Sox9 is associated with two distinct patterning events during snake lung morphogenesis.

The evolutionary forces that allowed species adaptation to different terrestrial environments and led to great diversity in body shape and size required acquisition of innovative strategies of pattern formation during organogenesis. An extreme example is the formation of highly elongated viscera in snakes. What developmental patterning strategies allowed to overcome the space constraints of the snake's body to meet physiological demands? Here we show that the corn snake uses a Sox2-Sox9 developmental tool kit common to other species to generate and shape the lung in two phases. Initially Sox9 was found at low levels at the tip of the primary lung bud during outgrowth and elongation of the bronchial bud, without driving branching programs characteristic of mammalian lungs. Later, Sox9 induction is recapitulated in the formation of an extensive network of radial septae emerging along the elongated bronchial bud that generates the respiratory region. We propose that altogether these represent key patterning events for formation of both the respiratory faveolar and non-respiratory posterior compartments of the snake's lung.

The mechanical and morphological properties of systemic and pulmonary arteries differ in the Madagascar ground boa, a snake without ventricular pressure separation.

The walls of the mammalian aorta and pulmonary artery are characterized by diverging morphologies and mechanical properties, which have been correlated with high systemic and low pulmonary blood pressure, as a result of intraventricular pressure separation. However, the relationship between intraventricular pressure separation and diverging aortic and pulmonary artery wall morphologies and mechanical characteristics is not understood. The snake cardiovascular system poses a unique model for the study of this relationship, as representatives both with and without intraventricular pressure separation exist. In this study, we performed uniaxial tensile testing on vessel samples taken from the aortas and pulmonary arteries of the Madagascar ground boa, Acrantophis madagascariensis, a species without intraventricular pressure separation. We then compared these morphological and mechanical characteristics with samples from the ball python, Python regius, and the yellow anaconda, Eunectes notaeus - species with and without intraventricular pressure separation, respectively. Our data suggest that although the aortas and pulmonary arteries of A. madagascariensis respond similarly to the same intramural blood pressure, they diverge in morphology, and that this attribute extends to E. notaeus. In contrast, P. regius aortas and pulmonary arteries diverge both morphologically and in terms of their mechanical properties. Our data indicate that intraventricular pressure separation cannot fully explain diverging aortic and pulmonary artery morphologies. Following the law of Laplace, we propose that pulmonary arteries of small luminal diameter represent a mechanism to protect the fragile pulmonary vasculature by reducing the blood volume that passes through, to which genetic factors may contribute more strongly than physiological parameters.

Hippo-Yap/Taz signaling: Complex network interactions and impact in epithelial cell behavior.

The Hippo pathway has emerged as a crucial integrator of signals in biological events from development to adulthood and in diseases. Although extensively studied in Drosophila and in cell cultures, major gaps of knowledge still remain on how this pathway functions in mammalian systems. The pathway consists of a growing number of components, including core kinases and adaptor proteins, which control the subcellular localization of the transcriptional co-activators Yap and Taz through phosphorylation of serines at key sites. When localized to the nucleus, Yap/Taz interact with TEAD transcription factors to induce transcriptional programs of proliferation, stemness, and growth. In the cytoplasm, Yap/Taz interact with multiple pathways to regulate a variety of cellular functions or are targeted for degradation. The Hippo pathway receives cues from diverse intracellular and extracellular inputs, including growth factor and integrin signaling, polarity complexes, and cell-cell junctions. This review highlights the mechanisms of regulation of Yap/Taz nucleocytoplasmic shuttling and their implications for epithelial cell behavior using the lung as an intriguing example of this paradigm. This article is categorized under: Gene Expression and Transcriptional Hierarchies > Regulatory Mechanisms Signaling Pathways > Cell Fate Signaling Establishment of Spatial and Temporal Patterns > Cytoplasmic Localization.

Use of hPSC-derived 3D organoids and mouse genetics to define the roles of YAP in the development of the esophagus.

Balanced progenitor activities are crucial for the development and maintenance of high turn-over organs such as the esophagus. However, the molecular mechanisms regulating these progenitor activities in the esophagus remain to be elucidated. Here, we demonstrated that Yap is required for the proliferation of esophageal progenitor cells (EPCs) in the developing murine esophagus. We found that Yap deficiency reduces EPC proliferation and stratification whereas persistent Yap activation increases cell proliferation and causes aberrant stratification of the developing esophagus. We further demonstrated that the role of YAP signaling is conserved in the developing human esophagus by utilizing 3D human pluripotent stem cell (hPSC)-derived esophageal organoid culture. Taken together, our studies combining loss/gain-of-function murine models and hPSC differentiation support a key role for YAP in the self-renewal of EPCs and stratification of the esophageal epithelium.

Yap and its subcellular localization have distinct compartment-specific roles in the developing lung.

Although the Hippo-yes-associated protein (Yap) pathway has been implicated in lung development, the specific roles for Yap and its nucleocytoplasmic shuttling in the developing airway and alveolar compartments remain elusive. Moreover, conflicting results from expression studies and differences in the lung phenotypes of Yap and Hippo kinase null mutants caused controversy over the dynamics and significance of Yap subcellular localization in the developing lung. Here, we show that the aberrant morphogenesis of Yap-deficient lungs results from the disruption of developmental events specifically in distal epithelial progenitors. We also show that activation of nuclear Yap is enough to fulfill the Yap requirements to rescue abnormalities in these lungs. Remarkably, we found that Yap nucleocytoplasmic shuttling is largely dispensable in epithelial progenitors for both branching morphogenesis and sacculation. However, if maintained transcriptionally active in airways, nuclear Yap profoundly alters proximal-distal identity and halts epithelial differentiation. Taken together, these observations provide novel insights into the crucial importance of Hippo-Yap signaling in the lung prenatally.

Heterochrony and early left-right asymmetry in the development of the cardiorespiratory system of snakes.

Snake lungs show a remarkable diversity of organ asymmetries. The right lung is always fully developed, while the left lung is either absent, vestigial, or well-developed (but smaller than the right). A 'tracheal lung' is present in some taxa. These asymmetries are reflected in the pulmonary arteries. Lung asymmetry is known to appear at early stages of development in Thamnophis radix and Natrix natrix. Unfortunately, there is no developmental data on snakes with a well-developed or absent left lung. We examine the adult and developmental morphology of the lung and pulmonary arteries in the snakes Python curtus breitensteini, Pantherophis guttata guttata, Elaphe obsoleta spiloides, Calloselasma rhodostoma and Causus rhombeatus using gross dissection, MicroCT scanning and 3D reconstruction. We find that the right and tracheal lung develop similarly in these species. By contrast, the left lung either: (1) fails to develop; (2) elongates more slowly and aborts early without (2a) or with (2b) subsequent development of faveoli; (3) or develops normally. A right pulmonary artery always develops, but the left develops only if the left lung develops. No pulmonary artery develops in relation to the tracheal lung. We conclude that heterochrony in lung bud development contributes to lung asymmetry in several snake taxa. Secondly, the development of the pulmonary arteries is asymmetric at early stages, possibly because the splanchnic plexus fails to develop when the left lung is reduced. Finally, some changes in the topography of the pulmonary arteries are consequent on ontogenetic displacement of the heart down the body. Our findings show that the left-right asymmetry in the cardiorespiratory system of snakes is expressed early in development and may become phenotypically expressed through heterochronic shifts in growth, and changes in axial relations of organs and vessels. We propose a step-wise model for reduction of the left lung during snake evolution.

The mechanical properties of the systemic and pulmonary arteries of Python regius correlate with blood pressures.

Pythons are unique amongst snakes in having different pressures in the aortas and pulmonary arteries because of intraventricular pressure separation. In this study, we investigate whether this correlates with different blood vessel strength in the ball python Python regius. We excised segments from the left, right, and dorsal aortas, and from the two pulmonary arteries. These were subjected to tensile testing. We show that the aortic vessel wall is significantly stronger than the pulmonary artery wall in P. regius. Gross morphological characteristics (vessel wall thickness and correlated absolute amount of collagen content) are likely the most influential factors. Collagen fiber thickness and orientation are likely to have an effect, though the effect of collagen fiber type and cross-links between fibers will need further study.