Toward a cell-type-specific lung HOX code.

Background: HOX genes encode transcription factors that are central regulators of cell fate during embryogenesis and of maintaining respective positional cell identity throughout life. Moreover, HOX factors shape the specification of regional properties of the respective tissues, but detailed information regarding the role of HOX genes in the adult human respiratory system is still lacking. Methods: We examined the HOX expression pattern in human lungs using global gene expression analyses of normal lung tissue specimens, followed by quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR). Cell-type specificity was investigated using published single-cell RNA sequencing (scRNAseq) datasets and by immunohistochemistry and immunofluorescence analyses. Results: The expressed HOX candidates HOXA3-HOXA7, HOXB5, HOXB7, and HOXB8, HOXC6-HOXC9, and HOXD1-D8 were localized in healthy lung specimens. Early and central HOXA and HOXB genes were found to be more highly expressed in adult lung tissue, predominantly localizing in stromal cells. While HOXA3 and HOXA5 were primarily localized in sub-bronchial and vascular smooth muscle cells (SMCs), HOXA4, HOXB5, and HOXB7 were predominantly found in vascular mural cells, whereas expression in sub-bronchial regions was more associated with connective tissue cells. HOXA6 was exclusively detected in sub-bronchial and adventitial connective tissue; a similar pattern was observed for HOXA7. HOXA7, HOXB5, and HOXB7, along with HOXD1, were found in the endothelial cells of predominantly larger vessels. Bronchial epithelial cells showed immunoreactivity for almost all the HOX proteins examined, although in some cases, this was limited to single cells. In the alveolar compartment, only HOXA6 (presumably in connective tissue cells), as well as HOXB6, HOXC6, and HOXC8, could be localized. A temporal analysis of the HOX expression development during lung organoid differentiation from induced pluripotent stem cells confirmed respective findings. Conclusion: Since the deregulation of HOX genes is increasingly observed in numerous (malignant) lung diseases, it is a necessary prerequisite to specify individual HOX factors and their cellular origin. This could not only provide better insights into the function of HOX genes in lung diseases but also offer the possibility of improving the regenerative potential through targeted modulation of (cell-type-specific) HOX genes.