MR microscopy of the human fetal upper extremity – a proof-of-principle study

Background

Bone morphogenetic proteins regulate multiple processes in embryonic development, including early dorso-ventral patterning and neural crest development. BMPs activate heteromeric receptor complexes consisting of type I and type II receptor-serine/threonine kinases. BMP receptors Ia and Ib, also known as ALK3 and ALK6 respectively, are the most common type I receptors that likely mediate most BMP signaling events. Since early expression patterns and functions in Xenopus laevis development have not been described, we have addressed these questions in the present study.

Results

Here we have analyzed the temporal and spatial expression patterns of ALK3 and ALK6; we have also carried out loss-of-function studies to define the function of these receptors in early Xenopus development. We detected both redundant and non-redundant roles of ALK3 and ALK6 in dorso-ventral patterning. From late gastrula stages onwards, their expression patterns diverged, which correlated with a specific, non-redundant requirement of ALK6 in post-gastrula neural crest cells. ALK6 was essential for induction of neural crest cell fate and further development of the neural crest and its derivatives.

Conclusions

ALK3 and ALK6 both contribute to the gene regulatory network that regulates dorso-ventral patterning; they play partially overlapping and partially non-redundant roles in this process. ALK3 and ALK6 are independently required for the spatially restricted activation of BMP signaling and msx2 upregulation at the neural plate border, whereas in post-gastrula development ALK6 exerts a highly specific, conserved function in neural crest development.

     

A new role for the Endothelin-1/Endothelin-A receptor signaling during early neural crest specification

The neural crest is induced at the border of the neural plate in a multistep process by signals emanated from the epidermis, neural plate and mesoderm. In this work we show for the first time the existence of a neural crest maintenance step which is dependent on signals released from the mesoderm. We identified Endothelin-1 (Edn1) and its receptor (Ednra) as key players of this signal and we show that Edn1/Ednra signaling is required for maintenance of the neural crest by a dual mechanism of cell specification and cell survival. We show that: (i) Ednra is expressed in prospective neural crest; (ii) loss-of-function experiments with antisense morpholino or with specific chemical inhibitor suppress the expression of early neural crest markers; (iii) gain-of-function experiments expand the neural crest territory; (iv) epistatic experiments show that Ednra/Edn1 is downstream of the early neural crest gene Msx1 and upstream of the late genes Sox9 and Sox10; and (v) Edn1/Ednra signaling inhibits apoptosis and controls cell specification of the neural crest. Together, our results provide insight on a new role of Edn1/Ednra cell signaling pathway during early neural crest development.     

The evolutionary origin of the vertebrate neural crest and its developmental gene regulatory network – insights from amphioxus

The neural crest is an embryonic cell population unique to vertebrates. During vertebrate embryogenesis, neural crest cells are first induced from the neural plate border; subsequently, they delaminate from the dorsal neural tube and migrate to their destination, where they differentiate into a wide variety of derivatives. The emergence of the neural crest is thought to be responsible for the evolution of many complex novel structures of vertebrates that are lacking in invertebrate chordates. Despite its central importance in understanding the origin of vertebrates, the evolutionary origin of the neural crest remains elusive. The basal chordate amphioxus (Branchiostoma floridae) occupies an outgroup position that is useful for investigating this question. In this review, I summarize recent genomic and comparative developmental studies between amphioxus and vertebrates and discuss their implications for the evolutionary origin of neural crest cells. I focus mainly on the origin of the gene regulatory network underlying neural crest development, and suggest several hypotheses regarding how this network could have been assembled during early vertebrate evolution.     

Draxin,an axon guidance protein,affects chick trunk neural crest migration

The neural crest is a multipotent population of migratory cells that arises in the central nervous system and subsequently migrates along defined stereotypic pathways. In the present work, we analyzed the role of a repulsive axon guidance protein, draxin, in the migration of neural crest cells. Draxin is expressed in the roof plate of the chick trunk spinal cord and around the early migration pathway of neural crest cells. Draxin modulates chick neural crest cell migration in vitro by reducing the polarization of these cells. When exposed to draxin, the velocity of migrating neural crest cells was reduced, and the cells changed direction so frequently that the net migration distance was also reduced. Overexpression of draxin also caused some early migrating neural crest cells to change direction to the dorsolateral pathway in the chick trunk region, presumably due to draxin’s inhibitory activity. These results demonstrate that draxin, an axon guidance protein, can also affect trunk neural crest migration in the chick embryo.     

Indian hedgehog signaling is required for proper formation, maintenance and migration of Xenopus neural crest

Neural crest induction is the result of the combined action at the neural plate border of FGF, BMP, and Wnt signals from the neural plate, mesoderm and nonneural ectoderm. In this work we show that the expression of Indian hedgehog (Ihh, formerly named Banded hedgehog) and members of the Hedgehog pathway occurs at the prospective neural fold, in the premigratory and migratory neural crest. We performed a functional analysis that revealed the requirement of Ihh signaling in neural crest development. During the early steps of neural crest induction loss of function experiments with antisense morpholino or locally grafted cyclopamine-loaded beads suppressed the expression of early neural crest markers concomitant with the increase in neural and epidermal markers. We showed that changes in Ihh activity produced no alterations in either cell proliferation or apoptosis, suggesting that this signal involves cell fate decisions. A temporal analysis showed that Hedgehog is continuously required not only in the early and late specification but also during the migration of the neural crest. We also established that the mesodermal source of Ihh is important to maintain specification and also to support the migratory process. By a combination of embryological and molecular approaches our results demonstrated that Ihh signaling drives in the migration of neural crest cells by autocrine or paracrine mechanisms. Finally, the abrogation of Ihh signaling strongly affected only the formation of cartilages derived from the neural crest, while no effects were observed on melanocytes. Taken together, our results provide insights into the role of the Ihh cell signaling pathway during the early steps of neural crest development.     

The initial development of the human brain.

An account of the early development of the human brain has been prepared from the data available for the Carnegie Collection, as well as from published information from other sources. Although the site of the neural plate can be discerned at stage 7, the first visible indication of the nervous system is the neural groove in certain embryos of stage 8, in which the embryonic disc measures more than 1 mm and the notochordal process at least 0.3 mm. The progressive fusion of the neural folds during stage 10, and the closure of the rostral and caudal neuropores at stages 11 and 12, respectively, are detailed with further precision than hitherto. It is emphasized that the major subdivisions of the human brain do not begin as vesicles, but as enlargements of the open neural folds at stage 9. The relationships of the neuromeres to the otic region, the somites, and the neural crest are clarified and illustrated. The early appearance of the telencephalon medium (before cerebral vesicles have formed) is stressed, and the terminological implications for the subdivisions of the brain are discussed.     

Zic2 is required for neural crest formation and hindbrain patterning during mouse development

The Zic genes are the vertebrate homologues of the Drosophila pair rule gene odd-paired. It has been proposed that Zic genes play several roles during neural development including mediolateral segmentation of the neural plate, neural crest induction, and inhibition of neurogenesis. Initially during mouse neural development Zic2 is expressed throughout the neural plate while later on expression in the neurectoderm becomes restricted to the lateral region of the neural plate. A hypomorphic allele of Zic2 has demonstrated that in the mouse Zic2 is required for the timing of neurulation. We have isolated a new allele of Zic2 that behaves as a loss of function allele. Analysis of this mutant reveals two further functions for Zic2 during early neural development. Mutation of Zic2 results in a delay of neural crest production and a decrease in the number of neural crest cells that are produced. These defects are independent of mediolateral segmentation of the neurectoderm and of dorsal neurectoderm proliferation, both of which occur normally in the mutant embryos. Additionally Zic2 is required during hindbrain patterning for the normal development of rhombomeres 3 and 5. This work provides the first genetic evidence that the Zic genes are involved in neural crest production and the first demonstration that Zic2 functions during hindbrain patterning.     

Zebrafish narrowminded suggests a genetic link between formation of neural crest and primary sensory neurons.

In the developing vertebrate nervous system, both neural crest and sensory neurons form at the boundary between non-neural ectoderm and the neural plate. From an in situ hybridization based expression analysis screen, we have identified a novel zebrafish mutation, narrowminded (nrd), which reduces the number of early neural crest cells and eliminates Rohon-Beard (RB) sensory neurons. Mosaic analysis has shown that the mutation acts cell autonomously suggesting that nrd is involved in either the reception or interpretation of signals at the lateral neural plate boundary. Characterization of the mutant phenotype indicates that nrd is required for a primary wave of neural crest cell formation during which progenitors generate both RB sensory neurons and neural crest cells. Moreover, the early deficit in neural crest cells in nrd homozygotes is compensated later in development. Thus, we propose that a later wave can compensate for the loss of early neural crest cells but, interestingly, not the RB sensory neurons. We discuss the implications of these findings for the possibility that RB sensory neurons and neural crest cells share a common evolutionary origin.     

Neural crest cells: From developmental biology to clinical interventions

Neural crest cells are multipotent cells, which are specified in embryonic ectoderm in the border of neural plate and epiderm during early development by interconnection of extrinsic stimuli and intrinsic factors. Neural crest cells are capable of differentiating into various somatic cell types, including melanocytes, craniofacial cartilage and bone, smooth muscle, and peripheral nervous cells, which supports their promise for cell therapy. In this work, we provide a comprehensive review of wide aspects of neural crest cells from their developmental biology to applicability in medical research. We provide a simplified model of neural crest cell development and highlight the key external stimuli and intrinsic regulators that determine the neural crest cell fate. Defects of neural crest cell development leading to several human disorders are also mentioned, with the emphasis of using human induced pluripotent stem cells to model neurocristopathic syndromes. Birth Defects Research (Part C) 102:263–274, 2014. © 2014 Wiley Periodicals, Inc.     

Tfap2a and Foxd3 regulate early steps in the development of the neural crest progenitor population

The neural crest is a stem cell-like population exclusive to vertebrates that gives rise to many different cell types including chondrocytes, neurons and melanocytes. Arising from the neural plate border at the intersection of Wnt and Bmp signaling pathways, the complexity of neural crest gene regulatory networks has made the earliest steps of induction difficult to elucidate. Here, we report that tfap2a and foxd3 participate in neural crest induction and are necessary and sufficient for this process to proceed. Double mutant tfap2a (mont blanc, mob) and foxd3 (mother superior, mos) mob;mos zebrafish embryos completely lack all neural crest-derived tissues. Moreover, tfap2a and foxd3 are expressed during gastrulation prior to neural crest induction in distinct, complementary, domains; tfap2a is expressed in the ventral non-neural ectoderm and foxd3 in the dorsal mesendoderm and ectoderm. We further show that Bmp signaling is expanded in mob;mos embryos while expression of dkk1, a Wnt signaling inhibitor, is increased and canonical Wnt targets are suppressed. These changes in Bmp and Wnt signaling result in specific perturbations of neural crest induction rather than general defects in neural plate border or dorso-ventral patterning. foxd3 overexpression, on the other hand, enhances the ability of tfap2a to ectopically induce neural crest around the neural plate, overriding the normal neural plate border limit of the early neural crest territory. Although loss of either Tfap2a or Foxd3 alters Bmp and Wnt signaling patterns, only their combined inactivation sufficiently alters these signaling gradients to abort neural crest induction. Collectively, our results indicate that tfap2a and foxd3, in addition to their respective roles in the differentiation of neural crest derivatives, also jointly maintain the balance of Bmp and Wnt signaling in order to delineate the neural crest induction domain.     

Repulsive guidance molecule A (RGM A) and its receptor neogenin during neural and neural crest cell development of Xenopus laevis

Background information. RGM A (repulsive guidance molecule A) is a GPI (glycosylphosphatidylinositol)‐anchored glycoprotein which has repulsive properties on axons due to the interaction with its receptor neogenin. In addition, RGM A has been demonstrated to function as a BMP (bone morphogenetic protein) co‐receptor. Results. In the present study, we provide the first analysis of early RGM A and neogenin expression and function in Xenopus laevis neural development. Tissue‐specific RGM A expression starts at stage 12.5 in the anterior neural plate. Loss‐of‐function analyses suggest a function of RGM A and neogenin in regulating anterior neural marker genes, as well as eye development and neural crest cell migration. Furthermore, overexpression of RGM A leads to ectopic expression of neural crest cell marker genes. Conclusions. These data indicate that RGM A and neogenin have important functions during early neural development, in addition to their role during axonal guidance and synapse formation.     

Early development of the central and peripheral nervous systems is coordinated by Wnt and BMP signals

The formation of functional neural circuits that process sensory information requires coordinated development of the central and peripheral nervous systems derived from neural plate and neural plate border cells, respectively. Neural plate, neural crest and rostral placodal cells are all specified at the late gastrula stage. How the early development of the central and peripheral nervous systems are coordinated remains, however, poorly understood. Previous results have provided evidence that at the late gastrula stage, graded Wnt signals impose rostrocaudal character on neural plate cells, and Bone Morphogenetic Protein (BMP) signals specify olfactory and lens placodal cells at rostral forebrain levels. By using in vitro assays of neural crest and placodal cell differentiation, we now provide evidence that Wnt signals impose caudal character on neural plate border cells at the late gastrula stage, and that under these conditions, BMP signals induce neural crest instead of rostral placodal cells. We also provide evidence that both caudal neural and caudal neural plate border cells become independent of further exposure to Wnt signals at the head fold stage. Thus, the status of Wnt signaling in ectodermal cells at the late gastrula stage regulates the rostrocaudal patterning of both neural plate and neural plate border, providing a coordinated spatial and temporal control of the early development of the central and peripheral nervous systems.     

Role of the transforming growth factor-β family in the expression of cranial neural crest-specific phenotypes

Cranial and trunk neural crest cells produce different derivatives in vitro. Cranial neural crest cultures produce large numbers of cells expressing fibronectin (FN) and procollagen I (PCol I) immunoreactivities, two markers expressed by mesenchymal derivatives in vivo. Trunk neural crest cultures produce relatively few FN or PCol I immunoreactive cells, but they produce greater numbers of melanocytes than do cranial cultures. Treatment of trunk neural crest cultures with transforming growth factor-β1 (TGF-β1) stimulates them to express both FN and PCol I immunoreactivities at levels comparable to those normally seen in cranial cultures and simultaneously decreases their expression of melanin. These observations raised the possibility that endogenous TGF-β is involved in specifying differences in the phenotypes expressed by cranial and trunk neural crest cells in vitro. Consistent with this idea, we found that treatment of cranial cultures with a function-blocking TGF-β antiserum inhibits the development of FN immunoreactive cells and stimulates the development of melanocytes. Cranial and trunk neural crest cells express approximately equal levels of TGF-β mRNA. However, trunk neural crest cells are significantly less sensitive to the FN-inducing effect of TGF-β1 than are cranial neural crest cells. These results suggest that: (1) endogenous TGF-β is required for the expression of mesenchymal phenotypes by cranial neural crest cells, and (2) differences in the phenotypes expressed by cranial and trunk neural crest cells in vitro result in part from differences in the sensitivities of these two cell populations to TGF-β. © 1995 John Wiley & Sons, Inc.     

Polymorphic microsatellites identified in an endangered dasyurid marsupial,the dibbler (Parantechinus apicalis)

Dibblers are carnivorous marsupials that were considered extinct for over 60 years. They now persist in small, highly fragmented populations. In this work we describe the characterization of seven polymorphic microsatellite loci that are being used to investigate population parameters. For 22 individuals, from the mainland population, these markers had allelic diversities from three to 11 (alleles per locus) and expected heterozygosities ranging from 0.53 to 0.89. Primers were also tested on a number of closely related dasyurids and in other marsupials, suggesting that their usefulness in population studies may go beyond investigations of the dibbler.     

Delta signaling mediates segregation of neural crest and spinal sensory neurons from zebrafish lateral neural plate

We examined the role of Delta signaling in specification of two derivatives in zebrafish neural plate: Rohon-Beard spinal sensory neurons and neural crest. deltaA-expressing Rohon-Beard neurons are intermingled with premigratory neural crest cells in the trunk lateral neural plate. Embryos homozygous for a point mutation in deltaA, or with experimentally reduced delta signalling, have supernumerary Rohon-Beard neurons, reduced trunk-level expression of neural crest markers and lack trunk neural crest derivatives. Fin mesenchyme, a putative trunk neural crest derivative, is present in deltaA mutants, suggesting it segregates from other neural crest derivatives as early as the neural plate stage. Cranial neural crest derivatives are also present in deltaA mutants, revealing a genetic difference in regulation of trunk and cranial neural crest development.     

Origins and Developmental Potential of the Neural Crest

Neural crest cells are a migratory population that forms most of the peripheral nervous system, facial skeleton, and numerous other derivatives. These cells arise from the neural ectoderm and are first recognizable as discrete cells after neural tube closure. In this review, I summarize the results of studies from our laboratory on neural crest cell lineage and origin. Our recent experiments demonstrate that interactions between the presumptive neural plate and the nonneural ectoderm are likely to be instrumental in the induction of the avian neural crest. Juxtaposition of these tissues at early stages results in the formation of neural crest cells at the interface. However, neural crest cells do not appear to be segregated from other neuroepithelial cells; cell lineage studies have demonstrated that individual precursor cells within the neural tube can give rise to both neural crest and neural tube derivatives as diverse as sensory, commissural, and motor neurons. This suggests that individual neuroectodermal cells are multipotent, such that a precursor within the neural tube has the ability to form both neural tube (central nervous system) and neural crest (peripheral nervous system and other) derivatives. Further support for flexibility in the developmental program of neuroepithelial cells comes from experiments in which the cranial neural folds are ablated; this results in regulation by the remaining ventral neural tube cells to form neural crest cells after the endogenous neural crest is removed. At later stage of development, this regulative capacity is lost. Following their emigration from the neural tube, neural crest cells become progressively restricted to defined embryonic states. Taken together, these experiments demonstrate that: (1) the neural crest is an induced population that arises by interactions within the ectoderm; (2) initially, progenitor cells are multipotent, having the potential to form multiple neural crest and neural tube derivatives; and (3) with time, the precursors become progressively restricted to form neural crest derivatives and eventually to individual phenotypes.     

Xenopus Id3 is required downstream of Myc for the formation of multipotent neural crest progenitor cells

Neural crest cells, a population of proliferative, migratory, tissue-invasive stem cells, are a defining feature of vertebrate embryos. These cells arise at the neural plate border during a time in development when precursors of the central nervous system and the epidermis are responding to the extracellular signals that will ultimately dictate their fates. Neural crest progenitors, by contrast, must be maintained in a multipotent state until after neural tube closure. Although the molecular mechanisms governing this process have yet to be fully elucidated, recent work has suggested that Myc functions to prevent premature cell fate decisions in neural crest forming regions of the early ectoderm. Here, we show that the small HLH protein Id3 is a Myc target that plays an essential role in the formation and maintenance of neural crest stem cells. A morpholino-mediated 'knockdown' of Id3 protein results in embryos that lack neural crest. Moreover, forced expression of Id3 maintains the expression of markers of the neural crest progenitor state beyond the time when they would normally be downregulated and blocks the differentiation of neural crest derivatives. These results shed new light on the mechanisms governing the formation and maintenance of a developmentally and clinically important cell population.     

Posteriorization by FGF, Wnt, and retinoic acid is required for neural crest induction.

The neural crest is a unique cell population induced at the lateral border of the neural plate. Neural crest is not produced at the anterior border of the neural plate, which is fated to become forebrain. Here, the roles of BMPs, FGFs, Wnts, and retinoic acid signaling in neural crest induction were analyzed by using an assay developed for investigating the posteriorization of the neural plate. Using specific markers for the anterior neural plate border and the neural crest, the posterior end of early neurula embryos was shown to be able to transform the anterior neural plate border into neural crest cells. In addition, tissue expressing anterior neural plate markers, induced by an intermediate level of BMP activity, was transformed into neural crest by posteriorizing signals. This transformation was mimicked by bFGF, Wnt-8, or retinoic acid treatment and was also inhibited by expression of the dominant negative forms of the FGF receptor, the retinoic acid receptor, and Wnt signaling molecules. The transformation of the anterior neural plate border into neural crest cells was also achieved in whole embryos, by retinoic acid treatment or by use of a constitutively active form of the retinoic acid receptor. By analyzing the expression of mesodermal markers and various graft experiments, the expression of the mutant retinoic acid receptor was shown to directly affect the ectoderm. We thereby propose a two-step model for neural crest induction. Initially, BMP levels intermediate to those required for neural plate and epidermal specification induce neural folds with an anterior character along the entire neural plate border. Subsequently, the most posterior region of this anterior neural plate border is transformed into the neural crest by the posteriorizing activity of FGFs, Wnts, and retinoic acid signals. We discuss a unifying model where lateralizing and posteriorizing signals are presented as two stages of the same inductive process required for neural crest induction.