Spatial mapping of tissue properties in vivo reveals a 3D stiffness gradient in the mouse limb bud.

Numerous hypotheses invoke tissue stiffness as a key parameter that regulates morphogenesis and disease progression. However, current methods are insufficient to test hypotheses that concern physical properties deep in living tissues. Here we introduce, validate, and apply a magnetic device that generates a uniform magnetic field gradient within a space that is sufficient to accommodate an organ-stage mouse embryo under live conditions. The method allows rapid, nontoxic measurement of the three-dimensional (3D) spatial distribution of viscoelastic properties within mesenchyme and epithelia. Using the device, we identify an anteriorly biased mesodermal stiffness gradient along which cells move to shape the early limb bud. The stiffness gradient corresponds to a Wnt5a-dependent domain of fibronectin expression, raising the possibility that durotaxis underlies cell movements. Three-dimensional stiffness mapping enables the generation of hypotheses and potentially the rigorous testing of mechanisms of development and disease.

Live tissue antibody injection: A novel method for imaging ECM in limb buds and other tissues.

Understanding the morphogenesis and differentiation of tissues and organs from progenitor fields requires methods to visualize this process. Despite an ever-growing recognition that ECM plays an important role in tissue development, studies of ECM movement, and patterns in live tissue are scarce. Here, we describe a method in which a living limb bud is immunolabeled prior to fixation using fluorescent antibodies that recognize two ECM constituents, fibronectin and fibrillin 2. The results show that undifferentiated mesenchyme in quail embryos can be distinguished from prechondrogenic cellular condensations, in situ, via ECM antibodies-indicating the developmental transition from naïve mesenchyme to committed skeletal tissue. We conclude that our live tissue injection method is a general approach that allows visualization of the structural characteristics and the distribution pattern of ECM scaffolds, in situ. With slight modifications, the method will produce robust fluorescence images of ECM scaffolds in any suitable tissue mass and allow multiple kinds of optical analyses including virtual 3D reconstructions.

Early onset of Runx2 expression caused craniosynostosis, ectopic bone formation, and limb defects.

RUNX2 is an essential transcription factor for osteoblast differentiation, because osteoblast differentiation is completely blocked in Runx2-deficient mice. However, it remains to be clarified whether RUNX2 is sufficient for osteoblast differentiation during embryogenesis. To address this issue, Runx2 transgenic mice were generated under the control of the Prrx1 promoter, which directs the transgene expression to mesenchymal cells before the onset of bone development. The transgene expression was detected in the cranium, limb buds, and the region from the mandible to anterior chest wall. The skull became small and the limbs were shortened depending on the levels of the transgene expression. Early onset of Runx2 expression in the cranial mesenchyme induced mineralization on E13.0, when no mineralization was observed in wild-type mice, and resulted in craniosynostosis as shown by the closure of sutures and fontanelles on E18.5. Col1a1 and Spp1 expressions were detected in the mineralized regions on E12.5-13.5. The limb bones were hypoplastic and fused, and ectopic bones were formed in the hands and feet. Col2a1 expression was inhibited but Col1a1 expression was induced in the limb buds on E12.5. In the anterior chest wall, ectopic bones were formed through the process of intramembranous ossification, interrupting the formation of cartilaginous anlagen of sternal manubrium. These findings indicate that RUNX2 is sufficient to direct mesenchymal cells to osteoblasts and lead to intramembranous bone formation during embryogenesis; Runx2 inhibits chondrocyte differentiation at an early stage; and that Runx2 expression at appropriate level, times and spaces during embryogenesis is essential for skeletal development.

Intrauterine ultrasonographic assessments of embryonic development.

OBJECTIVE: Our purpose was to describe embryonal anatomic structures by use of intrauterine ultrasonography with a 20 MHz flexible catheter-based, high-resolution, real-time miniature transducer. STUDY DESIGN: Thirty-four women about to undergo therapeutic abortion from 7 to 9.9 weeks' gestation were studied with specially developed catheter-based, high-resolution, real-time miniature (2.4 mm outer diameter) ultrasonography transducer (20 MHz). A percentage of anatomic structures visualized at each gestational age is presented. RESULTS: The number and the clarity of structures increased from 7 to 8 weeks of gestation; however, the image quality was degraded because of the increasing fetal size at 9 weeks. At 8 weeks secondary brain vesicles, spine, midgut herniation, liver, upper and lower limb buds, and sacral tail were visualized in all fetuses. The four-chamber view was first identified at 8 weeks, as were fingers or toes. The stomach was first noted at 9 weeks. The umbilical cord cyst was visualized in 8% of embryos at 7 weeks' gestation and in 29% of embryos at 8 weeks. One cystic hygroma was diagnosed at 8 weeks 5 days. CONCLUSION: Intrauterine ultrasonography provides information on the visualization of anatomic structures of the embryo. In this limited series one embryonic malformation was demonstrated, and thus there is a potential for its use in the detection of malformations. These results suggest that intrauterine ultrasonography has the potential to be a supplement to transvaginal ultrasonography during the first trimester in high-risk pregnancies.