Stable iPSC-derived NKX2-1+ lung bud tip progenitor organoids give rise to airway and alveolar cell types.

Bud tip progenitors (BTPs) in the developing lung give rise to all epithelial cell types found in the airways and alveoli. This work aimed to develop an iPSC organoid model enriched with NKX2-1+ BTP-like cells. Building on previous studies, we optimized a directed differentiation paradigm to generate spheroids with more robust NKX2-1 expression. Spheroids were expanded into organoids that possessed NKX2-1+/CPM+ BTP-like cells, which increased in number over time. Single cell RNA-sequencing analysis revealed a high degree of transcriptional similarity between induced BTPs (iBTPs) and in vivo BTPs. Using FACS, iBTPs were purified and expanded as induced bud tip progenitor organoids (iBTOs), which maintained an enriched population of bud tip progenitors. When iBTOs were directed to differentiate into airway or alveolar cell types using well-established methods, they gave rise to organoids composed of organized airway or alveolar epithelium, respectively. Collectively, iBTOs are transcriptionally and functionally similar to in vivo BTPs, providing an important model for studying human lung development and differentiation.

The master transcription factor SOX2, mutated in anophthalmia/microphthalmia, is post-transcriptionally regulated by the conserved RNA-binding protein RBM24 in vertebrate eye development.

Mutations in the key transcription factor, SOX2, alone account for 20% of anophthalmia (no eye) and microphthalmia (small eye) birth defects in humans-yet its regulation is not well understood, especially on the post-transcription level. We report the unprecedented finding that the conserved RNA-binding motif protein, RBM24, positively controls Sox2 mRNA stability and is necessary for optimal SOX2 mRNA and protein levels in development, perturbation of which causes ocular defects, including microphthalmia and anophthalmia. RNA immunoprecipitation assay indicates that RBM24 protein interacts with Sox2 mRNA in mouse embryonic eye tissue. and electrophoretic mobility shift assay shows that RBM24 directly binds to the Sox2 mRNA 3'UTR, which is dependent on AU-rich elements (ARE) present in the Sox2 mRNA 3'UTR. Further, we demonstrate that Sox2 3'UTR AREs are necessary for RBM24-based elevation of Sox2 mRNA half-life. We find that this novel RBM24-Sox2 regulatory module is essential for early eye development in vertebrates. We show that Rbm24-targeted deletion using a constitutive CMV-driven Cre in mouse, and rbm24a-CRISPR/Cas9-targeted mutation or morpholino knockdown in zebrafish, results in Sox2 downregulation and causes the developmental defects anophthalmia or microphthalmia, similar to human SOX2-deficiency defects. We further show that Rbm24 deficiency leads to apoptotic defects in mouse ocular tissue and downregulation of eye development markers Lhx2, Pax6, Jag1, E-cadherin and gamma-crystallins. These data highlight the exquisite specificity that conserved RNA-binding proteins like RBM24 mediate in the post-transcriptional control of key transcription factors, namely, SOX2, associated with organogenesis and human developmental defects.

The Nkd EF-hand domain modulates divergent wnt signaling outputs in zebrafish.

Wnt proteins regulate diverse biological responses by initiating two general outcomes: ß-catenin-dependent transcription and ß-catenin-independent activation of signaling cascades, the latter including modulation of calcium and regulation of cytoskeletal dynamics (Planar Cell Polarity, PCP). It has been difficult to elucidate the mechanisms by which Wnt signals are directed to effect one or the other outcome due to shared signaling proteins between the ß-catenin-dependent and -independent pathways, such as the Dishevelled binding protein Naked. While all Naked paralogs contain a putative calcium-binding domain, the EF-Hand, Drosophila Naked does not bind calcium. Here we find a lineage-specific evolutionary change within the Drosophila Naked EF-hand that is not shared with other insects or vertebrates. We demonstrate the necessary role of the EF-hand for Nkd localization changes in calcium fluxing cells and using in vivo assays, we identify a role for the zebrafish Naked EF-hand in PCP but not in ß-catenin antagonism. In contrast, Drosophila-like Nkd does not function in PCP, but is a robust antagonist of Wnt/ß-catenin signaling. This work reveals that the zebrafish Nkd1 EF-hand is essential to balance Wnt signaling inputs and modulate the appropriate outputs, while the Drosophila-like EF-Hand primarily functions in ß-catenin signaling.

Nascent matrix deposition supports alveolar organoid formation from aggregates in synthetic hydrogels.

Human induced pluripotent stem cell (iPSC) derived alveolar organoids have emerged as a system to model the alveolar epithelium in homeostasis and disease. However, alveolar organoids are typically grown in Matrigel, a mouse-sarcoma derived basement membrane matrix that offers poor control over matrix properties, prompting the development of synthetic hydrogels as a Matrigel alternative. Here, we develop a two-step culture method that involves pre-aggregation of organoids in hydrogel-based microwells followed by embedding in a synthetic hydrogel that supports alveolar organoid growth, while also offering considerable control over organoid and hydrogel properties. We find that the aggregated organoids secrete their own nascent extracellular matrix (ECM) both in the microwells and upon embedding in the synthetic hydrogels. Thus, the synthetic gels described here allow us to de-couple exogenous and nascent ECM in order to interrogate the role of ECM in organoid formation.

Functional Role of the RNA-Binding Protein Rbm24a and Its Target sox2 in Microphthalmia.

Congenital eye defects represent a large class of disorders affecting roughly 21 million children worldwide. Microphthalmia and anophthalmia are relatively common congenital defects, with approximately 20% of human cases caused by mutations in SOX2. Recently, we identified the RNA-binding motif protein 24a (Rbm24a) which binds to and regulates sox2 in zebrafish and mice. Here we show that morpholino knockdown of rbm24a leads to microphthalmia and visual impairment. By utilizing sequential injections, we demonstrate that addition of exogenous sox2 RNA to rbm24a-deplete embryos is sufficient to suppress morphological and visual defects. This research demonstrates a critical role for understanding the post-transcriptional regulation of genes needed for development.

A High-Throughput Assay for Congenital and Age-Related Eye Diseases in Zebrafish.

Debilitating visual impairment caused by cataracts or microphthalmia is estimated to affect roughly 20 million people in the United States alone. According to the National Eye Institute, by 2050 that number is expected to more than double to roughly 50 million. The identification of candidate disease-causing alleles for cataracts and microphthalmia has been accelerated with advanced sequencing technologies creating a need for verification of the pathophysiology of these genes. Zebrafish pose many advantages as a high-throughput model for human eye disease. By 5 days post-fertilization, zebrafish have quantifiable behavioral responses to visual stimuli. Their small size, many progeny, and external fertilization allows for rapid screening for vision defects. We have adapted the OptoMotor Response to assay visual impairment in zebrafish models of cataracts and microphthalmia. This research demonstrates an inexpensive, high-throughput method for analyzing candidate genes involved in visual impairment.