Development Of Neural Crest Derivatives Research Articles

Expression of Toll-like receptors 4 and 7 in murine peripheral nervous system development

BackgroundToll-Like Receptors (TLRs) play a critical role in the innate and adaptive immune system. They are the mammalian orthologs of Drosophila melanogaster protein Toll, which has been proved to have an early morphogenetic role in invertebrate embryogenesis that in the adult switches to an immune function. AimThe aim of this study was to evaluate the expression of TLR4 and TLR7 during dorsal root ganglia (DRG), paravertebral ganglia (PVG), and enteric nervous system (ENS) murine development. MethodsMouse embryos from different stages (i.e. E12 to E18) were processed for immunolocalization analysis on formalin-fixed paraffin-embedded sections, and isolated intestine were processed for whole-mount preparations. ResultsWe observed a differentially regulated expression of TLR4 and TLR7 during embryogenesis and an overall increased expression of both receptors during development. While TLR4 was detectable in neurons of DRG and PVG starting from E14 and only from E18 in the ENS, TLR7 was already expressed in scattered neurons of all the investigated regions at E12. ConclusionsTLR4 and TRL7 expression temporal patterns suggest a morphogenetic role for these receptors in the development of neural crest derivatives in mammals.

Pleiotropic effect of common PHOX2B variants in Hirschsprung disease and neuroblastoma.

Hirschsprung disease (HSCR) is a heterogeneous congenital disorder that affects the enteric nervous system, while neuroblastoma is an embryonal tumor of the sympathetic nervous system. Familial cases of both HSCR and neuroblastoma appear to be functionally linked to PHOX2B, which plays a key role in the development of neural crest derivatives. However, the association between common PHOX2B variants and disease risk is contested. Additionally, large-scale examination for pleiotropy or shared genetic susceptibility in sporadic HSCR and neuroblastoma cases lacks theoretical support. Here, we report the first examination of PHOX2B in 1470 HSCR and 469 neuroblastoma patients with matched healthy controls. The PHOX2B rs28647582 polymorphism was found to be associated with HSCR (P = 2.21E-03, OR = 1.26), and each subtype of the ailment (3.22E-03 ≤ P ≤ 0.43, 1.11 ≤ OR ≤ 2.32). The association between rs28647582 and NB risk was consistent with HSCR in a recessive model, though the P value was marginal (P = 0.06). These new genetic findings indicate the potential pleiotropic effects of PHOX2B in both HSCR and neuroblastoma, which could guide the development of therapeutic targets for the treatment of related neurodevelopmental disorders.

Zic Family Proteins in Emerging Biomedical Studies.

Zic family proteins have been investigated in various biomedical studies. Here we summarize the contact points between Zic proteins and recent medical research. The topics cover a wide range, reflecting the pleiotropic roles of these proteins in early embryogenesis and organogenesis. Zic1, Zic2, and Zic3 proteins play important roles in the development of axial and limb bones, and of muscles, among the derivatives of the notochord and somites. Zic1 is involved in bone's response to mechanical stress, and it also serves as a marker specific for brown adipocytes. Zic1, Zic2, Zic3, and Zic5 proteins are required for the development of neural crest derivatives, including the meningeal membrane and facial bones, and deficiency of these proteins causes cortical lamination defects resembling those in type II lissencephaly. In vascular systems, Zic3 is associated not only with normal cardiovascular development, failure of which causes congenital heart anomalies, but also controls maturation of the blood-brain barrier. Zic1 is also expressed in the brain pericytes possessing stem cell properties that control the blood-brain barrier activity and capillary hemodynamic responses. The possible involvement of Zic proteins in neuropsychiatric disorders has been indicated by the analyses of mutant mice behaviors. Zic1 and Zic3 mutant mice show hypotonia and decreased locomotor activities. Zic2 hypomorphic mutant mice exhibit schizophrenia-related behavioral abnormalities such as cognitive dysfunction and impaired sensorimotor gating and social behaviors, and ZIC2 mutations found in schizophrenia patients included a severely functionally defective one. Based on these facts, the application of Zic protein activities in translational medicine might be considered.

Contributions of PHOX2B in the Pathogenesis of Hirschsprung Disease

Hirschsprung disease (HSCR) is a congenital malformation of the hindgut resulting from a disruption of neural crest cell migration during embryonic development. It has a complex genetic aetiology with several genes involved in its pathogenesis. PHOX2B plays a key function in the development of neural crest derivatives, and heterozygous mutations cause a complex dysautonomia associating HSCR, Congenital Central Hypoventilation Syndrome (CCHS) and neuroblastoma (NB) in various combinations. In order to determine the role of PHOX2B in isolated HSCR, we performed a mutational screening in a cohort of 207 Spanish HSCR patients. Our most relevant finding has been the identification of a de novo and novel deletion (c.393_410del18) in a patient with HSCR. Results of in silico and functional assays support its pathogenic effect related to HSCR. Therefore our results support that PHOX2B loss-of-function is a rare cause of HSCR phenotype.

Hes1 is required for the development of the superior cervical ganglion of sympathetic trunk and the carotid body

Hes1 gene represses the expression of proneural basic helix-loop-helix (bHLH) factor Mash1, which is essential for the differentiation of the sympathetic ganglia and carotid body glomus cells. The sympathetic ganglia, carotid body, and common carotid artery in Wnt1-Cre/R26R double transgenic mice were intensely labeled by X-gal staining, i.e., the neural crest origin. The deficiency of Hes1 caused severe hypoplasia of the superior cervical ganglion (SCG). At embryonic day (E) 17.5-E18.5, the volume of the SCG in Hes1 null mutants was reduced to 26.4% of the value in wild-type mice. In 4 of 30 cases (13.3%), the common carotid artery derived from the third arch artery was absent in the null mutants, and the carotid body was not formed. When the common carotid artery was retained, the organ grew in the wall of the third arch artery and glomus cell precursors were provided from the SCG in the null mutants as well as in wild-types. However, the volume of carotid body in the null mutants was only 52.5% of the value in wild-types at E17.5-E18.5. These results suggest that Hes1 plays a critical role in regulating the development of neural crest derivatives in the mouse cervical region.

Comparative genomic sequence analysis coupled to chromatin immunoprecipitation: a screening procedure applied to search for regulatory elements at theRETlocus

RET gene expression is characterized by high tissue and stage specificity during the development of neural crest derivatives and in the pathogenesis of inherited cancer syndromes and Hirschsprung disease. Identifying all elements contributing to its transcriptional regulation might provide new clues to clarify both developmental and pathogenic mechanisms. We previously demonstrated that chromatin acetylation affects RET transcription; therefore, we have set up a strategy based on analysis of sequences conserved among species at the RET locus, combined with the characterization of their chromatin structure, to identify new potential regulatory elements. The histone acetylation level was evaluated by the chromatin immunoprecipitation method applied to cells displaying different degrees of endogenous RET expression. Real-time quantitative PCR of immunoprecipitated DNA-protein complexes and transfection experiments, with constructs expressing a reporter gene in which the putative regulatory regions are inserted, indicate a correlation between histone acetylation and endogenous RET expression and highlight conserved sequences with potential regulatory roles. This paper presents a reliable screening procedure to unearth elements able to affect gene regulation at the transcriptional level in a large genomic region.

Sox10 is required for the early development of the prospective neural crest in Xenopus embryos

The Sox family of transcription factors has been implicated in the development of different tissues during embryogenesis. Several mutations in humans, mice, and zebrafish have shown that depletion of Sox10 activity produces defects in the development of neural crest derivatives, such as melanocytes, ganglia of the peripheral nervous system, and some specific cell types as glia. We have isolated the Xenopus homologue of the Sox10 gene. It is expressed in prospective neural crest and otic placode regions from the earliest stages of neural crest specification and in migrating cranial and trunk neural crest cells. Loss-of-function experiments using morpholino antisense oligos against Sox10 produce a loss of neural crest precursors and an enlargement of the surrounding neural plate and epidermis. This effect of Sox10 depletion is produced during some of the earliest steps of neural crest specification, as is shown by the inhibition in the expression of Slug and FoxD3, which are early markers of neural crest specification. In addition, we show that Sox10 depletion leads to an increase in apoptosis and a decrease in cell proliferation in the neural folds, suggesting that Sox10 could work as a survival as well as a specification factor in neural crest precursors during premigratory stages. Although some of the deficiencies found in the Waardenburg syndrome and in the Hirschprung disease could be associated with a failure of the development of crest derivatives during the late phase of its development, or even during adulthood, our results suggest that inhibition of Sox10 activity produces an earlier failure of neural crest precursors. In experiments where melanocytes and ganglia were induced in vivo and in vitro, we were able to block their development by inhibiting Sox10 activity. These results are compatible with an additional late role of Sox10 on development of neural crest derivatives, as it has been previously proposed. We show that Sox10 expression is dependent on FGF and Wnt activity, both in the neural crest and in the otic placode territories. Finally, in order to establish the position of Sox10 in the hierarchical cascade of gene activation required for neural crest specification, we used inducible forms of the wild type and dominant negatives for the Snail and Slug genes. Our results show that Snail is able to control Sox10 expression. However, the overexpression of Slug was not able to upregulate Sox10 expression. Taken together, these results indicate that Sox10 may lie between Snail and Slug in the genetic cascade that controls neural crest development.

The SOX10 transcription factor: evaluation as a candidate gene for central and peripheral hereditary myelin disorders.

The SOX10 transcription factor is involved in development of neural crest derivatives and fate determination in glial cells. SOX10 mutations have been found in patients with intestinal aganglionosis and depigmentation with deafness (Waardenburg-Hirschsprung). Associated neurological signs have been reported in some cases, including a patient exhibiting a central and peripheral myelin deficiency. Therefore, we screened for SOX10 mutations in a large cohort of patients with peripheral and central myelin disorders. 56 were affected by classical demyelinating Charcot-Marie-Tooth disease without identified mutations in the genes encoding PNS myelin proteins (PMP22, P0), connexin 32 and the zinc-finger transcription factor, EGR2. 88 patients with undetermined leukodystrophy were selected from a large European prospective study. Associated clinical, magnetic resonance imaging and electrophysiological signs were consistent with a defect in CNS myelination in 83 and with an active degeneration of the CNS myelin in 5. No abnormalities in the proteolipid protein gene (PLP) were found. The absence of SOX100 mutation in this large cohort of patients suggests that this gene is not frequently involved in peripheral or central inherited myelin disorders.

Screen for mutations affecting development of Zebrafish neural crest.

The neural crest provides a useful model to learn how cell fate diversification is regulated during vertebrate development. Our approach is to isolate zebrafish mutations in which the development of neural crest derivatives is disrupted, in order to learn about the underlying genetic mechanisms. We describe a screen in which parthenogenetic diploid embryos are examined both for visible phenotypes and for cellular defects in neural crest-derived sensory neurons recognized immunohistochemically. We present preliminary results from this screen and briefly describe a few representative mutations. We also discuss the general utility of our strategy and comment on the future directions of this approach.

Screen for mutations affecting development of zebrafish neural crest

The neural crest provides a useful model to learn how cell fate diversification is regulated during vertebrate development. Our approach is to isolate zebrafish mutations in which the development of neural crest derivatives is disrupted, in order to learn about the underlying genetic mechanisms. We describe a screen in which parthenogenetic diploid embryos are examined both for visible phenotypes and for cellular defects in neural crest-derived sensory neurons recognized immunohistochemically. We present preliminary results from this screen and briefly describe a few representative mutations. We also discuss the general utility of our strategy and comment on the future directions of this approach. © 1996 Wiley-Liss, Inc.

Environmental influences in the development of neural crest derivatives: glucocorticoids, growth factors, and chromaffin cell plasticity

The neural crest gives rise to three major adrenergic cell types: sympathetic principal neurons, adrenal chromaffin cells, and small intensely fluorescent (SIF) cells. All of these derivatives synthesize and store catecholamines, but they differ in numerous other characteristics. SIF cells appear intermediate in phenotype between the other two. We have examined the role of several environmental factors in the differentiation of sympathetic principal neurons and adrenal chromaffin cells. In previous studies of young rat adrenal chromaffin cells in dissociated cell culture, differentiated characteristics such as the presence of the enzyme phenylethanolamine N-methyltransferase (PNMT), epinephrine (E) synthesis, and large catecholamine storage vesicles were not well maintained. Here we describe long-term culture of chromaffin cells which, in the presence of micromolar glucocorticoid, maintained all of these characteristics. In addition, chromaffin cells of a variety of ages were found to be dependent on glucocorticoid for long-term survival in culture. In the absence of glucocorticoid, many adrenal chromaffin cells from neonatal rats could be rescued by nerve growth factor (NGF) administration. They extended neurites, as previously described by Unsicker and colleagues (Unsicker, K., B. Krisch, U. Otten, and H. Thoenen (1978) Proc. Natl. Acad. Sci. U.S.A. 75: 3498-3502). In contrast to previous studies, however, with long-term exposure to NGF these cells became indistinguishable from mature sympathetic neurons, as judged by the following morphological and biochemical criteria: increased cell size and loss of intense CA fluorescence in their cell bodies; acquisition of characteristic neuronal ultrastructure, including morphologically specialized synapses; loss of chromaffin granules, PNMT, and E synthesis; and acquisition of neuron markers, including tetanus toxin labeling and immunoreactivity to neurofilament protein. This conversion to neurons was markedly enhanced by addition of a non-neuronal cell conditioned medium (CM) containing a neurite-promoting factor, which acted by increasing the NGF responsiveness of the chromaffin cells. Even chromaffin cells from adult rats, which are known to grow few processes in response to NGF alone, became neuronal in the presence of this CM plus NGF. While converting to neurons, adrenal chromaffin cells transiently assumed an intermediate phenotype resembling type I SIF cells, which suggests particular developmental relationships between the different cell types of the sympathoadrenal lineage.(ABSTRACT TRUNCATED AT 400 WORDS)

Control of tyrosinase gene expression and its relationship to neural crest induction in Rana pipiens. II. Measurement of tyrosinase mRNA accumulation during early embryogenesis using a specific cDNA probe.

The relationship between the process of neural induction and the control of tyrosinase gene expression in the cells that derive from the neural crest of amphibians has been examined at the molecular level. [3H] Tyrosinase cDNA was utilized as a probe to measure the levels of tyrosinase RNA transcripts present in Rana pipiens embryos from the time of fertilization through Stage 25 of cleavage (operculum complete, 240 h) and to correlate these levels with those published for the rate of tyrosinase protein synthesis. R. pipiens [3H]tyrosinase cDNA was synthesized from a purified tyrosinase mRNA template using avian myeloblastosis virus reverse transcriptase and was enriched for full length copies by preparative polyacrylamide gel electrophoresis. This cDNA product was estimated to represent greater than 90% by length of tyrosinase mRNA and hybridized to tyrosinase mRNA to greater than 97% within two orders of magnitude of R0t values. The extent of hybridization of [3H]tyrosinase cDNA to total embryonic RNA throughout early development paralleled the rate of synthesis of tyrosinase protein. Tyrosinase RNA transcripts were first detected at Stage 12-13 (0.0032% of total RNA) and rose to maximal levels by Stage 19 (0.011%). This represents a 50-fold increase from preinduction levels. These results are consistent with a model which predicts that one of the early events in the development of neural crest derivatives is the transcriptionally dependent accumulation of functionally mature tyrosinase mRNA.

Mutiple mucosal neuromas, dysautonomia and abnormal intradermal histamine reaction

With reference to a patient a description is given of the syndrome of multiple mucosal neuromas — a syndrome which seems to be caused by a disturbance in the development of neural crest derivatives. The syndrome was associated with a number of autonomie dysfunctions; this dysautonomia is correlated with the morphological findings in a sural nerve biopsy. The nerve was hypertrophic and showed few changes in the myelinated fibres; the unmyelinated fibres showed a chronic process of degeneration and regeneration, and small unmyelinated axons were found increased in number. It is suggested that the abnormal reaction to an intradermal histamine injection was due to the dysautonomia shown by this patient.