Skeletal morphogenesis in the urodele skull: III. Effect of hormone dosage in TH-induced remodeling

This study examines the dosage dependency of thyroid hormone (TH)-mediated remodelling in the cranial skeleton of the hemidactyliine plethodontid urodele, Eurycea bislineata. One set of experiments quantifies morphogenetic responses in 21 tissues for four size-age classes of larvae immersed in four different T₄ concentrations. A second set varies both the period and concentration of T₄ treatment to evaluate the effect of different TH profiles on adult tissue shape. The tissues surveyed in this study exhibit a 100-fold range in TH sensitivity. Those in regressive morphogenesis have tissue-specific sensitivities which correlate with the timing of their remodelling in natural development: bone resorption is more sentitive than cartilage resorption and is initiated earlier in metamorphosis. In contrast, the TH sensitivities of tissues in progressive morphogenesis vary within each tissue type and even within some tissues, and they do not correlate with timing in natural development. Some explanation for this discrepancy is offered by the constant spatial and temporal relationships between nasal cartilage and dermal bone, which suggest that some TH-mediated ossification may additionally require induction by cartilage. Also, the failure of nasolacrimal duct morphogenesis at all but the lowest dosage correlates with the inductdion of integumentary changes that may preclude duct formation. Variable T₄ treatments produce no effect upon the adult skull, other than loss of the nasolacrimal duct and/or foramen. These results have two developmental implicatons. First, the dosage dependencies of the nasolacrimal duct, ossification sequences, and cranial remodelling patterns all support a TH profile with exceptionally low levels at larval stages and at least a 100-fold increase at metamorphosis. Second, a small change in the rate of TH activity has the potential to effect a large-scale rearranggement and restructuring of TH-dependent remodelling. The lack of such transformations in metamorphic plethodontids suggests that TH activity is highly conserved in this group. © 1995 Wiley-Liss, Inc.     

From buds to follicles: matrix metalloproteinases in developmental tissue remodeling during feather morphogenesis

Organogenesis involves a series of dynamic morphogenesis and remodeling processes. Since feathers exhibit complex forms, we have been using the feather as a model to analyze how molecular pathways and cellular events are used. While several major molecular pathways have been studied, the roles of matrix degrading proteases and inhibitors in feather morphogenesis are unknown. Here we addressed this knowledge gap by studying the temporal and spatial expression of proteases and inhibitors in developing feathers using mammalian antibodies that cross react with chicken proteins. We also investigated the effect of protease inhibitors on feather development employing an in vitro feather bud culture system. The results show that antibodies specific for mammalian MMP2 and TIMP2 stained positive in both feather epithelium and mesenchyme. The staining co-localized in structures of E10-E13 developing feathers. Interestingly, MMP2 and TIMP2 exhibited a complementary staining pattern in developing E15 and E20 feathers and in maturing feather filaments. Although they exhibited a slight delay in feather bud development, similar patterns of MMP2 and TIMP2 staining were observed in in vitro culture explants. The broad spectrum pharmacological inhibitors AG3340 and BB103 (MMP inhibitors) but not Aprotinin (a plasmin inhibitor) showed a reversible effect on epithelium invagination and feather bud elongation. TIMP2, a physiological inhibitor to MMPs, exhibited a similar effect. Markers of feather morphogenesis showed that MMP activity was required for both epithelium invagination and mesenchymal cell proliferation. Inhibition of MMP activity led to an overall delay in the expression of molecules that regulate either early feather bud growth and/or differentiation and thereby produced abnormal buds with incomplete follicle formation. This work demonstrates that MMPs and their inhibitors are not only important in injury repair, but also in development tissue remodeling as demonstrated here for the formation of feather follicles.     

On the thyroid hormone-induced increase in respiratory capacity of isolated rat hepatocytes.

The respiratory capacities of hepatocytes, derived from hypothyroid, euthyroid and hyperthyroid rats, have been compared by measuring rates of oxygen uptake and by titrating components of the respiratory chain with specific inhibitors. Thyroid hormone increased the maximal rate of substrate-stimulated respiration and also increased the degree of ionophore-stimulated oxygen uptake. In titration experiments, similar concentrations of oligomycin or antimycin were required for maximal inhibition of respiration regardless of thyroid state, suggesting that the changes in respiratory capacity were not the result of variation in the amounts of ATP synthase or cytochrome b. However, less rotenone was required for maximal inhibition of respiration in the hypothyroid state than in cells from euthyroid or hyperthyroid rats, implying that hepatocytes from hypothyroid animals contain less NADH dehydrogenase. The concentration of carboxyatractyloside necessary for maximal inhibition of respiration was 100 microM in hepatocytes from hypothyroid rats, but 200 microM and 300 microM in hepatocytes from euthyroid and hyperthyroid rats, respectively, indicating a possible correlation between levels of thyroid hormone and the amount or activity of adenine nucleotide translocase. The increased capacity for coupled respiration in response to thyroid hormone is not associated with an increase in the components of the electron transport chain or ATP synthase, but correlates with an increased activity of adenine nucleotide translocase.     

Relationships between neuronal cell adhesion molecule and LHRH neurons in the urodele brain: a developmental immunohistochemical study.

Polysialic acid (PSA), a homopolymer attached to neural cell adhesion molecule (NCAM) is considered a major hallmark of vertebrate cell migration. We studied the distribution of PSA-NCAM by immunohistochemistry, during brain development, in two urodele amphibians, Pleurodeles waltl and the neotenic newt Ambystoma mexicanum. In both species a gradual increase of immunolabelling was observed throughout the brain from developmental stage 30 to stage 52. At the onset of metamorphosis, some differences became evident: in Pleurodeles immunostaining was gradually restricted to the olfactory system while in Ambystoma, PSA-NCAM maintained a more extended distribution (for example throughout the telencephalic walls) suggesting, for the brain of this latter species, a rather preserved neuronal plasticity. The aim of the present work was to correlate the above described PSA-NCAM-immunoreactivity (IR) with the distribution of luteinizing hormone-releasing hormone (LH-RH) containing neurons, which represent a well known example of neural elements migrating from the olfactory placode. LHRH-IR, undetectable till stage 30, was later found together with PSA-NCAM-IR in both the olfactory system and septo-hypothalamic areas. Such observations further support a role of PSA in providing a migration route toward the establishment of a part, at least, of the urodele LHRH system. The possible functional meaning of the LHRH-containing neurons localized between dorsal and ventral thalamus of Ambystoma, never reported before in this area, almost devoid of PSA-NCAM-IR, is discussed.     

Setting the stage: developmental biology in pre-college classrooms

Exercises that employ dynamic living material have proved highly successful at generating interest in science among young students. Developing embryos and larvae are especially well suited for such endeavors, for they can be handled without expensive or elaborate equipment, and their changing nature engages students. Using amphibian embryos, which are relatively large and exhibit profound, easily observed morphological changes, and amphibian larvae, which are easily kept and observed, captures the attention of children. By designing inquiry-based exercises and focused discussion sessions, a high intellectual content can be integrated into these endeavors. The long-term implications for generating an informed citizenry, improving the participation of women in science, and empowering elementary school teachers are profound. Professional developmental biology researchers should feel encouraged to participate in these types of activities.     

Effect of iodine on early stage thyroid autonomy

Thyroid autonomy is a frequent cause of thyrotoxicosis in regions with iodine deficiency. Epidemiological data suggest that iodide may influence the course of pre-existing thyroid autonomy. Making use of FRTL-5 cells stably expressing a constitutively activating TSH receptor mutation as an in vitro model of thyroid autonomy, we investigated the impact of iodide on proliferation, function and changes in global gene expression. We demonstrate that iodine inhibits growth in TSHR WT and L629F mutant FRTL-5 cells and downregulates e.g. protocadherin cluster (Pcdha1-13) and thyroid responsive element (Thrsp). In addition functional genes e.g. iodotyrosine deiodinase (iyd) and oncogen junB are upregulated, while sodium-iodide-symporter (Nis) and thyroid peroxidase (Tpo) are downregulated by iodide. Iodide tunes down the biological activity of autonomous thyrocytes and may thus be of therapeutic benefit not only to prevent the occurrence of somatic TSHR mutations, causing thyroid autonomy, but also to slow down the development of clinically relevant disease.     

Formation of the digestive system in zebrafish. II. Pancreas morphogenesis

Recent studies have suggested that the zebrafish pancreas develops from a single pancreatic anlage, located on the dorsal aspect of the developing gut. However, using a transgenic zebrafish line that expresses GFP throughout the endoderm, we report that, in fact, two pancreatic anlagen join to form the pancreas. One anlage is located on the dorsal aspect of the developing gut and is present by 24 h postfertilization (hpf), the second anlage is located on the ventral aspect of the developing gut in a position anterior to the dorsal anlage and is present by 40 hpf. These two buds merge by 52 hpf to form the pancreas. Using heart and soul mutant embryos, in which the pancreatic anlagen most often do not fuse, we show that the posterior bud generates only endocrine tissue, while the anterior bud gives rise to the pancreatic duct and exocrine cells. Interestingly, at later stages, the anterior bud also gives rise to a small number of endocrine cells usually present near the pancreatic duct. Altogether, these studies show that in zebrafish, as in the other model systems analyzed to date, the pancreas arises from multiple buds. To analyze whether other features of pancreas development are conserved and investigate the influence of surrounding tissues on pancreas development, we examined the role of the vasculature in this process. Contrary to reports in other model systems, we find that, although vascular endothelium is in contact with the posterior bud throughout pancreas development, its absence in cloche mutant embryos does not appear to affect the early morphogenesis or differentiation of the pancreas.     

Expression of type II iodothyronine deiodinase marks the time that a tissue responds to thyroid hormone-induced metamorphosis in Xenopus laevis

The thyroid gland synthesizes thyroxine (T4), which passes through the larval tadpole's circulatory system. The enzyme type II iodothyronine deiodinase (D2) converts thyroxine (T4) to the active hormone 3,5,3'-triiodothyronine (T3) in peripheral tissues. An early response to thyroid hormone (TH) in the Xenopus laevis tadpole is the stimulation of cell division in cells that line the brain ventricles, the lumen of the spinal cord, and the limb buds. These cells express constitutively high levels of D2 mRNA. Exogenous T4 induces early DNA synthesis in brain, spinal cord, and limb buds as efficiently as T3. The deiodinase inhibitor iopanoic acid blocks T4- but not T3-induced cell division. At metamorphic climax, both TH-induced cell division and D2 expression decrease in the brain. Then D2 expression appears in late-responding tissues including the anterior pituitary, the intestine, and the tail where cell division is reduced or absent. Therefore, constitutive expression of D2 occurs in the earliest target tissues of TH that will grow and differentiate, while TH-induced expression of D2 takes place in late-responding tissues that will remodel or die. This pattern of constitutive and induced D2 expression contributes to the timing of metamorphic changes in these tissues.     

Effect of developmental stage on arginase and urea production in the liver of rat

Arginase activity in the liver, brain, and testis of rats was examined during the different phases of life span. When expressed as specific activity (micromoles of L-arginine hydrolyzed per minute per gram of whole homogenate protein), the enzyme activity in the brain and testis decreased markedly during the early stage of life and stayed low during the remainder of the life span. On the other hand, the arginase in the liver showed a great dependency on the developmental phase of the animal, showing two distinct peaks: one during the early phase (20 days after birth) and the other at a later time (3 months of age). This pattern of change in the hepatic arginase activity closely coincided with the pattern of the rate of urea synthesis determined with liver slices and expressed in terms of micromoles of urea formed per hour per gram of tissue slice. In contrast to the above observations, however, curves obtained by plotting the total liver arginase or urea synthetic activity vs the developmental stage of rats showed no measurable discontinuity. Further studies revealed that the observed pattern of specific activity of hepatic arginase was, in part, due to the change in the relative concentration of arginase protein in the liver.     

Mouse models and the evolutionary developmental biology of the skull

Understanding development is relevant to understanding evolutionbecause developmental processes structure the expression ofphenotypic variation upon which natural selection acts. Advancesin developmental biology are fueling a new synthesis of developmentaland evolutionary biology, but it remains unclear how to usedevelopmental information that largely derives from a few modelorganisms to test hypotheses about the evolutionary developmentalbiology of taxa such as humans and other primates that havenot been or are not amenable to direct study through experimentaldevelopmental biology. In this article, we discuss how and whenmodel organisms like mice are useful for studying the evolutionarydevelopmental biology of even rather distantly related and morphologicallydifferent groups like primates. A productive approach is tofocus on processes that are likely to play key roles in producingevolutionarily significant phenotypic variation across a largephylogenetic range. We illustrate this approach by applyingthe analysis of craniofacial variation in mouse mutant modelsto primate and human evolution.