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Despite the fundamental role of Hoxa5 in mouse development revealed by the well-characterized phenotypes of Hoxa5 mutant mice, HOXA5-dependent regulatory networks remain ill-defined. We generated a Hoxa5FLAG epitope-tagged mouse line to perform ChIP-seq experiments and uncover genome-wide occupancy of the HOXA5 protein. This was done in the developing lung tissue, in which Hoxa5 plays a predominant role since Hoxa5-/- mouse mutants die at birth from respiratory defects. ChIP-seq allowed us to define an in vivo HOXA5 binding motif and its widespread genome distribution in the embryonic lung. Combined with ATAC-seq assays and epigenetic analyses, HOXA5 targets were identified. They include Hox genes known to show expression changes in lungs from Hoxa5 null mutant embryos. Moreover, several key actors of lung morphogenesis were found to possess HOXA5-binding sites and appeared as potential targets of HOXA5. Impact of the loss of Hoxa5 function on their expression was confirmed by in situ hybridization. These targets include members of the FGF10, SHH, BMP4 and WNT2 signaling pathways. Altogether, these data unveil the crucial role of HOXA5 in the coordinated control of the signaling networks instructing lung development. 

Abstract Hoxa5plays numerous roles in development, but its downstream molecular effects are mostly unknown. We applied bulk RNA-seq assays to characterize the transcriptional impact of the loss ofHoxa5gene function in seven different biological contexts, including developing respiratory and musculoskeletal tissues that present phenotypes inHoxa5mouse mutants. This global analysis revealed few common transcriptional changes, suggesting that HOXA5 acts mainly via the regulation of context-specific effectors. However,Hoxgenes themselves appeared as potentially conserved targets of HOXA5 across tissues. Notably, a trend toward reduced expression ofHoxAgenes was observed inHoxa5null mutants in several tissue contexts. Comparative analysis of epigenetic marks along theHoxAcluster in lung tissue from two differentHoxa5mutant mouse lines revealed limited effect of either mutation indicating thatHoxa5gene targeting did not significantly perturb the chromatin landscape of the surroundingHoxAcluster. Combined with the shared impact of the twoHoxa5mutant alleles on phenotype andHoxexpression, these data argue against the contribution of localciseffects toHoxa5mutant phenotypes and support the notion that the HOXA5 protein acts intransin the control ofHoxgene expression. 

The skeletal system derives from multiple embryonic sources whose derivatives must develop in coordination to produce an integrated whole. In particular, interactions across the lateral somitic frontier, where derivatives of the somites and lateral plate mesoderm come into contact, are important for proper development. Many questions remain about genetic control of this coordination, and embryological information is incomplete for some structures that incorporate the frontier, including the sternum. Hox genes act in both tissues as regulators of skeletal pattern. Here, we used conditional deletion to characterize the tissue-specific contributions of Hoxa5 to skeletal patterning. We found that most aspects of the Hoxa5 skeletal phenotype are attributable to its activity in one or the other tissue, indicating largely additive roles. However, multiple roles are identified at the junction of the T1 ribs and the anterior portion of the sternum, or presternum. The embryology of the presternum has not been well described in mouse. We present a model for presternum development, and show that it arises from multiple, paired LPM-derived primordia. We show evidence that HOXA5 expression marks the embryonic precursor of a recently identified lateral presternum structure that is variably present in therians. 

Brown adipose tissue (BAT) plays critical thermogenic, metabolic and endocrine roles in mammals, and aberrant BAT function is associated with metabolic disorders including obesity and diabetes. The major BAT depots are clustered at the neck and forelimb levels, and arise largely within the dermomyotome of somites, from a common progenitor with skeletal muscle. However, many aspects of BAT embryonic development are not well understood. Hoxa5 patterns other tissues at the cervical and brachial levels, including skeletal, neural and respiratory structures. Here, we show that Hoxa5 also positively regulates BAT development, while negatively regulating formation of epaxial skeletal muscle. HOXA5 protein is expressed in embryonic preadipocytes and adipocytes as early as embryonic day 12.5. Hoxa5 null mutant embryos and rare, surviving adults show subtly reduced iBAT and sBAT formation, as well as aberrant marker expression, lower adipocyte density and altered lipid droplet morphology. Conversely, the epaxial muscles that arise from a common dermomyotome progenitor are expanded in Hoxa5 mutants. Conditional deletion of Hoxa5 with Myf5/Cre can reproduce both BAT and epaxial muscle phenotypes, indicating that HOXA5 is necessary within Myf5- positive cells for proper BAT and epaxial muscle development. However, recombinase-based lineage tracing shows that Hoxa5 does not act cell-autonomously to repress skeletal muscle fate. Interestingly, Hoxa5 -dependent regulation of adipose-associated transcripts is conserved in lung and diaphragm, suggesting a shared molecular role for Hoxa5 in multiple tissues. Together, these findings establish a role for Hoxa5 in embryonic BAT development.