Both Vegf-A alleles were deleted in TECs by a complementation strategy termed nude mouse [mutated in the transcription factor Foxn1 (forkhead box N1)] blastocyst complementation. Injection of Foxn1 ؉/؉ ES cells into Foxn1 nu/nu blastocysts reconstituted a functional thymus. By dissecting thymus stromal cell subsets, we have defined, in addition to medullary TECs (mTECs) and cortical TECs (cTECs), another prominent stromal cell subset designated cortical mesenchymal cells (cMes). In chimeric thymi, mTECs and cTECs but not cMes were exclusively ES cell-derived. According to this distinct origin, the Vegf-A gene was deleted in mTECs and cTECs, whereas cMes still expressed Vegf-A. This genetic mosaic was associated with hypovascularization and disruption of the organ-typical network of vascular arcades. Thus, vascular growth factor production by TECs is required for normal thymus vascular architecture. These experiments provide insights into Foxn1-dependent and Foxn1-independent stromal cell development and demonstrate the value of this chimeric approach to analyzing gene function in thymus epithelium.mesenchyme ͉ nude mice ͉ thymus development ͉ vascular endothelial growth factor ͉ nude mouse blastocyst complementation