Using the phage P1-derived Cre/loxP recombination system, we have developed a strategy for efficient mammary tissue specific inactivation of floxed genes. Transgenic mice were generated which express Cre DNA-recombinase under the control of the mammary gland specific promoter of the ovine beta-lactoglobulin (BLG) gene. To test the specificity of Cre mediated recombination, we crossed these mice to animals harbouring a floxed DNA ligase I allele. We show that the BLG-Cre construct specifies mammary specific gene deletion, and furthermore that it is temporally regulated, predominantly occurring during lactation. We fully characterised the extent of gene deletion in one line (line 74). In this strain the virgin gland is characterised by low levels (7%) of Cre mediated deletion, whereas 70-80% of cells within the lactating mammary gland have undergone recombination. Immunohistochemistry and indirect in situ PCR were used respectively to demonstrate that both Cre protein and Cre activity were evenly distributed throughout the population of secretory epithelial cells. The level of background recombination in non-mammary tissues was found to be < or = 1.1%, irrespective of mammary gland developmental status. Crossing the transgenic BLG-Cre strain described here to mice harbouring other floxed alleles will facilitate the functional analysis of those genes during differentiation and development of the mammary gland.
While humans have limited potential for limb regeneration, some vertebrates can regenerate bony appendages following amputation. During zebrafish fin regeneration, mature osteoblasts at the amputation stump dedifferentiate and migrate to the blastema, where they re-enter the cell cycle and then re-differentiate to form new bone. Osteoblastic cells exhibit dual mesenchymal and epithelial characteristics during fin regeneration, however little is known about why or how this occurs. Using single-cell RNA-sequencing, we found osteoprogenitors are enriched with components associated with the epithelial-to-mesenchymal transition (EMT) and its reverse, mesenchymal-to-epithelial transition (MET). In trajectory analyses, osteoblastic cells solely expressed EMT components, or transiently expressed MET components prior to expressing those for EMT. We found that cdh11, a cancer EMT marker, is expressed during osteoblast dedifferentiation. We also found that esrp1, a regulator of alternative splicing in epithelial cells whose expression is important for MET, is expressed in a subset of osteoprogenitors during outgrowth. This study provides a valuable single cell resource for the study of osteoblast differentiation during zebrafish fin regeneration, and identifies MET- and EMT-associated components which may be important for successful appendage regeneration.
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