Francisca Ferreira Vasconcelos

Adaptation to breathing is a critical step in lung function and it is crucial for organismal survival. Alveoli are the lung gas exchange units and their development, from late embryonic to early postnatal stages, requires feedbacks between multiple cell types. However, how the crosstalk between the alveolar cell types is modulated to anticipate lung adaptation to breathing is still unclear. Here, we uncovered a synchronous alternative splicing switch in multiple genes in the developing mouse lungs at the transition to birth, and we identified hnRNP A1, Cpeb4, and Elavl2/HuB as putative splicing regulators of this transition. Notably, we found thatVegfaswitches from theVegfa164 isoform to the longerVegfa188 isoform exclusively in lung alveolar epithelial AT1 cells. Functional analysis revealed that VEGFA 188 (and not VEGFA 164) drives the specification of Car4-positive aerocytes, a subtype of alveolar endothelial cells specialized in gas exchanges. Our results reveal that the cell type–specific regulation ofVegfaalternative splicing just before birth modulates the epithelial-endothelial crosstalk in the developing alveoli to promote lung adaptation to breathing.

Gas exchanges are ensured by lung alveoli, which are mainly composed by epithelial alveolar type 1 (AT1), alveolar type 2 (AT2) and capillary endothelial cells (ECs). Alveologenesis starts during late embryonic development and continues after birth and relies on extensive biochemical crosstalk between these cell types. How this crosstalk is modulated to anticipate and accommodate the radical changes occurring at birth is still unclear. We investigated the alternative splicing (AS) changes occurring during lung development at the embryonic to postnatal transition by performing RNAseq of mouse lungs at distinct developmental stages. We found that most of the AS changes occur at the embryonic to postnatal transition. In addition, we identified hnRNP A1, Cpeb4 and Elavl2/HuB as putative splicing regulators of this transition. We show that the AS of a major pro- angiogenic chemokine, vascular endothelial growth factor A (VEGFA), is differentially regulated at this transition. Remarkably, we found that there is a switch from the predominance of Vegfa 164 to Vegfa 188 just before birth specifically in AT1 cells, whilst in other cell populations Vegfa does not undergo AS changes. Moreover, we identified a novel Vegfa isoform generated by the retention of intron 5, Vegfa i5 . Our results reveal a cell type-specific regulation of Vegfa AS that may constitute a pre- birth adaptation mechanism of the epithelial-endothelial crosstalk, which may be fundamental for the adaptation to breathing and may have implications for pathological conditions.

Significance: In this report, we describe how endothelial cells, the cells lining the interior of blood vessels, invade into tissues to form new vessels through sprouting angiogenesis. We found that endothelial cells use a specific lamellipodia-related membrane protrusion for invasion, which we termed dactylopodia. These protrusions have a special morphology, originate from filopodia, are linked to membrane-ruffling activity, and are specialized in invading into avascular extracellular matrix. Our work lays the foundations for drug discovery targeting sprouting angiogenesis.

Sprouting angiogenesis is fundamental for development and contributes to multiple diseases, including cancer, diabetic retinopathy and cardiovascular diseases. Sprouting angiogenesis depends on the invasive properties of endothelial tip cells. However, there is very limited knowledge on the mechanisms that endothelial tip cells use to invade into tissues. Here, we prove that endothelial tip cells use long lamellipodia projections (LLPs) as the main cellular protrusion for invasion into non-vascular extracellular matrix. We show that LLPs and filopodia protrusions are balanced by myosin-IIA (MIIA) and actin-related protein 2/3 (Arp2/3) activity. Endothelial cell-autonomous ablation of MIIA promotes excessive LLPs formation in detriment of filopodia. Conversely, endothelial cell-autonomous ablation of Arp2/3 prevents LLPs development and leads to excessive filopodia formation. We further show that MIIA inhibits Rac1-dependent activation of Arp2/3, by regulating the maturation state of focal adhesions. Our discoveries establish the first comprehensive model of how endothelial tip cells regulate its protrusive activity and will pave the way towards new strategies to block invasive tip cells during sprouting angiogenesis.

Morphogenesis of hierarchical vascular networks depends on the integration of multiple biomechanical signals by endothelial cells, the cells lining the interior of blood vessels. Expansion of vascular networks arises through sprouting angiogenesis, a process involving extensive cell rearrangements and collective cell migration. Yet, the mechanisms controlling angiogenic collective behavior remain poorly understood. Here, we show this collective cell behavior is regulated by non-canonical Wnt signaling. We identify that Wnt5a specifically activates Cdc42 at cell junctions downstream of ROR2 to reinforce coupling between adherens junctions and the actin cytoskeleton. We show that Wnt5a signaling stabilizes vinculin binding to alpha-catenin, and abrogation of vinculin in vivo and in vitro leads to uncoordinated polarity and deficient sprouting angiogenesis in Mus musculus. Our findings highlight how non-canonical Wnt signaling coordinates collective cell behavior during vascular morphogenesis by fine-tuning junctional mechanocoupling between endothelial cells.

Summary: Cell migration is essential during development, regeneration, homeostasis, and disease. Depending on the microenvironment, cells use different mechanisms to migrate. Yet, all modes of migration require the establishment of an intracellular front–rear polarity axis for directional movement. Although front–rear polarity can be easily identified in in vitro conditions, its assessment in vivo by live‐imaging is challenging due to tissue complexity and lack of reliable markers. Here, we describe a novel and unique double fluorescent reporter mouse line to study front–rear cell polarity in living tissues, called GNrep. This mouse line simultaneously labels Golgi complexes and nuclei allowing the assignment of a nucleus‐to‐Golgi axis to each cell, which functions as a readout for cell front–rear polarity. As a proof‐of‐principle, we validated the efficiency of the GNrep line using an endothelial‐specific Cre mouse line. We show that the GNrep labels the nucleus and the Golgi apparatus of endothelial cells with very high efficiency and high specificity. Importantly, the features of fluorescent intensity and localization for both mCherry and eGFP fluorescent intensity and localization allow automated segmentation and assignment of polarity vectors in complex tissues, making GNrep a great tool to study cell behavior in large‐scale automated analyses. Altogether, the GNrep mouse line, in combination with different Cre recombinase lines, is a novel and unique tool to study of front–rear polarity in mice, both in fixed tissues or in intravital live imaging. This new line will be instrumental to understand cell migration and polarity in development, homeostasis, and disease.

Proneural transcription factors such as Ascl1 function as master regulators of neurogenesis in vertebrates, being both necessary and sufficient for the activation of a full program of neuronal differentiation. Novel insights into the dynamics of Ascl1 expression at the cellular level, combined with the progressive characterization of its transcriptional program, have expanded the classical view of Ascl1 as a differentiation factor in neurogenesis. These advances resulted in a new model, whereby Ascl1 promotes sequentially the proliferation and differentiation of neural/stem progenitor cells. The multiple activities of Ascl1 are associated with the activation of distinct direct targets at progressive stages along the neuronal lineage. How this temporal pattern is established is poorly understood. Two modes of Ascl1 expression recently described (oscillatory versus sustained) are likely to be of importance, together with additional mechanistic determinants such as the chromatin landscape and other transcriptional pathways. Here we revise these latest findings, and discuss their implications to the gene regulatory functions of Ascl1 during neurogenesis.