Patterning the vertebrate neuraxis

Neuraxial patterning is a continuous process that extends over a protracted period of development. During gastrulation a crude anteroposterior pattern, detectable by molecular markers, is conferred on the neuroectoderm by signals from the endomesoderm that are largely inseparable from those of neural induction itself. This coarse-grained pattern is subsequently reinforced and refined by diverse, locally acting mechanisms. Segmentation and long-range signaling from organizing centers are prominent among the emerging principles governing regional pattern.     

Characterization of two patched receptors for the vertebrate hedgehog protein family

The multitransmembrane protein Patched (PTCH) is the receptor for Sonic Hedgehog (Shh), a secreted molecule implicated in the formation of embryonic structures and in tumorigenesis. Current models suggest that binding of Shh to PTCH prevents the normal inhibition of the seven-transmembrane-protein Smoothened (SMO) by PTCH. According to this model, the inhibition of SMO signaling is relieved after mutational inactivation of PTCH in the basal cell nevus syndrome. Recently, PTCH2, a molecule with sequence homology to PTCH, has been identified. To characterize both PTCH molecules with respect to the various Hedgehog proteins, we have isolated the human PTCH2 gene. Biochemical analysis of PTCH and PTCH2 shows that they both bind to all hedgehog family members with similar affinity and that they can form a complex with SMO. However, the expression patterns of PTCH and PTCH2 do not fully overlap. While PTCH is expressed throughout the mouse embryo, PTCH2 is found at high levels in the skin and in spermatocytes. Because Desert Hedgehog (Dhh) is expressed specifically in the testis and is required for germ cell development, it is likely that PTCH2 mediates its activity in vivo. Chromosomal localization of PTCH2 places it on chromosome 1p33-34, a region deleted in some germ cell tumors, raising the possibility that PTCH2 may be a tumor suppressor in Dhh target cells.     

Isolation of a Drosophila homolog of the vertebrate homeobox gene Rx and its possible role in brain and eye development

Vertebrate and invertebrate eye development require the activity of several evolutionarily conserved genes. Among these the Pax-6 genes play a major role in the genetic control of eye development. Mutations in Pax-6 genes affect eye development in humans, mice, and Drosophila, and misexpression of Pax-6 genes in Drosophila can induce ectopic eyes. Here we report the identification of a paired-like homeobox gene, DRx, which is also conserved from flies to vertebrates. Highly conserved domains in the Drosophila protein are the octapeptide, the identical homeodomain, the carboxyl-terminal OAR domain, and a newly identified Rx domain. DRx is expressed in the embryo in the procephalic region and in the clypeolabrum from stage 8 on and later in the brain and the central nervous system. Compared with eyeless, the DRx expression in the embryo starts earlier, similar to the pattern in vertebrates, where Rx expression precedes Pax-6 expression. Because the vertebrate Rx genes have a function during brain and eye development, it was proposed that DRx has a similar function. The DRx expression pattern argues for a conserved function at least during brain development, but we could not detect any expression in the embryonic eye primordia or in the larval eye imaginal discs. Therefore DRx could be considered as a homolog of vertebrate Rx genes. The Rx genes might be involved in brain patterning processes and specify eye fields in different phyla.     

Patterning of the neural ectoderm of Xenopus laevis by the amino-terminal product of hedgehog autoproteolytic cleavage

The patterns of embryonic expression and the activities of Xenopus members of the hedgehog gene family are suggestive of role in neural induction and patterning. We report that these hedgehog polypeptides undergo autoproteolytic cleavage. Injection into embryos of mRNAs encoding Xenopus banded-hedgehog (X-bhh) or the amino-terminal domain (N) demonstrates that the direct inductive activities of X-bhh are encoded by N. In addition, both N and X-bhh pattern neural tissue by elevating expression of anterior neural genes. Unexpectedly, an internal deletion of X-bhh (delta N-C) was found to block the activity of X-bhh and N in explants and to reduce dorsoanterior structures in embryos. As elevated hedgehog activity increases the expression of anterior neural genes, and as delta N-C reduces dorsoanterior structures, these complementary data support a role for hedgehog in neural induction and anteroposterior patterning.     

Intrinsic programmes of growth and survival in developing vertebrate neurons

Neurons become dependent on a supply of target-derived neurotrophins for survival when their axons reach their targets in development. Because the distance axons have to grow varies from one population of neurons to another, the timing of dependence on neurotrophins likewise varies. Although it would be expected that the simplest way of getting the timing right would be for the target to provide a suitable signal to the arriving axons, detailed studies on developing cranial sensory neurons suggest that these neurons are programmed, before differentiation, to acquire dependence at the correct time independently of external signals. Neurons not only partly compensate for different target distances by extending axons more rapidly the further they have to grow, but possess an intrinsic clock that switches on dependence at the right time in accordance with the time it normally takes their axons to reach their targets. Here the experimental evidence for these intrinsic programmes of growth and survival is reviewed, the rationale that might have favoured their evolution is discussed, and parallels are drawn with other developing neuronal systems.     

A regional study of developing rat brain: the accumulation and distribution of proteolipid proteins

The accumulation and distribution of proteolipid proteins in rat brain and selected brain regions (cerebellum, cerebral cortex, basal ganglia, and hippocampus) were studied during early postnatal development. In whole brain an eightfold increase of proteolipid was observed between ten and 33 days after birth. This was reflected in the separate regions examined where the proteolipid protein content increased six- to ten-fold during the same period. The basal ganglia and cerebral cortex contributed the greatest amount to the total proteolipid present. However, at 28-33 days the greatest concentration (mg/g tissue) was observed in the basal ganglia and hippocampus. When the proteolipid protein preparations were examined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, distinctive, heterogeneous patterns for each brain region were obtained. Proteolipid from basal ganglia (the region richest in white matter) consisted primarily of two major protein bands with apparent molecular weights of approximately 21,500 and 26,000. Both of these bands dramatically increased in quantity during myelination, and the larger protein coelectrophoresed with isolated myelin proteolipid protein. Both bands were also found present in proteolipid preparations from the other brain regions but in varying amounts relative to the total. The data suggest that the increase in proteolipid observed during this developmental period was due in large measure to the accumulation of myelin-specific proteolipids, but also that a significant proportion of the increase was due to the accumulation of nonmyelin components.     

Gene-expression screens in vertebrate embryos: more than meets the eye

The health control of the travellers and medical assistance for them and their families is one of the oldest and most fundamental missions of the "Office des migrations internationales" (OMI) as defined by the reglementary treaties of November 1945 and June 1946. More recent reglementations have broadened the competence of the OMI to include certain categories of foreigners who had been exempt from all controls. Finally, the ministerial order of November the 7th 1994 specifies means of the health screening. Medical examinations are carried in various French and foreign institutions. It nowadays has become a consultation aiming at prevention and orientation. The medical examination has three principal objectives: the detection of little known abnormalities; the contact between the medical service of the OMI and regional services in charge of the first check-up; the health education of the examined persons. The clinical and paraclinical results are communicated to the patient who is also informed of the most serious health questions, as well as ways of gaining access to medical care in France; this is done by taking into account all medical parameters and health conditions prevailing in the patient's native country. All detected abnormalities are brought to the attention of the head physician of the OMI who in turn informs the medical inspector of the "Direction départementale des affires sanitaires et sociales" (DDASS) in charge of making sure that the migrant benefits from health and social assistance and receives medical treatment. All pathological results are given to the examined persons in form of a written and confidential report, enabling them to visit a doctor of their choice. A network has thus been built up throughout the various departments involved in the first medical examination and the DDASS has made available for the OMI medical staff listings of public institutions likely to welcome the migrants.     

Choline availability modulates the expression of TGFbeta1 and cytoskeletal proteins in the hippocampus of developing rat brain

Choline availability influences long-term memory in concert with changes in the spatial organization and morphology of septal neurons, however little is known concerning the effects of choline on the hippocampus, a region of the brain also important for memory performance. Pregnant rats on gestational day 12 were fed a choline control (CT), choline supplemented (CS), or choline deficient (CD) diet for 6 days and fetal brain slices were prepared on embryonic day 18 (E18). The hippocampus in these brain slices was studied for the immunohistochemical localization of the growth-related proteins transforming growth factor beta type 1 (TGFbeta1) and GAP43, the cytoskeletal proteins vimentin and microtubule associated protein type 1 (MAP1), and the neuronal cell marker neuron specific enolase (NSE). In control hippocampus, there was weak expression of TGFbeta1 and vimentin proteins, but moderately intense expression of MAP1 protein. These proteins were not homogeneously distributed, but were preferentially localized to cells with large cell bodies located in the central (approximately CA1-CA3) region of the hippocampus, and to the filamentous processes of small cells in the fimbria region. Feeding a choline-supplemented diet decreased, whereas a choline-deficient diet increased the intensity of immunohistochemical labeling for these proteins in E18 hippocampus. GAP43 and NSE were localized to peripheral nervous tissue but not hippocampus, indicating that the maturation of axons and neurite outgrowth in embryonic hippocampus were unaffected by the availability of choline in the diet. These data suggest that the availability of choline affects the differentiation of specific regions of developing hippocampus.     

Emerging roles for SH2/PTB-containing Shc adaptor proteins in the developing mammalian brain

In mammalian systems, SH2-containing cytoplasmic signalling molecules are known to play an important role in determining cell responsiveness to the environment. In particular, following activation of a receptor protein tyrosine kinase (RPTK), proteins like Shc and Grb2 bind to phosphotyrosine residues of stimulated receptors, thereby activating downstream components of specific signalling pathways. The ShcA gene was identified in 1992 and was found to encode three proteins with properties of adaptor molecules coupling RPTKs to Ras. Early data obtained in non-neuronal cells have revealed that Shc and Grb2 proteins are highly expressed and activated in all cells. However, recent analyses of ShcA mRNA and protein in the developing brain revealed progressive downregulation of their expression during differentiation from neuroblasts to neurons. Conversely, the two newly identified Shc homologues (ShcB/Sli and ShcC/Rai) are highly expressed in the mature brain.Thus, variations in the intracellular levels of adaptor proteins might represent one of the mechanisms by which a differentiating cell changes its ability to respond to a given factor, allowing a cell to choose between proliferation and differentiation.     

Sexual differentiation of the vertebrate brain: principles and mechanisms

A wide variety of sexual dimorphisms, structural differences between the sexes, have been described in the brains of many vertebrate species, including humans. In animal models of neural sexual dimorphism, gonadal steroid hormones, specifically androgens, play a crucial role in engendering these differences by masculinizing the nervous system of males. Usually, the androgen must act early in life, often during the fetal period to masculinize the nervous system and behavior. However, there are a few examples of androgen, in adulthood, masculinizing both the structure of the nervous system and behavior. In the modal pattern, androgens are required both during development and adulthood to fully masculinize brain structure and behavior. In rodent models of neural sexual dimorphism, it is often the aromatized metabolites of androgen, i.e., estrogens, which interact with estrogen receptors to masculinize the brain, but there is little evidence that aromatized metabolites of androgen play this role in primates, including humans. There are other animal models where androgens themselves masculinize the nervous system through interaction with androgen receptors. In the course of masculinizing the nervous system, steroids can affect a wide variety of cellular mechanisms, including neurogenesis, cell death, cell migration, synapse formation, synapse elimination, and cell differentiation. In animal models, there are no known examples where only a single neural center displays sexual dimorphism. Rather, each case of sexual dimorphism seems to be part of a distributed network of sexually dimorphic neuronal populations which normally interact with each other. Finally, there is ample evidence of sexual dimorphism in the human brain, as sex differences in behavior would require, but there has not yet been any definitive proof that steroids acting early in development directly masculinize the human brain.     

Pax proteins and eye development

Male and female rats were used to investigate the effects of type of dietary carbohydrate (CHO), copper, and ethanol consumption on lung antioxidant enzyme activities and levels of phosphorylated compounds in whole blood. Copper-deficient female rats exhibited a greater degree of copper deficiency than males, as assessed by hepatic copper concentration and hepatic copper superoxide dismutase (CuSOD) activity. However, copper-deficient male rats fed fructose-containing diets exhibited greater growth retardation, anemia, and heart hypertrophy than females consuming the same diets and males fed starch. In addition, one of 10 copper-deficient male rats that ate a fructose-based diet and drank water and one of 10 copper-deficient male rats that ate a starch-based diet and drank ethanol died. Copper-deficient, starch-fed males exhibited the highest activities of glutathione peroxidase (GSH-Px) and catalase as compared with fructose-fed rats. Ethanol consumption elevated the activities of GSH-Px and catalase. Copper-deficient female rats exhibited higher catalase but lower GSH-Px activities than males. It is suggested that in copper deficiency, the ability to increase antioxidant enzyme activities in rats consuming starch is greater than in rats consuming fructose. Rats fed starch are provided with a greater degree of protection against oxidative damage than rats fed fructose. In addition, polyphosphorylated compounds in blood were reduced in copper-deficient male rats that consumed fructose-based diets. This may impair supply of oxygen to tissues.     

Regulation of netrin-1a expression by hedgehog proteins

Netrins, a family of growth cone guidance molecules, are expressed both in the ventral neural tube and in subsets of mesodermal cells. In an effort to better understand the regulation of netrins, we examined the expression of netrin-1a in mutant cyclops, no tail, and floating head zebrafish embryos, in which axial midline structures are perturbed. Netrin-1a expression requires signals present in notochord and floor plate cells. In the myotome, but not the neural tube, netrin-1a expression requires sonic hedgehog. In embryos lacking sonic hedgehog, the sonic-you locus, netrin-1a expression is reduced or absent in the myotomes but present in the neural tube. Embryos lacking sonic hedgehog express tiggy-winkle hedgehog in the floor plate, suggesting that, in the neural tube, tiggy-winkle hedgehog can compensate for the lack of sonic hedgehog in inducing netrin-1a expression. Ectopic expression of sonic hedgehog, tiggy-winkle hedgehog, or echidna hedgehog induces ectopic netrin-1a expression in the neural tube, and ectopic expression of sonic hedgehog or tiggy-winkle hedgehog, but not echidna hedgehog, induces ectopic netrin-1a expression in somites. These data demonstrate that in vertebrates netrin expression is regulated by Hedgehog signaling.     

Patterning of mammalian somites by surface ectoderm and notochord: evidence for sclerotome induction by a hedgehog homolog

An early step in the development of vertebrae, ribs, muscle, and dermis is the differentiation of the somitic mesoderm into dermomyotome dorsally and sclerotome ventrally. To analyze this process, we have developed an in vitro assay for somitic mesoderm differentiation. We show that sclerotomal markers can be induced by a diffusible factor secreted by notochord and floor plate and that heterologous cells expressing Sonic hedgehog (shh/vhh-1) mimic this effect. In contrast, expression of dermomyotomal markers can be caused by a contact-dependent signal from surface ectoderm and a diffusible signal from dorsal neural tube. Our results extend previous studies by suggesting that dorsoventral patterning of somites involves the coordinate action of multiple dorsalizing and ventralizing signals and that a diffusible form of Shh/Vhh-1 mediates sclerotome induction.     

Characterization of glucose transporter isoforms in the adult and developing human eye

The expression of glucose transporter isoforms (Glut 1, Glut 3, Glut 4, and Glut 5) in the human eye was investigated at various ages ranging between 8 weeks gestation (first trimester) and adult using Western blot and immunohistochemical analyses. Glut 1 and Glut 3 expression and cellular localization patterns were similar to those of human brain. Glut 1 (50-kilodalton protein) was expressed by epithelial cells (retinal pigmented epithelium, choroidal, iridial, and pars planus), which form the blood-eye barrier, retinal Mueller cells, the lens fiber cells, iridial microvascular endothelial cells, and to a lesser extent by the outer segments of the photoreceptor cells in the adult eye. This pattern was conserved throughout development and was evident as early as 8 weeks gestation. In addition, the endothelial cells of vitreous hyaloid vessels expressed Glut 1 at 8 weeks gestation. Glut 3 (50 to 55-kilodalton protein) immunoreactivity was observed only in the adult inner synaptic layer of the retina. Neither Glut 4 nor Glut 5 was expressed in any occular tissue at any age examined. These results suggest that Glut 1 is the main glucose transporter of the human eye and that it is ontogenically conserved. In contrast, Glut 3 is associated with selective neuronal processes, and its expression is developmentally altered.     

Alteration in 5'-nucleotidase activities and composition of liver and brain microsomes of developing rats fed different dietary fats

Four groups of male weanling rats were fed during three months, diets different in the nature of fats and the activity of 5' nucleotidase and fatty acid composition of brain and liver microsomes were studied. Group A were fed a standard commercial diet, group B a fat free-diet and group C and D a fat free-diet, containing respectively 10% of peanut-rapeseed oil and 10% of salmon oil. In brain and liver microsomes, 5'-nucleotidase activity increased throughout the development for all diets (except for the fat-free diet). Slight differences were found in rats fed the peanut-rapeseed oil diet compared to controls estimated at the same time. However, in animals fed the fish-oil diet, 5' nucleotidase had the highest activity in both brain and liver microsomes. Marked changes occurred in the fatty acid patterns of brain and liver microsomes among the various groups. The greatest alterations were found in the liver microsomes. In brain and liver microsomal membranes the fat-free diet induced an increase in monounsaturated fatty acids, an synthesis of eicosatrienoic acid, and a decrease in (n-6) and (n-3) polyunsaturated fatty acids. Animals fed a peanut-rapeseed oil and control diet showed similar fatty acid patterns in liver and brain microsomes. However, when rats were fed a fish-oil diet, the liver microsomal membranes were highly enriched in eicosapentaenoic and docosahexaenoic acids, and simultaneously there was a decrease in arachidonic acid. These results suggest that manipulation of the lipid environment influences 5'-nucleotidase activity by the interaction of the enzyme with specific membrane lipids.     

Androgen and estrogen receptors in the developing mouse brain

Specific binding of the androgens, 5alpha-dihydrotestosterone (DHT) and testosterone, and of 17beta-estradiol by brain cytosol from mice at 3-5,9-11, and 18-23 days of age was measured by charcoal assay and glycerol gradient centrifugation and analyzed by Scatchard plots. The immature mouse brain contains putative receptors for these steroids which migrate at 8 S in gradients at low ionic strength and at 5 S in 0.5 M KCl. Investigation of estradiol binding was complicated by the presence in cytosol from 3-5 day-old mice, and to a lesser extent from 9-11 day-old mice, of the high capacity, fetoneonatal estradiol binding protein (FEBP) which is no longer detectable at 3 weeks. The rapid dissociation of the FEBP-estradiol complex under non-equilibrium conditions probably led to over-estimation of free steroid concentration and thus to an apparent increase in the affinity of 8 S receptor for estradiol with age (for female brain cytosol KD=9.5 X 10(-10)M at 3-5 days and 2.7 X 10(-10)M at 18-23 days). The number of estradiol binding sites remains relatively constant during the first 3 weeks at 7-9 fmol/mg protein, while the number of DHT binding sites in female brain increases from 3.2+/-0.3 to 6.6+/-0.9 to 9.6+/-0.3 (mean+/-SE) fmol/mg protein in the 3 age groups. Dissociation constants and numbers of sites for both DHT and estradiol binding are similar in brain cytosol from male and female mice. Testosterone and DHT compete for the same binding site, but its affinity for DHT is about twice that for testosterone. The high affinity of the brain receptor for DHT (KD=4-5 X 10(-10)M) may reflect the slow metabolism of DHT to 5alpha-androstanediols, amounting to less than 10% after 2 h at 0 C. Binding of DHT and estradiol to cytosol from brain regions was also investigated. DHT receptors increase in parallel in various regions with age; the concentration of sites in the hypothalamus-preoptic area (HPOA) is 1.2-3.4 times that in the cerebral cortex (C). The concentration of estradiol binding sites in HPOA to that in C increases about 12-fold from neonatal to adult stages, reflecting both an increase in HPOA sites and a decrease in C sites, while the concentration in the remainder of the brain shows little change. Androgen and estrogen receptors in brain cytosol from immature mice can be distinguished by their different specificities and developmental patterns in whole brain and brain regions. The presence and properties of these receptors in the brain of neonatal mice are discussed with respect to their possible role in sexual differentiation of the brain.     

Organising activities of engrailed, hedgehog, wingless and decapentaplegic in the genital discs of Drosophila melanogaster

The genes engrailed (en), hedgehog (hh), wingless (wg) and decapentaplegic (dpp) have been shown to play vital organising roles in the development and differentiation of thoracic imaginal discs. We have analysed the roles of these genes in organising the development and differentiation of the genital discs, which are bilaterally symmetrical and possess different primordia, namely, the male and female genital primordia and an anal primordium. Our results suggest that the organising activity of en in genital discs programs the normal development and differentiation of the genital disc by regulating the expression of hh. Hh in turn induces wg and dpp, the genes whose products act as secondary signalling molecules. Moreover, the complementary patterns of wg and dpp expression are essential for the bilateral symmetry and are maintained by mutual repression.