Intravitreal injection of FGF and TGF-ß inhibitors disrupts cranial cartilage development.

Cartilage development is a tightly regulated process that requires the interaction of epithelial and mesenchymal tissues layers to initiate the aggregation of mesenchyme in a condensation. Several signaling molecules have been implicated in cartilage formation including FGFs, WNTs, and members of the TGF-ß super family. However, little is known about the earliest signals involved in these initial phases of development. Here we aimed to investigate whether direct intravitreal injection of pharmaceutical inhibitors for FGF and TGF-ß signaling would perturb cranial cartilages in zebrafish. Via wholemount bone and cartilage staining, we found effects on multiple cranial cartilage elements. We found no effect on scleral cartilage development, however, the epiphyseal bar, basihyal, and basicapsular cartilages were disrupted. Interestingly, the epiphyseal bar arises from the same progenitor pool as the scleral cartilage, namely, the periocular ectomesenchyme. This study adds to the foundational knowledge about condensation induction of cranial cartilage development and provides insight into the timing and signaling involved in the early development of several craniofacial cartilage elements in zebrafish.

Quantification and comparison of teleost scleral cartilage development and growth.

The ocular skeleton is composed of the scleral cartilage and the scleral ossicles. Teleost scleral cartilage is composed of a single layer of chondrocytes embedded in the sclera of the eye. The teleost scleral cartilage ring can vary in depth across teleost families and species, from a narrow ring a few cells wide to a deeper ring that resembles a cup and surrounds the entire sclera. However, very little research has been conducted on the development and morphology of teleost scleral cartilage. Thus, this study aims to characterize the development of the scleral cartilage in the zebrafish and Mexican tetra, with respect to the timing of emergence, depth throughout development, and positioning within the eye. We hypothesized that the scleral cartilage would first emerge in the scleral tissue closely abutting the ora serrata, and that growth would proceed in an anterior-to-posterior direction, resulting in differences in scleral cartilage depth between different fish species. We found that the scleral cartilage ring does not develop uniformly along its circumference, and that its relationship to the ora serrata varies between the rostral and caudal regions. Furthermore, distinct differences in the growth trajectory of the scleral cartilage indicate that the deep scleral cartilage of the Pachón cavefish is the result of both decreased eye size and prolonged cartilage growth. A significant difference in the size of the scleral chondrocytes was also noted. Overall, this study provides the first characterization of early scleral cartilage development in teleost fish and indicates that some aspects of scleral cartilage development and morphology are highly conserved while others are not.

Elucidating the early signaling cues involved in zebrafish chondrogenesis and cartilage morphology.

Across the teleost skeleton, cartilages are diverse in their composition suggesting subtle differences in their developmental mechanisms. This study aims to elucidate the regulatory role of bone morphogenetic protein (BMPs) during the morphogenesis of two cartilage elements in zebrafish: the scleral cartilage in the eye and the caudal fin endoskeleton. Zebrafish larvae were exposed to a BMP inhibitor (LDN193189) at a series of timepoints preceding the initial appearance of the scleral cartilage and caudal fin endoskeleton. Morphological assessments of the cartilages in later stages, revealed that BMP-inhibited fish harbored striking disruptions in caudal fin endoskeletal morphology, regardless of the age at which the inhibitor treatment was performed. In contrast, scleral cartilage morphology was unaffected in all age groups. Morphometric and principal component analysis, performed on the caudal fin endoskeleton, revealed differential clustering of principal components one and two in BMP-inhibited and control fish. Additionally, the expression of sox9a and sox9b were reduced in BMP-inhibited fish when compared to controls, indicating that LDN193189 acts via a Sox9-dependent pathway. Further examination of notochord flexion also revealed a disruptive effect of BMP inhibition on this process. This study provides a detailed characterization of the effects of BMP inhibition via LDN193189 on zebrafish cartilage morphogenesis and development. It highlights the specific, localized role of the BMP-signaling pathways during the development of different cartilage elements and sheds some light on the morphological characteristics of fossil teleosts that together suggest an uncoupling of the developmental processes between the upper and lower lobes of the caudal fin.

Accurate whole-mount bone and cartilage staining requires acid-free conditions.

Bone and cartilage staining has provided anatomists with the ability to generate detailed descriptions of the adult and developing skeleton. Typically, Alizarin red S and Alcian blue are used for the staining of bone and cartilage, respectively. The binding of Alizarin red S and calcium is most stable at basic conditions, however, Alcian blue exhibits specific binding to polyanionic substances such as mucopolysaccharides under acidic conditions. Typical bone and cartilage staining protocols are conducted under acidic conditions. Because of this discrepancy in optimal pH, issues can arise in the staining of small specimens such as larval fish. Specifically, staining embryonic or larval specimens under acidic conditions can cause decalcification of small bones. Decalcification can completely inhibit the uptake of Alizarin red S in small bones. In order to mitigate this issue, researchers have developed an acid-free staining protocol that utilizes the concept of critical electrolyte concentration. While many researchers have adopted acid-free bone and cartilage staining, some researchers continue to stain these small specimens with acidic staining protocols. To ensure the reliability and validity of our skeletal descriptions, we urge scientists to utilize acid-free staining protocols when analyzing the skeletons of larval or embryonic specimens.

An improved rapid mixing device for time-resolved electrospray mass spectrometry measurements.

Time series data can provide valuable insight into the complexity of biological reactions. Such information can be obtained by mass-spectrometry-based approaches that measure pre-steady-state kinetics. These methods are based on a mixing device that rapidly mixes the reactants prior to the on-line mass measurement of the transient intermediate steps. Here, we describe an improved continuous-flow mixing apparatus for real-time electrospray mass spectrometry measurements. Our setup was designed to minimize metal-solution interfaces and provide a sheath flow of nitrogen gas for generating stable and continuous spray that consequently enhances the signal-to-noise ratio. Moreover, the device was planned to enable easy mounting onto a mass spectrometer replacing the commercial electrospray ionization source. We demonstrate the performance of our apparatus by monitoring the unfolding reaction of cytochrome C, yielding improved signal-to-noise ratio and reduced experimental repeat errors.