Homeobox genes are regulators of placedependent morphogenesis and play important roles in controlling the expression patterns of cell adhesion molecules (CAMs). To identify proteins that bind to a regulatory element common to the genes for two neural CAMs, Ng-CAM and L1, we screened a mouse cDNA expression library with a concatamer of the sequence CCATTAGPyGA and found a new homeobox gene, which we have called Barx2. The homeodomain encoded by Barx2 is 87% identical to that of Barx1, and both genes are related to genes at the Bar locus of Drosophila melanogaster. Barx1 and Barx2 also encode an identical stretch of 17 residues downstream of the homeobox; otherwise, they share no appreciable homology. In vitro, Barx2 stimulated activity of an L1 promoter construct containing the CCATTAGPyGA motif but repressed activity when this sequence was deleted. Localization studies showed that expression of Barx1 and Barx2 overlap in the nervous system, particularly in the telencephalon, spinal cord, and dorsal root ganglia. Barx2 was also prominently expressed in the f loor plate and in Rathke's pouch. During craniofacial development, Barx1 and Barx2 showed complementary patterns of expression: whereas Barx1 appeared in the mesenchyme of the mandibular and maxillary processes, Barx2 was observed in the ectodermal lining of these tissues. Intense expression of Barx2 was observed in small groups of cells undergoing tissue remodeling, such as ectodermal cells within indentations surrounding the eye and maxillo-nasal groove and in the first branchial pouch, lung buds, precartilagenous condensations, and mesenchyme of the limb. The localization data, combined with Barx2's dual function as activator and repressor, suggest that Barx2 may differentially control the expression of L1 and other target genes during embryonic development.

Bovine mammary tissue was collected by surgical biopsy at intervals during involution for histological and ultrastructural observation. In lactating tissue (d 0 of involution, collected 8 h after the final milking), alveolar epithelial cells had marked ultrastructural evidence of lactation, including protein-containing secretory vesicles, lipid droplets, extensive rough endoplasmic reticulum, and numerous mitochondria. By d 2 of involution, alveolar epithelial cells contained large vacuoles apparently formed by coalescing of protein-containing secretory vesicles and lipid droplets. Large vacuoles were observed in epithelial cells until about the 3rd wk of involution. By d 2 of involution, the Golgi apparatus generally was not apparent. Rough endoplasmic reticulum and mitochondria were observed throughout the period studied, although in reduced amounts compared with their presence in lactating tissue. A marked increase in lysosomal or cytosegresomal structures in epithelial cells was not observed. There was no evidence of extensive sloughing of epithelial cells from the basement membrane. There was a progressive increase in the interalveolar area and a concurrent decrease in the alveolar luminal area as involution progressed. Ultrastructural examination showed that alveolar epithelial cells at d 21 and 30 of involution appear to be functionally active but not secreting milk components.

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Scores of homeobox gene-encoded transcription factors are expressed in a definite spatiotemporal pattern during embryogenesis and regulate a series of as yet unidentified target genes to help coordinate the morphogenetic process. We have suggested that homeobox gene products modulate the expression of adhesion molecule genes and have shown in cotransfection experiments that the promoters for the neural cell adhesion molecule (N-CAM) and cytotactin/tenascin genes respond to cues from different homeobox-containing genes. In this study, we show that the HoxC6 (Hox-3.3)-encoded homeoprotein binds to a DNA sequence in the N-CAM promoter CCTAATTATTAA, designated homeodomain binding site I (HBS-I). To test whether HoxC6 regulated N-CAM promoter activity, we cotransfected the Long and Short reading frame variants of Xenopus HoxC6 (CMV-HoxC6-L and CMV-HoxC6-S) driven by the human cytomegalovirus (CMV) promoter together with a chloramphenicol acetyltransferase (CAT) reporter gene driven by the mouse N-CAM promoter (N-CAM-Pro-CAT). Cotransfection of NIH 3T3 cells with either of the CMV-HoxC6 expression vectors stimulated N-CAM promoter-driven CAT expression. A 47-bp region from the N-CAM promoter that included HBS-I and an adjacent potential HBS, HBS-II, conferred HoxC6 regulation on a simian virus 40 minimal promoter. HBS-I was sufficient for transactivation of the minimal promoter by CMV-HoxC6-S. However, transcriptional activation by CMV-HoxC6-L required both HBS-I and HBS-II, inasmuch as mutation of either HBS-I, HBS-II, or both motifs abolished the response. These studies suggest that HBS-I is a target site for binding and transcriptional control of the N-CAM promoter by homeoproteins, although accessory DNA sequences (such as HBS-II) may also be required. Together with previous studies, these results support the notion that N-CAM gene expression may be controlled by different combinations of homeoproteins that appear in a place-dependent manner during embryogenesis.