Alexander M. Schreiber

Diverse developmental programs of Xenopus laevis metamorphosis are inhibited by a dominant negative thyroid hormone receptor

Metamorphosis of anuran tadpoles is controlled by thyroid hormone (TH). Here we demonstrate that transgenic Xenopus laevis tadpoles expressing a dominant negative form of TH receptor-α are resistant to a wide variety of the metamorphic changes induced by TH. This result confirms that TH receptors mediate both early and late developmental programs of metamorphosis as diverse as growth in the brain, limb buds, nose and Meckels cartilage, remodeling of the intestine, and death and resorption of the gills and tail.

Multiple thyroid hormone-induced muscle growth and death programs during metamorphosis in Xenopus laevis

Xenopuslaevis tadpole tails contain fast muscle fibers oriented in chevrons and two pairs of slow muscle “cords” along the length of the tail. When tail resorption is inhibited by a number of different treatments, fast muscle but not the slow cord muscle still is lost, demonstrating that the fast tail muscle is a direct target of the thyroid hormone-induced death program. Expression of a dominant negative form of the thyroid hormone receptor (TRDNα) was restricted to tadpole muscle by means of a muscle-specific promoter. Even though the transgene protects fast tail muscle from thyroid hormone (TH)-induced death, the tail shortens, and the distal muscle chevrons at the tail tip are degraded. This default pathway for muscle death is probably caused by the action of proteolytic enzymes secreted by neighboring fibroblasts. Non-muscle tissues that are sensitive to TH, such as the fibroblasts, are not protected by the transgene when it is expressed solely in muscle. If allowed to develop to metamorphosis, these transgenic animals die at the climax of metamorphosis before tail resorption has begun. Their limbs have very little muscle even though the rest of limb morphology is normal. Thus, fast tail muscle and limb muscle have their own cell autonomous death and growth programs, respectively, that are independent of the fate of the other neighboring cell types. In contrast, death of the slow muscle is controlled by the other cell types of the tail.

Asymmetric craniofacial remodeling and lateralized behavior in larval flatfish

SUMMARY Flatfishes, such as flounder, are the worlds most asymmetric vertebrates. It is unknown if the development of lateralized swimming behavior during metamorphosis is an adaptive response to bilaterally asymmetric eye positioning, or if this results from a vestibular response to thyroid hormone. This study describes larval development in left-sided, right-sided and bilaterally symmetric variants of southern flounder (Paralichthys lethostigma). Behavior and skull asymmetries precede metamorphosis, and the development of lateralized behaviors was independent of eye position in larvae treated with thyroid hormone and in symmetrical variants. Therefore, lateralized behavior is not an adaptive response to eye translocation, but rather must result from changing vestibular responses to thyroid hormone.

Tadpole skin dies autonomously in response to thyroid hormone at metamorphosis

Transgenic tadpoles that express a dominant negative thyroid hormone (TH) receptor specifically in their skin undergo normal metamorphosis, with one exception: they retain a larval epidermis over the developing adult epithelium. TH-induced death of the tadpole epidermis is inhibited by the dominant negative TH receptor whereas the TH-induced response of the neighboring fibroblasts and the cells that form the adult skin occur normally. Therefore death of the tadpole skin is a direct and cell autonomous target of TH, and its protection has no detectable influence on TH-induced changes of other cell types.

Metamorphosis in the summer flounder, Paralichthys dentatus: thyroidal status influences salinity tolerance.

Metamorphosis in the summer flounder (Paralichthys dentatus) is controlled by thyroid hormones (TH) and takes place as the larvae move from a salinity of about 35 parts per thousand (ppt) in the ocean to salinity ranging from 0-35 ppt in estuaries. Historically, the role of TH in juvenile and adult teleost osmoregulation has been ambiguous, and it is not known if TH influences larval teleost osmoregulatory development. This study addresses the influence of thyroxine (T4) on the development of tolerance to low (5 ppt) and high salinity (45 and 50 ppt) as determined by salinity tolerance tests. In untreated larvae, tolerance to both low and high salinity was high during early premetamorphosis (early pre-M) and decreased or was very low from late prometamorphosis (late pro-M) through mid-metamorphic climax (mid-MC). Salinity tolerance increased 2-3-fold during late MC when whole-animal T4 levels are highest, and reached maximum tolerance at the juvenile stage. The early induction of metamorphosis by exposing larvae in pre-M to exogenous T4 reduced tolerance to low salinity during early and mid-MC, though tolerance of fish that had developed into juveniles was not impaired. In contrast, T4 increased high salinity tolerance during early and mid-MC, and the juvenile stage. This T4-induced heterochrony in salinity tolerance with regards to developmental stage suggests that the effects of T4 on salinity tolerance may be uncoupled from accelerated metamorphosis. Treatment of larvae with thiourea (TU, an inhibitor of T4 synthesis) inhibited metamorphosis and reduced tolerance to high salinity, but did not affect tolerance to low salinity. Reduced tolerance to high salinity by TU was only partially counteracted by T4 treatment, suggesting that TU also affects hypoosmoregulatory activity by an extrathyroidal mechanism. Our findings suggest that in the summer flounder T4 plays a more important role in the development of hypoosmoregulatory ability than hyperosmoregulatory ability. J. Exp. Zool. 284:414-424, 1999.

Metamorphosis and early larval development of the flatfishes (Pleuronectiformes): an osmoregulatory perspective

Flatfish (Pleuronectiformes) distribution in the environment is influenced by salinity, and varies among species and with developmental stage. Osmoregulatory ability likely plays an important role in defining species and developmental stage-specific distribution. Although the mechanisms of osmoregulation in adult and juvenile teleosts have been widely addressed, far less is known about their larval osmoregulatory physiology. Much of our current understanding of larval fish ion-regulation stems from studies using flatfishes, and this article reviews advances in this field, primarily from the point of view of the developing flatfishes. Addressed here are the ontogeny of salinity tolerance, the development of several important osmoregulatory tissues (the skin, gut, and gill), and the influence of the endocrine system on osmoregulation during early larval development and metamorphosis.

Cell-cell interactions during remodeling of the intestine at metamorphosis in Xenopus laevis

Amphibian metamorphosis is accompanied by extensive intestinal remodeling. This process, mediated by thyroid hormone (TH) and its nuclear receptors, affects every cell type. Gut remodeling in Xenopus laevis involves epithelial and mesenchymal proliferation, smooth muscle thickening, neuronal aggregation, formation of intestinal folds, and shortening of its length by 75%. Transgenic tadpoles expressing a dominant negative TH receptor (TRDN) controlled by epithelial-, fibroblast-, and muscle-specific gene promoters were studied. TRDN expression in the epithelium caused abnormal development of virtually all cell types, with froglet guts displaying reduced intestinal folds, thin muscle and mesenchyme, absence of neurons, and reduced cell proliferation. TRDN expression in fibroblasts caused abnormal epithelia and mesenchyme development, and expression in muscle produced fewer enteric neurons and a reduced inter-muscular space. Gut shortening was inhibited only when TRDN was expressed in fibroblasts. Gut remodeling results from both cell-autonomous and cell-cell interactions.

Metamorphosis in summer flounder: effects of acclimation to low and high salinities

The aim of these studies was to provide larviculturists with information about the effect of low and high salinities on the survival, growth, and development of summer flounder (Paralichthys dentatus) with the goal of enhancing commercial success of land-based flounder culture. Larvae were transferred after first feeding and prior to the onset of metamorphosis and monitored for 3 to 7 weeks. In experiments 1 and 2, larvae were raised in 2-l bowls containing seawater altered to salinities of 14, 30, or 38 ppt at starting densities of 3 premetamorphic and 6 prometamorphic larvae/l, respectively. Survival and development were not affected, nor was growth in experiment 1. Growth was affected in experiment 2, and larvae in 14 ppt had greater total lengths than larvae in 38 ppt. In experiments 3 and 4, larvae were raised in 38-l tanks at starting densities 4 and 3 prometamorphic larvae/l, respectively, containing water with salinities of 8, 30, or 38 ppt or in salinities of 8 and 30 ppt. Again, survival was not significantly affected by salinity. In experiment 3, 8 ppt resulted in better growth and more advanced development; however, no differences were found in experiment 4. Overall, 38 ppt may tend to result in adverse effects. Larval summer flounder are not adversely affected by low salinity (8 or 14 ppt) and were observed in some cases to grow and develop better in environments more closely approximating their internal environment.

Flatfish: An Asymmetric Perspective on Metamorphosis

The most asymmetrically shaped and behaviorally lateralized of all the vertebrates, the flatfishes are an endless source of fascination to all fortunate enough to study them. Although all vertebrates undergo left-right asymmetric internal organ placement during embryogenesis, flatfish are unusual in that they experience an additional period of postembryonic asymmetric remodeling during metamorphosis, and thus deviate from a bilaterally symmetrical body plan more than other vertebrates. As with amphibian metamorphosis, all the developmental programs of flatfish metamorphosis are ultimately under the control of thyroid hormone. At least one gene pathway involved in embryonic organ lateralization (nodal-lefty-pitx2) is re-expressed in the larval stage during flatfish metamorphosis. Aspects of modern flatfish ontogeny, such as the gradual translocation of one eye to the opposite side of the head and the appearance of key neurocranial elements during metamorphosis, seem to elegantly recapitulate flatfish phylogeny. This chapter highlights the current state of knowledge of the developmental biology of flatfish metamorphosis with emphases on the genetic, morphological, behavioral, and evolutionary origins of flatfish asymmetry.

Thyroid hormone-responsive genes mediate otolith growth and development during flatfish metamorphosis

Flatfish begin life as up-right swimming, bilaterally symmetrical larvae that metamorphose into asymmetrically shaped juveniles that swim with a highly lateralized posture. We have previously shown that TH induces abrupt growth and mineralization of one component of the vestibular system, the otoliths, during early larval development and metamorphosis. Here we report that four of five vestibular-specific genes that we tested (alpha-tectorin, otogelin, otolith matrix protein, and otopetrins 1 and 2 that are known to be associated with otolith development in other vertebrates are up-regulated 1.5- to 7-fold in larval flatfish during spontaneous metamorphosis and/or following 72 h of TH treatment. These findings suggest that otolith growth and development are mediated by diverse TH-responsive genes during flatfish metamorphosis.