Essential omega-3 fatty acids tune microglial phagocytosis of synaptic elements in the mouse developing brain.

Omega-3 fatty acids (n-3 PUFAs) are essential for the functional maturation of the brain. Westernization of dietary habits in both developed and developing countries is accompanied by a progressive reduction in dietary intake of n-3 PUFAs. Low maternal intake of n-3 PUFAs has been linked to neurodevelopmental diseases in Humans. However, the n-3 PUFAs deficiency-mediated mechanisms affecting the development of the central nervous system are poorly understood. Active microglial engulfment of synapses regulates brain development. Impaired synaptic pruning is associated with several neurodevelopmental disorders. Here, we identify a molecular mechanism for detrimental effects of low maternal n-3 PUFA intake on hippocampal development in mice. Our results show that maternal dietary n-3 PUFA deficiency increases microglia-mediated phagocytosis of synaptic elements in the rodent developing hippocampus, partly through the activation of 12/15-lipoxygenase (LOX)/12-HETE signaling, altering neuronal morphology and affecting cognitive performance of the offspring. These findings provide a mechanistic insight into neurodevelopmental defects caused by maternal n-3 PUFAs dietary deficiency.

Effects of low dose fluticasone/salmeterol combination on surrogate inflammatory markers in moderate persistent asthma.

BACKGROUND: Noninvasive surrogate markers provide valuable information on the asthmatic inflammatory process. We wished to examine the effects of low dose fluticasone/salmeterol combination on different commonly used inflammatory markers in moderate persistent asthma. METHODS: Twenty-five moderate persistent atopic asthmatics were enrolled of whom 20 completed an open label study. Following an initial 4 week steroid washout period in which patients took salmeterol 50 microg dry powder inhaler 1 puff BD, they received the addition of fluticasone as fluticasone 100 microg/salmeterol 50 microg combination dry powder inhaler 1 puff BD for the next 2 weeks. Exhaled nitric oxide, spirometry, methacholine PD20, sputum/blood eosinophils and sputum/serum eosinophil cationic protein (ECP) were measured following the salmeterol only and fluticasone/salmeterol combination treatment periods. RESULTS: Compared to salmeterol alone (i.e. after the steroid washout), the use of fluticasone/salmeterol combination conferred significant improvements (P < 0.05) in all surrogate markers of inflammation apart from serum ECP. Geometric mean fold changes were 4.3-fold/1.3-fold for sputum/blood eosinophils, 2.2-fold/1.2-fold for sputum/serum ECP, 2.3-fold for methacholine PD20 and 1.8-fold for exhaled nitric oxide. CONCLUSIONS: Surrogate markers apart from serum ECP may be used as a guide to evaluate the anti-inflammatory effects of low dose inhaled corticosteroids. Sputum markers tend to be more sensitive than blood when assessing the anti-inflammatory response.

Adrenal gland SNAP-25 expression is altered in thyroid hormone receptor knock-out mice.

SNAP-25 is a protein in neurons and neuroendocrine cells, which is involved, together with syntaxin and VAMP, in neurotransmitter release and neurite outgrowth. Since the thyroid hormone receptors TR alpha and TR beta are essential for nervous system development, their possible role in regulating the expression of these vesicle trafficking proteins was examined by analysing SNAP-25 levels in TR alpha and TR beta knock-out mice. Immunoblotting and RT-PCR showed that SNAP-25 levels are increased in the adrenal gland, but not in cerebellum, in knock-out mice, while syntaxin-1 and VAMP-2 are unaffected in either tissue. Treatment of the pheochromocytoma-derived cell line PC12 with the thyroid hormone L-3,5,3'-triiodothyronine (T3) decreased SNAP-25 expression. Together, these data suggest that thyroid hormones exert a negative regulatory effect on SNAP-25 in adrenal medullary neuroendocrine cells.

NGF enhances depolarization effects on SNAP-25 expression: induction of SNAP-25b isoform.

The 25 kDa synaptosomal associated protein (SNAP-25), which is implicated in neuronal plasticity and neurosecretion, exists as two isoforms generated by alternative splicing of exons 5a and 5b. The aim of the present study was to characterize factors influencing isoform expression. We report that chronic depolarization of PC12 cells alone or in the presence of NGF induces the expression of isoform-b, in addition to a 1.8- to 3-fold increase in SNAP-25 mRNA and protein as determined by immunoblotting and combined RT-PCR and Southern blot analysis. When cerebellar granule neurons were cultured in elevated K+, the predominant isoform switched from SNAP-25a to SNAP-25b. Taken together these results suggested that chronic depolarization regulates the transcription and processing of SNAP-25 mRNA.

The hypophysis controls expression of SNAP-25 and other SNAREs in the adrenal gland.

SNAP-25 (Synaptosomal Associated Protein of 25 kDa), in association with two other SNARE (soluble NSF attachment protein receptor) proteins, syntaxin and Vesicle Associated Membrane Protein, VAMP, is implicated in regulated and constitutive exocytosis in neurones and neuroendocrine cells. Our previous studies have shown that it is expressed more by noradrenergic than adrenergic chromaffin cells in the rat adrenal gland. Since certain hormones under hypophyseal control play an essential role in determining chromaffin cell phenotype, the present study examined the effect of hypophysectomy on SNAP-25 expression. Hypophysectomy was found by immunoblotting and RT-PCR analysis to increase adrenal gland SNAP-25, syntaxin-1 and VAMP-2 levels, without modifying the relative expression of SNAP-25 isoforms: immunocytochemistry showed a dramatic increase in SNAP-25 expression in former adrenergic chromaffin cells. Since adrenal glucocorticoids are considerably reduced by hypophysectomy, the effect of corticosterone replacement therapy was investigated. This did not change levels of SNAP-25, syntaxin-1 or VAMP-2. SNARE expression was also unmodified in pheochromocytoma cells treated with a synthetic glucocorticoid. In contrast, subcutaneous injection of hypophysectomized rats with thyroid hormone decreased adrenal SNAP-25, demonstrating the potential importance of the pituitary-thyroid axis. The current data thus demonstrate that the hypophysis exerts an inhibitory control on adrenal gland SNARE proteins. They suggest that glucocorticoids are unlikely to be directly responsible for this but provide evidence that thyroid hormones are implicated in this phenomenon. The putative role of hormonal regulation on SNARE function is discussed.

SNAP-25 regulation during adrenal gland development: comparison with differentiation markers and other SNAREs.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is one of a limited number of soluble N-ethylmaleimide-sensitive fusion attachment protein receptors (SNAREs) that play a major role in membrane docking of synaptic vesicles and secretory granules during regulated exocytosis. We have previously shown that SNAP-25 levels differ between noradrenergic and adrenergic chromaffin cell populations of the adult adrenal gland. We examine SNAP-25 expression by immunofluoresence in cells of the sympathoadrenal lineage in the rat during late embryonic and postnatal development. In parallel, tyrosine hydroxylase was used to identify sympathoadrenal cells, phenylethanolamine N-methyltransferase to distinguish adrenergic from noradrenergic chromaffin cells, and chromogranin A to define the presence of secretory granules. In addition, SNAP-25 protein and mRNA levels were followed in adrenal gland extracts by immunoblotting and reverse transcription-polymerase chain reaction (RT-PCR). Protein levels were compared with those of other molecules also implicated in organelle trafficking, including syntaxin 1 and vesicle-associated membrane protein (VAMP-2) and the nonneuronal analogues SNAP-23 and cellubrevin. This study provides evidence that SNAP-25 is expressed early during development in sympathoadrenal neurons and migrating cells. It is detected in intra-adrenal chromoblasts as soon as they enter the adrenal primordium. Its differential expression between catecholamine chromaffin cell phenotypes is already evident from the 17th embryonic day, future noradrenergic cells appearing to express higher levels than adrenergic cells. The granule maturation marker chromogranin A is expressed in chromaffin cells later than SNAP-25. Both SNAP-25 protein and mRNA increased rapidly in the adrenal gland in the perinatal period to peak during the first postnatal week, after which levels dropped dramatically to adult values. In contrast, levels of both syntaxin and SNAP-23 appeared to remain fairly constant throughout adrenal gland development. VAMP-2 expression increased gradually around birth to reach maximal levels during the first two postnatal weeks, and then decreased slightly. Cellubrevin levels also appeared to increase gradually until adult values were attained by the end of the second postnatal week. The threefold increase of SNAP-25 mRNA shortly after birth compared to the low adult levels suggests that during this period SNAP-25 is implicated in additional functions than regulated secretion, possibly associated with cellular growth or maturation.

How botulinum and tetanus neurotoxins block neurotransmitter release.

Botulinum neurotoxins (BoNT, serotypes A-G) and tetanus neurotoxin (TeNT) are bacterial proteins that comprise a light chain (M(r) approximately 50) disulfide linked to a heavy chain (M(r) approximately 100). By inhibiting neurotransmitter release at distinct synapses, these toxins cause two severe neuroparalytic diseases, tetanus and botulism. The cellular and molecular modes of action of these toxins have almost been deciphered. After binding to specific membrane acceptors, BoNTs and TeNT are internalized via endocytosis into nerve terminals. Subsequently, their light chain (a zinc-dependent endopeptidase) is translocated into the cytosolic compartment where it cleaves one of three essential proteins involved in the exocytotic machinery: vesicle associated membrane protein (also termed synaptobrevin), syntaxin, and synaptosomal associated protein of 25 kDa. The aim of this review is to explain how the proteolytic attack at specific sites of the targets for BoNTs and TeNT induces perturbations of the fusogenic SNARE complex dynamics and how these alterations can account for the inhibition of spontaneous and evoked quantal neurotransmitter release by the neurotoxins.

Molecular markers of sympathoadrenal cells.

Cells constituting the sympathoadrenal (SA) cell lineage originate from the neural crest and acquire a catecholaminergic fate following migration to the dorsal aorta. Subsequently, SA cells migrate to sites widely dispersed throughout the body. In addition to endocrine chromaffin and "small intensely fluorescent" cells in adrenal glands and in extra-adrenal tissues such as the paraganglia, this lineage also includes neurones located in sympathetic ganglia and in the adrenal gland. It is widely assumed that these cells are all derived from the same precursors, which then differentiate along divergent pathways in response to different external stimuli. During embryonic differentiation, SA cells lose some of their early traits and acquire other distinguishing features. To help understand how the lineage diverges in terms of phenotype and function, this article examines the cellular expression of a variety of "marker" proteins that characterize the individuals of the lineage. In particular, differences between adrenal medullary adrenergic and noradrenergic chromaffin cells in the expression of proteins, such as the neural adhesion molecule L1, the growth-associated protein GAP-43 and molecules involved in the secretory process, are emphasized. Factors that might differentially regulate such molecular markers in these cells are discussed.

Cultured glial cells express the SNAP-25 analogue SNAP-23.

Astrocytes release glutamate and aspartate in response to elevated intracellular calcium levels, and it has been proposed that this occurs by a vesicular release mechanism, in which SNARE proteins are implicated. Although syntaxin, synaptobrevin, and cellubrevin have been shown to be expressed by cultured astrocytes, SNAP-25 has not been detected. By using immunocytochemical, immunoblotting, and polymerase chain reaction techniques, the present study demonstrates that SNAP-23, an analogue of SNAP-25, is expressed by astrocytes both in culture and in rat cerebellum. These findings provide additional evidence that astrocytes release excitatory amino acids by a vesicular mechanism involving SNARE proteins. SNAP-23 and also syntaxin 1 and cellubrevin were found to be expressed in glial precursor cells, oligodendrocytes, and microglia. These data suggest that the t-SNAREs SNAP-23 and syntaxin 1 and the v-SNARE cellubrevin participate in general membrane insertion mechanisms involved in diverse glial cell functions such as secretion, phagocytosis, and myelinogenesis.

Differential expression of SNAP-25 isoforms and SNAP-23 in the adrenal gland.

In the rat adrenal gland, we previously observed that SNAP-25 is not restricted to the plasmalemma in noradrenergic cells as it is in adrenergic cells, and hypothesized that SNAP-25 isoform expression is different in the two phenotypes. Expression of SNAP-25 isoforms and SNAP-23 was examined by immunoblotting, immunofluorescence, and RT-PCR. Amplifications of SNAP-25 mRNAs were combined with Southern hybridization, restriction enzyme analysis, and sequencing of cloned PCR products to compare SNAP-25 isoform expression in rat and bovine adrenal glands. SNAP-25 and SNAP-23 mRNA and protein are expressed in the glands; SNAP-23 is enriched in the adrenal cortex, whereas SNAP-25 is restricted to the adrenal medulla. Furthermore, high levels of SNAP-25 and low levels of SNAP-23 are observed in the PC12 cells, whereas both SNAP-25 and SNAP-23 are expressed in adrenal medullary cultures. In all extracts, the SNAP-23 mRNA corresponded to SNAP-23a. SNAP-25a is the major form expressed in rat adrenal glands (75%), as it is in PC12 cells (80%), but both SNAP-25a and SNAP-25b (40% vs. 60%) are expressed in bovine adrenal medulla in situ and in culture. In addition, an enriched population of adrenergic cells (93%) expressed a higher level of SNAP-25b (70%), suggesting that this isoform may not be restricted to fast neurotransmission.

Shoot regeneration from leaves of Prunus serotina Ehrh. (black cherry) and P. avium L. (wild cherry).

Leaves excised from shoot cultures of Prunus avium cvs. F12/1 and Charger and genotype 1908, and from five genotypes of P. serotina and two hybrids of P. avium×P. sargentii developed shoots on Woody Plant medium (WPM) supplemented with either benzyladenine (BA) or thidiazuron (TDZ). Regeneration in both P. avium 1908 and a genotype of P. serotina was improved using TDZ rather than BA in the medium. Regeneration occurred more frequently in P. serotina if leaves were cultured on medium with WPM rather than modified Driver and Kuniyuki walnut medium. The proportions of leaves that regenerated varied between genotypes of the same species. Regenerated shoots of both P. avium and P. serotina developed into shoot cultures following transfer to the media used to produce the shoot cultures used as explant sources.

Are exocytosis mechanisms neurotransmitter specific?

Neurotransmission is a multistage regulated process in which a variety of active molecules contained in vesicles are liberated in response to specific stimuli from different types of neurone or related cells. This includes the release of fast neurotransmitters such as amino acids and acetylcholine from central and peripheral synapses, but also that of relatively slow-acting polypeptides from central and peripheral neurones or neuroendocrine cells. Considerable progress has been made over recent years in the understanding at a molecular level of the mechanism of regulated exocytosis, a crucial phase in this phenomenon. The currently proposed overall mechanism, which incorporates the "SNARE" hypothesis for vesicle-membrane docking and fusion, is based on data from experimental models ranging from brain synaptosomes to mast cells. Since the kinetics of the models studied and the physiological effects of the neurotransmitters implicated vary so much, it is pertinent to question whether a general mechanism can be proposed from such experimental data. This review examines known differences in putative exocytotic mechanisms for the various systems studied and attempts to relate these to the nature of the active substances released. Differences exist in each step of the exocytosis process and include the channel through which Ca2+ enters to trigger it or the internal Ca2- source, the type of vesicle in which the transmitter is packaged, the way vesicles are translocated to the surface membrane or how they dock and fuse with it. Major differences have been reported in release mechanisms of different types of vesicle, but minor differences also exist within the same vesicle class. Thus small synaptic vesicles and large dense core vesicles are translocated by distinct processes and the Ca2+ channels, Ca2+ sensors and docking proteins involved in other steps are not identical in all neuronal phenotypes. It may be concluded that each of these differences has evolved to accommodate the different physiological requirements of the neuromodulator released.

Mucosal inflammation in pediatric diversion colitis: a quantitative analysis.

BACKGROUND: Diversion colitis commonly occurs in bypassed segments of colorectum, and has been described qualitatively in Hirschsprung's disease patients with colostomies. The objective of this study was to characterize quantitatively the changes in the inflammatory cell population in the mucosa of children with diversion colitis. METHODS: Paraffin blocks of well-oriented, full-thickness colorectal tissues were obtained from 15 children with diversion colitis (all with Hirschsprung's disease), four pediatric controls and four adult controls. Sections were immunostained for B and T lymphocytes, macrophages, IgG, IgM, and IgA. Measurements were made referent to a standard length of muscularis mucosae. Lymphoid follicles were counted and the areas occupied by B and T cells were determined by image analysis. Cells in the interfollicular lamina propria were counted separately, but IgA-containing plasma cells were too abundant to enumerate. RESULTS: Pediatric diversion colitis was characterized by enlarged and more numerous lymphoid follicles with approximately four times as many B lymphocytes and twice as many T lymphocytes in the follicular compartment of the mucosa when compared to pediatric controls. The interfollicular mucosa was thickened (499 +/- 27 versus 380 +/- 56 microns) and contained approximately six times as many B cells and eight times as many T cells as controls. Macrophages and plasma cells containing IgG and IgM were not significantly increased. CONCLUSIONS: These findings extend the qualitative observations of increased follicular and lamina propria lymphoid tissue in bypassed segments of colon, and are consistent with the hypothesis of persistent antigenic stimulation of the mucosa-associated lymphoid tissue.

SNAP-25 is differentially expressed by noradrenergic and adrenergic chromaffin cells.

This study examines chromaffin cell expression of the synaptosomal-associated protein SNAP-25 in the adrenal medulla by immunoblotting, immunocytochemistry and PCR. Both mRNAs coding for the SNAP-25 isoforms a and b were detected and SNAP-25 was found to be present in all chromaffin cells in adult rat adrenal gland sections. It was essentially restricted to a zone close to the cytoplasmic face of the plasma membrane in the majority of cells, but located extensively throughout the cytoplasm in a chromaffin cell sub-population, identified by double immunofluorescence labelling to have a noradrenergic phenotype. This differential SNAP-25 expression may reflect different stages in the phenotypic development of the sympathoadrenal lineage and be related to an additional functional role in noradrenergic chromaffin cells not associated with secretion.

Glucocorticoids and nerve growth factor differentially modulate cell adhesion molecule L1 expression in PC12 cells.

The differential expression of the cell adhesion molecule L1 by chromaffin cells has recently been suggested to be responsible for the segregation of chromaffin cells into homotypic catecholaminergic groups in the adrenal gland. The present study was undertaken to test the hypothesis that glucocorticoids, which increase in the adrenal gland during development, could be responsible for the repression of L1 in adrenergic chromaffin cells. PC12 cells were used as the experimental model, and relative L1 protein and mRNA levels were examined after treating the cells with glucocorticoids or NGF. Analysis of western blots indicated that glucocorticoids decreased the L1 protein levels by one-half, whereas NGF increased L1 protein levels approximately 2.3-fold. In addition, the glucocorticoids inhibited both the NGF induction of the neurite outgrowth and the increase in L1 expression. Analysis of the mRNA levels by PCR and northern blots indicated that glucocorticoids reduced the L1 mRNA, whereas NGF increased the level of L1 mRNA. Maximal inhibition of L1 expression was observed at concentrations of 10(-7) M dexamethasone, and the decrease occurred during the second day of treatment. The effects of dibutyryl cyclic AMP and phorbol ester on the glucocorticoid and NGF regulation of L1 protein were also examined. This is the first report indicating that L1 expression can be down-regulated by glucocorticoids. The results support the hypothesis that during development the repression of L1 in adrenergic chromaffin cells may be, in part, linked to the increase in glucocorticoid levels in the adrenal gland.

Expression of GAP-43 (neuromodulin) during the development of the rat adrenal gland.

The 'growth-associated protein', GAP-43 was originally considered to be a neuron-specific protein associated with plasticity. However, we have recently shown that GAP-43 is expressed by noradrenergic, but not by adrenergic chromaffin cells in the adult rat adrenal gland. In this study, we examine the expression of GAP-43 during embryonic and post-natal development of the adrenal gland using immunohistochemical techniques. In parallel, antibodies directed against two neuroendocrine markers, the catecholamine-synthesizing enzymes, tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) were employed to permit identification of the developing chromaffin cell phenotypes. At embryonic day 15.5, GAP-43 was predominately localized in sympathoadrenergic precursor cells in the extra-adrenal blastema, and also in nerve fibers within the adrenal gland. At later embryonic stages, GAP-43 was expressed by nearly all intra-adrenal chromoblasts. Two subsets of chromoblasts can be distinguished even at early stages. A strong GAP-43-positive immunoreaction was observed in those chromoblasts organized in a few large compact clusters which weakly expressed TH and did not express PNMT. A generally weaker GAP-43 immunoreaction was observed in a second type of intra-adrenal chromoblasts which were organized in small isolated groups and characterized by a PNMT-positive, and strong TH-positive immunoreactivity. GAP-43 immunoreactivity was still associated with many PNMT-positive adrenergic chromoblasts at birth, but decreased to undetectable levels during the first post-natal week. By the second post-natal week, GAP-43 was restricted, as in the adult, to noradrenergic chromaffin cells which expressed TH, but not PNMT, in addition to nerve fibers and their associated glial cells in the gland. An immunoblot analysis confirmed a decrease in GAP-43 protein during the post-natal period. In agreement with these observations, a three-fold decrease in GAP-43 mRNA in the adrenal gland was measured between late embryogenesis and the second post-natal week. During development, the spatiotemporal expression of GAP-43 suggests a possible role in the migration and aggregation of chromaffin cell precursors into the medullary region of the adrenal gland.

Expression of cell adhesion molecules and catecholamine synthesizing enzymes in the developing rat adrenal gland.

Cell adhesion molecules play a major role in determining tissue architecture during histogenesis. This immunocytochemical study of the adrenal gland examines the embryonic and early postnatal cellular expression of two neural cell adhesion molecules, NCAM and L1, which are widely expressed in brain and have been found also to be expressed in the adult rat adrenal gland. In parallel, antibodies directed against two neuroendocrine cell markers, tyrosine hydroxylase and phenylethanolamine N-methyltransferase, were employed to verify the phenotypic nature of developing chromaffin cells in order to correlate cell adhesion molecule expression with the state of chromaffin cell differentiation. NCAM was found to be expressed by chromoblasts within extra-adrenal blastema (i.e. before their migration into the cortical primordium) at the 16th day of embryonic life. It continued to be expressed by all developing chromaffin cells after their infiltration into the developing adrenal gland at all ages. L1 was also expressed by chromoblasts in extra-adrenal sites, but was found only in a subpopulation of chromaffin cells within the cortical primordium from the 16th embryonic day onwards. Those chromoblasts which expressed L1 constituted relatively large compact cell clusters within the gland at this stage, while intra-adrenal chromaffin cells not expressing L1 were dispersed in small cell groups. L1 was also strongly expressed by nerve fibres (and their surrounding Schwann cells) which appeared to innervate cell groups as early as the 16th embryonic day. Both extra- and intra-adrenal chromoblasts expressed tyrosine hydroxylase, but the large L1-positive cell aggregates were less intensely immunoreactive for tyrosine hydroxylase than were cells in small groups. PNMT expression was restricted to L1-negative intra-adrenal chromoblasts present in small groups. Ultrastructural observations demonstrated that cells expressing L1 contained few secretory granules at the 18th embryonic day. It is concluded from these data that these chromoblasts are the precursors of the noradrenergic cells found in the mature gland. In addition, the arrangement of noradrenergic chromaffin cells in the form of homotypic cell groups throughout the course of histogenesis of the adrenal medulla is likely to be a direct consequence of the exclusive co-expression of both NCAM and L1 by this subpopulation of maturing chromaffin cells.

Cellular localization of the neural cell adhesion molecule L1 in adult rat neuroendocrine and endocrine tissues: comparisons with NCAM.

The tissue distribution and cellular localization of the neural cell adhesion molecule L1 was determined by immunocytochemistry at the optical and ultrastructural levels in adult rat neuroendocrine tissues and pancreatic endocrine cells. L1 was found to be abundant in the neurohypophysis but undetectable in the rest of the pituitary gland. It was barely detectable in the normal rat endocrine pancreas, but a rat pancreatic insulinoma cell line was found by immunofluorescence to express low levels of L1. In the adrenal medulla, it was present on a sub-population of chromaffin cells and its density appeared to be lower on surfaces exposed to the extracellular matrix. Double immunolabelling showed this sub-population to consist of noradrenergic chromaffin cells. Adrenergic chromaffin cells were found not to express L1. In addition, the tissue distribution and cellular localization of NCAM mRNAs was determined by in situ hybridization, extending our previous studies on the cellular expression of NCAM proteins in endocrine and neuroendocrine tissues. This confirmed that the NCAM message has a wider cellular distribution than L1 within the hypophysis and the adrenal gland. In addition to secretory cells, L1 immunoreactivity was detected in glial cells, in particular in the pituicytes of the neurohypophysis, which further distinguishes them from astrocytes, their counterparts in the central nervous system. These data are discussed in terms of the different embryological origins of the various endocrine tissues examined and also in terms of the specific design constraints imposed on these tissues during their development.