Villus myofibroblasts are developmental and adult progenitors of mammalian gut lymphatic musculature.

Inside the finger-like intestinal projections called villi, strands of smooth muscle cells contract to propel absorbed dietary fats through the adjacent lymphatic capillary, the lacteal, sending fats into the systemic blood circulation for energy production. Despite this vital function, mechanisms of formation, assembly alongside lacteals, and maintenance of villus smooth muscle are unknown. By combining single-cell RNA sequencing and quantitative lineage tracing of the mouse intestine, we identified a local hierarchy of subepithelial fibroblast progenitors that differentiate into mature smooth muscle fibers via intermediate contractile myofibroblasts. This continuum persists as the major mechanism for villus musculature renewal throughout adult life. The NOTCH3-DLL4 signaling axis governs the assembly of smooth muscle fibers alongside their adjacent lacteals and is required for fat absorption. Our studies identify the ontogeny and maintenance of a poorly defined class of intestinal smooth muscle, with implications for accelerated repair and recovery of digestive function following injury.

Origin and adult renewal of the gut lacteal musculature from villus myofibroblasts.

Intestinal smooth muscles are the workhorse of the digestive system. Inside the millions of finger-like intestinal projections called villi, strands of smooth muscle cells contract to propel absorbed dietary fats through the adjacent lymphatic vessel, called the lacteal, sending fats into the blood circulation for energy production. Despite this vital function, how villus smooth muscles form, how they assemble alongside lacteals, and how they repair throughout life remain unknown. Here we combine single-cell RNA sequencing of the mouse intestine with quantitative lineage tracing to reveal the mechanisms of formation and differentiation of villus smooth muscle cells. Within the highly regenerative villus, we uncover a local hierarchy of subepithelial fibroblast progenitors that progress to become mature smooth muscle fibers, via an intermediate contractile myofibroblast-like phenotype. This continuum persists in the adult intestine as the major source of renewal of villus smooth muscle cells during adult life. We further found that the NOTCH3-DLL4 signaling axis governs the assembly of villus smooth muscles alongside their adjacent lacteal, and we show that this is necessary for gut absorptive function. Overall, our data shed light on the genesis of a poorly defined class of intestinal smooth muscle and pave the way for new opportunities to accelerate recovery of digestive function by stimulating muscle repair.

Visualizing and manipulating the production and accumulation of hyaluronan for functional assessment in chicken embryos.

Hyaluronan (HA) accumulates in the extracellular matrix to regulate organ morphogenesis. The spatiotemporal dynamics of its production and function are poorly understood due to its instability. Here, we present a protocol using the embryonic chicken intestine as a binary in vivo system for HA visualization and manipulation. We describe steps for pharmacological manipulation and in ovo electroporation to target HA production and accumulation. We then detail HA-binding protein assay to detect HA accumulation and quantification of tissue morphology changes. For complete details on the use and execution of this protocol, please refer to Sivakumar et al. (2018)1 and Sanketi et al. (2022).2.

Inhibition of ATM kinase rescues planarian regeneration after lethal radiation.

As stem cells divide, they acquire mutations that can be passed on to daughter cells. To mitigate potentially deleterious outcomes, cells activate the DNA damage response (DDR) network, which governs several cellular outcomes following DNA damage, including repairing DNA or undergoing apoptosis. At the helm of the DDR are three PI3-like kinases including Ataxia-Telangiectasia Mutated (ATM). We report here that knockdown of ATM in planarian flatworms enables stem cells to withstand lethal doses of radiation which would otherwise induce cell death. In this context, stem cells circumvent apoptosis, replicate their DNA, and recover function using homologous recombination-mediated DNA repair. Despite radiation exposure, atm knockdown animals survive long-term and regenerate new tissues. These effects occur independently of ATM's canonical downstream effector p53. Together, our results demonstrate that in planarians, ATM promotes radiation-induced apoptosis. This acute, ATM-dependent apoptosis is a key determinant of long-term animal survival. Our results suggest that inhibition of ATM in these organisms could, therefore, potentially favor cell survival after radiation without obvious effects on stem cell behavior.

Pitx2 patterns an accelerator-brake mechanical feedback through latent TGFß to rotate the gut.

The vertebrate intestine forms by asymmetric gut rotation and elongation, and errors cause lethal obstructions in human infants. Rotation begins with tissue deformation of the dorsal mesentery, which is dependent on left-sided expression of the Paired-like transcription factor Pitx2. The conserved morphogen Nodal induces asymmetric Pitx2 to govern embryonic laterality, but organ-level regulation of Pitx2 during gut asymmetry remains unknown. We found Nodal to be dispensable for Pitx2 expression during mesentery deformation. Intestinal rotation instead required a mechanosensitive latent transforming growth factor-ß (TGFß), tuning a second wave of Pitx2 that induced reciprocal tissue stiffness in the left mesentery as mechanical feedback with the right side. This signaling regulator, an accelerator (right) and brake (left), combines biochemical and biomechanical inputs to break gut morphological symmetry and direct intestinal rotation.

In Ovo Gain- and Loss-of-Function Approaches to Study Gut Morphogenesis.

The polarity of cellular components is essential for cellular shape changes, oriented cell migration, and modulating intra- and intercellular mechanical forces. However, many aspects of polarized cell behavior-especially dynamic cell shape changes during the process of morphogenesis-are almost impossible to study in cells cultured in plastic dishes. Avian embryos have always been a treasured model system to study vertebrate morphogenesis for developmental biologists. Avian embryos recapitulate human biology particularly well in the early stages due to their flat disc gastruloids. Since avian embryos can be manipulated in ovo they present paramount opportunities for highly localized targeting of genetic mechanisms during cellular and developmental processes. Here, we review the application of these methods for both gain of function and loss of function of a gene of interest at a specific developmental stage during left-right (LR) asymmetric gut morphogenesis. These tools present a powerful premise to investigate various polarized cellular activities and molecular processes in vivo in a reproducible manner.

Avian Embryos as a Model to Study Vascular Development.

The use of live imaging is indispensable for advancing our understanding of vascular morphogenesis. Imaging fixed embryos at a series of distinct developmental time points, although valuable, does not reveal the dynamic behavior of cells, as well as their interactions with the underlying ECM. Due to the easy access of chicken embryos to manipulation and high-resolution imaging, this model has been at the origin of key discoveries. In parallel, known through its extensive use in quail-chick chimera studies, the quail embryo is equally poised to genetic manipulations and paramount to direct imaging of transgenic reporter quails. Here we describe ex ovo time-lapse confocal microscopy of transgenic quail embryo slices to image vascular development during gut morphogenesis. This technique is powerful as it allows direct observation of the dynamic endothelial cell behaviors along the left-right (LR) axis of the dorsal mesentery (DM), the major conduit for blood and lymphatic vessels that serve the gut. In combination with in ovo plasmid electroporation and quail-chick transplantation, these methods have allowed us to study the molecular mechanisms underlying blood vessel assembly during the formation of the intestine. Below we describe our protocols for the generation of embryo slices, ex ovo time-lapse imaging of fluorescently labeled cells, and quail-chick chimeras to study the early stages of gut vascular development.

Changes in Nkx2.1, Sox2, Bmp4, and Bmp16 expression underlying the lung-to-gas bladder evolutionary transition in ray-finned fishes.

The key to understanding the evolutionary origin and modification of phenotypic traits is revealing the responsible underlying developmental genetic mechanisms. An important organismal trait of ray-finned fishes is the gas bladder, an air-filled organ that, in most fishes, functions for buoyancy control, and is homologous to the lungs of lobe-finned fishes. The critical morphological difference between lungs and gas bladders, which otherwise share many characteristics, is the general direction of budding during development. Lungs bud ventrally and the gas bladder buds dorsally from the anterior foregut. We investigated the genetic underpinnings of this ventral-to-dorsal shift in budding direction by studying the expression patterns of known lung genes (Nkx2.1, Sox2, and Bmp4) during the development of lungs or gas bladder in three fishes: bichir, bowfin, and zebrafish. Nkx2.1 and Sox2 show reciprocal dorsoventral expression patterns during tetrapod lung development and are important regulators of lung budding; their expression during bichir lung development is conserved. Surprisingly, we find during gas bladder development, Nkx2.1 and Sox2 expression are inconsistent with the hypothesis that they regulate the direction of gas bladder budding. Bmp4 is expressed ventrally during lung development in bichir, akin to the pattern during mouse lung development. During gas bladder development, Bmp4 is not expressed. However, Bmp16, a paralogue of Bmp4, is expressed dorsally in the developing gas bladder of bowfin. Bmp16 is present in the known genomes of Actinopteri (ray-finned fishes excluding bichir) but absent from mammalian genomes. We hypothesize that Bmp16 was recruited to regulate gas bladder development in the Actinopteri in place of Bmp4.