Swim-Training Changes the Spatio-Temporal Dynamics of Skeletogenesis in Zebrafish Larvae (Danio rerio)

Fish larvae experience many environmental challenges during development such as variation in water velocity, food availability and predation. The rapid development of structures involved in feeding, respiration and swimming increases the chance of survival. It has been hypothesized that mechanical loading induced by muscle forces plays a role in prioritizing the development of these structures. Mechanical loading by muscle forces has been shown to affect larval and embryonic bone development in vertebrates, but these investigations were limited to the appendicular skeleton. To explore the role of mechanical load during chondrogenesis and osteogenesis of the cranial, axial and appendicular skeleton, we subjected zebrafish larvae to swim-training, which increases physical exercise levels and presumably also mechanical loads, from 5 until 14 days post fertilization. Here we show that an increased swimming activity accelerated growth, chondrogenesis and osteogenesis during larval development in zebrafish. Interestingly, swim-training accelerated both perichondral and intramembranous ossification. Furthermore, swim-training prioritized the formation of cartilage and bone structures in the head and tail region as well as the formation of elements in the anal and dorsal fins. This suggests that an increased swimming activity prioritized the development of structures which play an important role in swimming and thereby increasing the chance of survival in an environment where water velocity increases. Our study is the first to show that already during early zebrafish larval development, skeletal tissue in the cranial, axial and appendicular skeleton is competent to respond to swim-training due to increased water velocities. It demonstrates that changes in water flow conditions can result into significant spatio-temporal changes in skeletogenesis

Influence of the Testa on Seed Dormancy, Germination, and Longevity in Arabidopsis

The testa of higher plant seeds protects the embryo against adverse environmental conditions. Its role is assumed mainly by controlling germination through dormancy imposition and by limiting the detrimental activity of physical and biological agents during seed storage. To analyze the function of the testa in the model plant Arabidopsis, we compared mutants affected in testa pigmentation and/or structure for dormancy, germination, and storability. The seeds of most mutants exhibited reduced dormancy. Moreover, unlike wild-type testas, mutant testas were permeable to tetrazolium salts. These altered dormancy and tetrazolium uptake properties were related to defects in the pigmentation of the endothelium and its neighboring crushed parenchymatic layers, as determined by vanillin staining and microscopic observations. Structural aberrations such as missing layers or a modified epidermal layer in specific mutants also affected dormancy levels and permeability to tetrazolium. Both structural and pigmentation mutants deteriorated faster than the wild types during natural aging at room temperature, with structural mutants being the most strongly affected

The TRANSPARENT TESTA12 Gene of Arabidopsis Encodes a Multidrug Secondary Transporter-like Protein Required for Flavonoid Sequestration in Vacuoles of the Seed Coat Endothelium

Phenolic compounds that are present in the testa interfere with the physiology of seed dormancy and germination. We isolated a recessive Arabidopsis mutant with pale brown seeds, transparent testa12 (tt12), from a reduced seed dormancy screen. Microscopic analysis of tt12 developing and mature testas revealed a strong reduction of proanthocyanidin deposition in vacuoles of endothelial cells. Double mutants with tt12 and other testa pigmentation mutants were constructed, and their phenotypes confirmed that tt12 was affected at the level of the flavonoid biosynthetic pathway. The TT12 gene was cloned and found to encode a protein with similarity to prokaryotic and eukaryotic secondary transporters with 12 transmembrane segments, belonging to the MATE (multidrug and toxic compound extrusion) family. TT12 is expressed specifically in ovules and developing seeds. In situ hybridization localized its transcript in the endothelium layer, as expected from the effect of the tt12 mutation on testa flavonoid pigmentation. The phenotype of the mutant and the nature of the gene suggest that TT12 may control the vacuolar sequestration of flavonoids in the seed coat endothelium

Swim-training had a differential effect on on the age at appearance of bone structures between control and trained fish.

<p><b>A,B</b>) BF50<i>_age</i> values visualized in the corresponding structures in control fish (A) and trained fish (B). The branchial region is indicated separately, ventral view. <b>C</b>) Differences in BF50<i>_age</i> values between control and trained fish. Positive values indicate that structures appear earlier in the trained fish. Structures with a difference less than twice the standard error are indicated in grey.</p

Schematic representation of the swim-training set-up in lateral view and critical flow velocity.

<p><b>A</b>) Water was pumped (1) to the top aquarium and flowed back into the reservoir via the training tubes (4), outflow tubes (5) and outflow hoses (6) due to gravity. The difference in water level between the top aquarium and the outflow tubes (5) (indicated with up down black arrow) determined the flow velocity in the training tubes (4). Control fish were kept in similar tubes in the same set-up (7). Both the training and control section consisted of five tubes placed parallel to each other (not visible in drawing). Each tube had its own outflow tube and hose. <b>B</b>) Critical flow velocity (<i>U_crit</i>) over time and during swim-training experiments (<i>U_critse</i>). Zebrafish were subjected to 50% (<i>U_crit</i>_50%) of the moving average <i>U_crit</i> (<i>U_critma</i>).</p

Swim-training increased burst frequency and growth in trained fish.

<p><b>A</b>) Average number of bursts per second ± standard deviation as a function of age in days post fertilization (dpf). <b>B</b>) Average standard lengths (dots) ± standard deviation (with quadratic regression fit) as a function of age (dpf).</p

Number of fish sampled during each stage in a swim-experiment.

<p>Number of fish sampled during each stage in a swim-experiment.</p