Identification of an Intronic Regulatory Element Necessary for Tissue-Specific Expression of Foxn1 in Thymic Epithelial Cells.

The thymus is critical for the establishment of the adaptive immune system and the development of a diverse T cell repertoire. T cell development depends upon cell-cell interactions with epithelial cells in the thymus. The thymus is composed of two different types of epithelial cells: cortical and medullary epithelial cells. Both of these express and critically depend on the transcription factor Foxn1 Foxn1 is also expressed in the hair follicle, and disruption of Foxn1 function in mice results in severe thymic developmental defects and the hairless (nude) phenotype. Despite its importance, little is known about the direct regulation of Foxn1 expression. In this study, we identify a cis-regulatory element (RE) critical for expression of Foxn1 in mouse thymic epithelial cells but dispensable for expression in hair follicles. Analysis of chromatin accessibility, histone modifications, and sequence conservation identified regions within the first intron of Foxn1 that possessed the characteristics of REs. Systematic knockout of candidate regions lead us to identify a 1.6 kb region that, when deleted, results in a near total disruption of thymus development. Interestingly, Foxn1 expression and function in the hair follicle were unaffected. RNA fluorescent in situ hybridization showed a near complete loss of Foxn1 mRNA expression in the embryonic thymic bud. Our studies have identified a genomic RE with thymic-specific control of Foxn1 gene expression.

Mesenchymal Hox6 function is required for mouse pancreatic endocrine cell differentiation.

Despite significant advances in our understanding of pancreatic endocrine cell development, the function of the pancreatic mesodermal niche in this process is poorly understood. Here we report a novel role for mouse Hox6 genes in pancreatic organogenesis. Hox6 genes are expressed exclusively in the mesoderm of the developing pancreas. Genetic loss of all three Hox6 paralogs (Hoxa6, Hoxb6 and Hoxc6) leads to a dramatic loss of endoderm-derived endocrine cells, including insulin-secreting ß-cells, and to mild delays and disruptions in pancreatic branching and exocrine differentiation. Ngn3-expressing pan-endocrine progenitor cells are specified normally in Hox6 mutant pancreata, but fail to mature into hormone-producing cells. Reduced expression of Wnt5a is observed in mutant pancreatic mesenchyme, leading to subsequent loss of expression of the crucial Wnt inhibitors Sfrp3 and Dkk1 in endocrine progenitor cells. These results reveal a key role for Hox6 genes in establishing Wnt mesenchymal-epithelial crosstalk in pancreatic development.

Protocol for drug screening of patient-derived tumor organoids using high-content fluorescent imaging.

High-content imaging of tumor organoids (TOs) treated with therapeutic agents provides detailed cell viability readouts at the organoid level. In contrast, most used protocols provide one number per well. While requiring the use of inverted microscopy with an automated stage, this protocol can provide critical information about heterogeneous responses of TOs to various treatments. This protocol describes a technique for culturing and drug testing TOs using fluorescent indicators of cell viability with high reproducibility. For complete details on the use and execution of this protocol, please refer to Larsen et al. (2021).

A pan-cancer organoid platform for precision medicine.

Patient-derived tumor organoids (TOs) are emerging as high-fidelity models to study cancer biology and develop novel precision medicine therapeutics. However, utilizing TOs for systems-biology-based approaches has been limited by a lack of scalable and reproducible methods to develop and profile these models. We describe a robust pan-cancer TO platform with chemically defined media optimized on cultures acquired from over 1,000 patients. Crucially, we demonstrate tumor genetic and transcriptomic concordance utilizing this approach and further optimize defined minimal media for organoid initiation and propagation. Additionally, we demonstrate a neural-network-based high-throughput approach for label-free, light-microscopy-based drug assays capable of predicting patient-specific heterogeneity in drug responses with applicability across solid cancers. The pan-cancer platform, molecular data, and neural-network-based drug assay serve as resources to accelerate the broad implementation of organoid models in precision medicine research and personalized therapeutic profiling programs.

Hox6 genes modulate in vitro differentiation of mESCs to insulin-producing cells.

The differentiation of glucose-responsive, insulin-producing cells from ESCs in vitro is promising as a cellular therapy for the treatment of diabetes, a devastating and common disease. Pancreatic ß-cells are derived from the endoderm in vivo and therefore most current protocols attempt to generate a pure population of first endoderm, then pancreas epithelium, and finally insulin-producing cells. Despite this, differentiation protocols result in mixed populations of cells that are often poorly defined, but also contain mesoderm. Using an in vitro mESC-to-ß cell differentiation protocol, we show that expression of region-specific Hox genes is induced. We also show that the loss of function of the Hox6 paralogous group, genes expressed only in the mesenchyme of the pancreas (not epithelium), affect the differentiation of insulin-producing cells in vitro. This work is consistent with the important role for these mesoderm-specific factors in vivo and highlights contribution of supporting mesenchymal cells in in vitro differentiation.

Hox5 Genes Regulate the Wnt2/2b-Bmp4-Signaling Axis during Lung Development.

Hox genes are required for proper anteroposterior axial patterning and the development of several organ systems. Here, we show that all three Hox5 paralogous genes play redundant roles in the developing lung. Hoxa5;Hoxb5;Hoxc5 triple-mutant embryos develop severely hypoplastic lungs with reduced branching and proximal-distal patterning defects. Hox5 genes are exclusively expressed in the lung mesoderm; however, defects are observed in both lung mesenchyme and endodermally derived epithelium, demonstrating that Hox5 genes act to regulate mesodermal-epithelial crosstalk during development. We show that Hox5 loss of function leads to loss of Wnt2/2b expression in the distal lung mesenchyme and the downregulation of previously identified downstream targets of Wnt2/2b signaling, including Lef1, Axin2, and Bmp4. Wnt2/2b-enriched media rescue proper Sox2/Sox9 patterning and restore Bmp4 expression in Hox5 triple-mutant lung explants. Taken together, these data show that Hox5 genes are key upstream mesenchymal regulators of the Wnt2/2b-Bmp4-signaling axis critical for proper lung patterning.