Congenital microcoria deletion in mouse links Sox21 dysregulation to disease and suggests a role for TGFB2 in glaucoma and myopia.

Congenital microcoria (MCOR) is a rare hereditary developmental defect of the iris dilator muscle frequently associated with high axial myopia and high intraocular pressure (IOP) glaucoma. The condition is caused by submicroscopic rearrangements of chromosome 13q32.1. However, the mechanisms underlying the failure of iris development and the origin of associated features remain elusive. Here, we present a 3D architecture model of the 13q32.1 region, demonstrating that MCOR-related deletions consistently disrupt the boundary between two topologically associating domains (TADs). Deleting the critical MCOR-causing region in mice reveals ectopic Sox21 expression precisely aligning with Dct, each located in one of the two neighbor TADs. This observation is consistent with the TADs' boundary alteration and adoption of Dct regulatory elements by the Sox21 promoter. Additionally, we identify Tgfb2 as a target gene of SOX21 and show TGFΒ2 accumulation in the aqueous humor of an MCOR-affected subject. Accumulation of TGFB2 is recognized for its role in glaucoma and potential impact on axial myopia. Our results highlight the importance of SOX21-TGFB2 signaling in iris development and control of eye growth and IOP. Insights from MCOR studies may provide therapeutic avenues for this condition but also for glaucoma and high myopia conditions, affecting millions of people.

The Emerging Roles of the Cephalic Neural Crest in Brain Development and Developmental Encephalopathies.

The neural crest, a unique cell population originating from the primitive neural field, has a multi-systemic and structural contribution to vertebrate development. At the cephalic level, the neural crest generates most of the skeletal tissues encasing the developing forebrain and provides the prosencephalon with functional vasculature and meninges. Over the last decade, we have demonstrated that the cephalic neural crest (CNC) exerts an autonomous and prominent control on the development of the forebrain and sense organs. The present paper reviews the primary mechanisms by which CNC can orchestrate vertebrate encephalization. Demonstrating the role of the CNC as an exogenous source of patterning for the forebrain provides a novel conceptual framework with profound implications for understanding neurodevelopment. From a biomedical standpoint, these data suggest that the spectrum of neurocristopathies is broader than expected and that some neurological disorders may stem from CNC dysfunctions.

The hedgehog pathway and ocular developmental anomalies.

Mutations in effectors of the hedgehog signaling pathway are responsible for a wide variety of ocular developmental anomalies. These range from massive malformations of the brain and ocular primordia, not always compatible with postnatal life, to subtle but damaging functional effects on specific eye components. This review will concentrate on the effects and effectors of the major vertebrate hedgehog ligand for eye and brain formation, Sonic hedgehog (SHH), in tissues that constitute the eye directly and also in those tissues that exert indirect influence on eye formation. After a brief overview of human eye development, the many roles of the SHH signaling pathway during both early and later morphogenetic processes in the brain and then eye and periocular primordia will be evoked. Some of the unique molecular biology of this pathway in vertebrates, particularly ciliary signal transduction, will also be broached within this developmental cellular context.

LKB1 signaling in cephalic neural crest cells is essential for vertebrate head development.

Head development in vertebrates proceeds through a series of elaborate patterning mechanisms and cell-cell interactions involving cephalic neural crest cells (CNCC). These cells undergo extensive migration along stereotypical paths after their separation from the dorsal margins of the neural tube and they give rise to most of the craniofacial skeleton. Here, we report that the silencing of the LKB1 tumor suppressor affects the delamination of pre-migratory CNCC from the neural primordium as well as their polarization and survival, thus resulting in severe facial and brain defects. We further show that LKB1-mediated effects on the development of CNCC involve the sequential activation of the AMP-activated protein kinase (AMPK), the Rho-dependent kinase (ROCK) and the actin-based motor protein myosin II. Collectively, these results establish that the complex morphogenetic processes governing head formation critically depends on the activation of the LKB1 signaling network in CNCC.

The facial neural crest controls fore- and midbrain patterning by regulating Foxg1 expression through Smad1 activity.

The facial neural crest (FNC), a pluripotent embryonic structure forming craniofacial structures, controls the activity of brain organisers and stimulates cerebrum growth. To understand how the FNC conveys its trophic effect, we have studied the role of Smad1, which encodes an intracellular transducer, to which multiple signalling pathways converge, in the regulation of Foxg1. Foxg1 is a transcription factor essential for telencephalic specification, the mutation of which leads to microcephaly and mental retardation. Smad1 silencing, based on RNA interference (RNAi), was performed in pre-migratory FNC cells. Soon after electroporation of RNAi molecules, Smad1 inactivation abolished the expression of Foxg1 in the chick telencephalon, resulting in dramatic microcephaly and partial holoprosencephaly. In addition, the depletion of Foxg1 activity altered the expression Otx2 and Foxa2 in di/mesencephalic neuroepithelium. However, when mutated forms of Smad1 mediating Fgf and Wnt signalling were transfected into FNC cells, these defects were overcome. We also show that, downstream of Smad1 activity, Dkk1, a Wnt antagonist produced by the FNC, initiated the specification of the telencephalon by regulating Foxg1 activity. Additionally, the activity of Cerberus in FNC-derived mesenchyme synergised with Dkk1 to control Foxg1 expression and maintain the balance between Otx2 and Foxa2.

A conserved role for non-neural ectoderm cells in early neural development.

During the early steps of head development, ectodermal patterning leads to the emergence of distinct non-neural and neural progenitor cells. The induction of the preplacodal ectoderm and the neural crest depends on well-studied signalling interactions between the non-neural ectoderm fated to become epidermis and the prospective neural plate. By contrast, the involvement of the non-neural ectoderm in the morphogenetic events leading to the development and patterning of the central nervous system has been studied less extensively. Here, we show that the removal of the rostral non-neural ectoderm abutting the prospective neural plate at late gastrulation stage leads, in mouse and chick embryos, to morphological defects in forebrain and craniofacial tissues. In particular, this ablation compromises the development of the telencephalon without affecting that of the diencephalon. Further investigations of ablated mouse embryos established that signalling centres crucial for forebrain regionalization, namely the axial mesendoderm and the anterior neural ridge, form normally. Moreover, changes in cell death or cell proliferation could not explain the specific loss of telencephalic tissue. Finally, we provide evidence that the removal of rostral tissues triggers misregulation of the BMP, WNT and FGF signalling pathways that may affect telencephalon development. This study opens new perspectives on the role of the neural/non-neural interface and reveals its functional relevance across higher vertebrates.

Combinatorial activity of Six1-2-4 genes in cephalic neural crest cells controls craniofacial and brain development.

The combinatorial expression of Hox genes is an evolutionarily ancient program underlying body axis patterning in all Bilateria. In the head, the neural crest (NC)--a vertebrate innovation that contributes to evolutionarily novel skeletal and neural features--develops as a structure free of Hox-gene expression. The activation of Hoxa2 in the Hox-free facial NC (FNC) leads to severe craniofacial and brain defects. Here, we show that this condition unveils the requirement of three Six genes, Six1, Six2, and Six4, for brain development and morphogenesis of the maxillo-mandibular and nasofrontal skeleton. Inactivation of each of these Six genes in FNC generates diverse brain defects, ranging from plexus agenesis to mild or severe holoprosencephaly, and entails facial hypoplasia or truncation of the craniofacial skeleton. The triple silencing of these genes reveals their complementary role in face and brain morphogenesis. Furthermore, we show that the perturbation of the intrinsic genetic FNC program, by either Hoxa2 expression or Six gene inactivation, affects Bmp signaling through the downregulation of Bmp antagonists in the FNC cells. When upregulated in the FNC, Bmp antagonists suppress the adverse skeletal and cerebral effects of Hoxa2 expression. These results demonstrate that the combinatorial expression of Six1, Six2, and Six4 is required for the molecular programs governing craniofacial and cerebral development. These genes are crucial for the signaling system of FNC origin, which regulates normal growth and patterning of the cephalic neuroepithelium. Our results strongly suggest that several congenital craniofacial and cerebral malformations could be attributed to Six genes' misregulation.

Cubilin, a high affinity receptor for fibroblast growth factor 8, is required for cell survival in the developing vertebrate head.

Cubilin (Cubn) is a multiligand endocytic receptor critical for the intestinal absorption of vitamin B12 and renal protein reabsorption. During mouse development, Cubn is expressed in both embryonic and extra-embryonic tissues, and Cubn gene inactivation results in early embryo lethality most likely due to the impairment of the function of extra-embryonic Cubn. Here, we focus on the developmental role of Cubn expressed in the embryonic head. We report that Cubn is a novel, interspecies-conserved Fgf receptor. Epiblast-specific inactivation of Cubn in the mouse embryo as well as Cubn silencing in the anterior head of frog or the cephalic neural crest of chick embryos show that Cubn is required during early somite stages to convey survival signals in the developing vertebrate head. Surface plasmon resonance analysis reveals that fibroblast growth factor 8 (Fgf8), a key mediator of cell survival, migration, proliferation, and patterning in the developing head, is a high affinity ligand for Cubn. Cell uptake studies show that binding to Cubn is necessary for the phosphorylation of the Fgf signaling mediators MAPK and Smad1. Although Cubn may not form stable ternary complexes with Fgf receptors (FgfRs), it acts together with and/or is necessary for optimal FgfR activity. We propose that plasma membrane binding of Fgf8, and most likely of the Fgf8 family members Fgf17 and Fgf18, to Cubn improves Fgf ligand endocytosis and availability to FgfRs, thus modulating Fgf signaling activity.

Integrative analysis of molecular pathways and morphological anomalies associated with congenital Zika syndrome.

Congenital Zika syndrome (CZS) comprises a set of clinical manifestations that can be presented by neonates born to mothers infected by the Zika virus (ZIKV). CZS-associated phenotypes include neurological, skeletal, and systemic alterations and long-term developmental sequelae. One of the most frequently reported clinical conditions is microcephaly characterized by a reduction in head circumference and cognitive complications. Nevertheless, the associations among the diverse signaling pathways underlying CZS phenotypes remain to be elucidated. To shed light on CZS, we have extensively reviewed the morphological anomalies resulting from ZIKV infection, as well as genes and proteins of interest obtained from the published literature. With this list of genes or proteins, we performed computational analyses to explore the cellular processes, molecular mechanisms, and molecular pathways related to ZIKV infection. Therefore, in this review, we comprehensively describe the morphological abnormalities caused by congenital ZIKV infection and, through the analysis noted above, propose common molecular pathways altered by ZIKV that could explain both central nervous system and craniofacial skeletal alterations.

The neural crest is a powerful regulator of pre-otic brain development.

The role of the neural crest (NC) in the construction of the vertebrate head was demonstrated when cell tracing techniques became available to follow the cells exiting from the cephalic neural folds in embryos of various vertebrate species. Experiments carried out in the avian embryo, using the quail/chick chimera system, were critical in showing that the entire facial skeleton and most of the skull (except for he occipital region) were derived from the NC domain of the posterior diencephalon, mesencephalon and rhombomeres 1 and 2 (r1, r2). This region of the NC was designated FSNC (for Facial Skeletogenic NC). One characteristic of this part of the head including the neural anlage is that it remains free of expression of the homeotic genes of the Hox-clusters. In an attempt to see whether this rostral Hox-negative domain of the NC has a specific role in the development of the skeleton, we have surgically removed it in chick embryos at 5-6 somite stages (5-6 ss). The operated embryos showed a complete absence of facial and skull cartilages and bones showing that the Hox expressing domain of the NC caudally located to the excision did not regenerate to replace the anterior NC. In addition to the deficit in skeletal structures, the operated embryos exhibited severe brain defects resulting in anencephaly. Experiments described here have shown that the neural crest cells regulate the amount of Fgf8 produced by the two brain organizers, the Anterior Neural Ridge (ANR) and the isthmus. This regulation is exerted via the secretion of anti-BMP signaling molecules (e.g. Gremlin and Noggin), which decrease BMP production hence enhancing the amount of Fgf8 synthesized in the ANR (also called "Prosencephalic organizer") and the isthmus. In addition to its role in building up the face and skull, the NC is therefore an important signaling center for brain development.

Regulation of pre-otic brain development by the cephalic neural crest.

Emergence of the neural crest (NC) is considered an essential asset in the evolution of the chordate phylum, as specific vertebrate traits such as peripheral nervous system, cephalic skeletal tissues, and head development are linked to the NC and its derivatives. It has been proposed that the emergence of the NC was responsible for the formation of a "new head" characterized by the spectacular development of the forebrain and associated sense organs. It was previously shown that removal of the cephalic NC (CNC) prevents the formation of the facial structures but also results in anencephaly. This article reports on the molecular mechanisms whereby the CNC controls cephalic neurulation and brain morphogenesis. This study demonstrates that molecular variations of Gremlin and Noggin level in CNC account for morphological changes in brain size and development. CNC cells act in these processes through a multi-step control and exert cumulative effects counteracting bone morphogenetic protein signaling produced by the neighboring tissues (e.g., adjacent neuroepithelium, ventro-medial mesoderm, superficial ectoderm). These data provide an explanation for the fact that acquisition of the NC during the protochordate-to-vertebrate transition has coincided with a large increase of brain vesicles.

Neural crest contribution to forebrain development.

The neural crest (NC), a defining feature of vertebrate embryo, generates most of the skeletal tissues encasing the developing forebrain and provides the prosencephalon with functional vasculature and meninges. Recent findings show that early in development, the cephalic NC is also essential for the pre-otic neural tube closure and promotes the development of the prosencephalic alar plate by regulating the morphogenetic activities of forebrain organizers.

Deciphering the Neural Crest Contribution to Cephalic Development with Avian Embryos.

For decades, the quail-chick system has been a gold standard approach to track cells and their progenies over complex morphogenetic movements and long-range migrations as well as to unravel their dialogue and interplays in varied processes of cell induction. More specifically, this model became decisive for the systematic explorations of the neural crest and its lineages and allowed a tremendous stride in understanding the wealth and complexity of this fascinating cell population. Much of our knowledge on craniofacial morphogenesis and vertebrate organogenesis was first gained in avian chimeras and later extended to mammalian models and humans. In addition, this system permits tissue and gene manipulations to be performed at once in the same cell population. Through the use of in ovo electroporation, this model became tractable for functional genomics, hence being even more resourceful for functional studies. Due to the ease of access and the possibility to combine micromanipulation of tissue anlagen and gene expression, this model offers the prospect of decrypting instructive versus permissive tissue interactions, to identify and crack the molecular codes underlying cell positioning and differentiation, with an unparalleled spatiotemporal accuracy.

Role of the neural crest in face and brain development.

Since the time of Ramon y Cajal, very significant progress has been accomplished in our knowledge of the fate of the early neural primordium. The origin of the peripheral nervous system from the transient and pluripotent embryonic structure, the neural crest has been fully deciphered using appropriate cell marking techniques. Most of the pioneer work in this field was carried out in lower vertebrates up to 1950 and later on in the avian embryo. New techniques which allow the genetic labelling of embryonic cells by transgenesis are now applied in mammals and fish. One of the highlights of neural crest studies was its paramount role in head and face morphogenesis. Work pursued in our laboratory for the last fifteen years or so has analysed at both cellular and molecular levels the contribution of the NCCs to the construction of the facial and cranial structures. Recently, we have found that the cephalic neural crest plays also a key role in the formation of the fore- and mid-brain.

A New Wnt1-CRE TomatoRosa Embryonic Stem Cell Line: A Tool for Studying Neural Crest Cell Integration Capacity.

Neural crest (NC) cells are a migratory, multipotent population giving rise to numerous lineages in the embryo. Their plasticity renders attractive their use in tissue engineering-based therapies, but further knowledge on their in vivo behavior is required before clinical transfer may be envisioned. We here describe the isolation and characterization of a new mouse embryonic stem (ES) line derived from Wnt1-CRE-R26 RosaTomatoTdv blastocyst and show that it displays the characteristics of typical ES cells. Further, these cells can be efficiently directed toward an NC stem cell-like phenotype as attested by concomitant expression of NC marker genes and Tomato fluorescence. As native NC progenitors, they are capable of differentiating toward typical derivative phenotypes and interacting with embryonic tissues to participate in the formation of neo-structures. Their specific fluorescence allows purification and tracking in vivo. This cellular tool should facilitate a better understanding of the mechanisms driving NC fate specification and help identify the key interactions developed within a tissue after in vivo implantation. Altogether, this novel model may provide important knowledge to optimize NC stem cell graft conditions, which are required for efficient tissue repair.

The contribution of the neural crest to the vertebrate body.

As a transitory structure providing adult tissues of the vertebrates with very diverse cell types, the neural crest (NC) has attracted for long the interest of developmental biologists and is still the subject of ongoing research in a variety of animal models. Here we review a number of data from in vivo cell tracing and in vitro single cell culture experiments, which gained new insights on the mechanisms of cell migration, proliferation and differentiation during NC ontogeny. We put emphasis on the role of Hox genes, morphogens and interactions with neighbouring tissues in specifying and patterning the skeletogenic NC cells in the head. We also include advances made towards characterizing multipotent stem cells in the early NC as well as in various NC derivatives in embryos and even in adult.

Neural crest derivatives in ocular and periocular structures.

In vertebrates, the eye is an ectodermal compound structure associating neurectodermal and placodal anlagen. In addition, it benefits early on from a mesenchymal ectoderm-derived component, the neural crest. In this respect, the construction of chimeras between quail and chick has been a turning point, instrumental in appraising the contribution of the cephalic neural crest to the development of ocular and periocular structures. Given the variety of crest derivatives underscored in the developing eye, this study illustrates the fascinating ability of this unique structure to finely adapt its differentiation to microenvironmental cues. This analysis of neural crest cell contribution to ocular development emphasizes their paramount role to design the anterior segment of the eye, supply refracting media and contribute to the homeostasy of the anterior optic chamber.

In vivo methods to analyze cell origins, migrations, homing, and interactions in the blood, vascular, and immune systems of the avian and mammalian embryo.

In vivo experimental approaches that have been designed to study the ontogeny of the hematopoietic system in higher vertebrates are described in the present chapter. The avian embryo is directly available to manipulations in ovo during gastrulation and organogenesis. This permissiveness has led to the design of various approaches that provided crucial insights into the ontogeny of the hematopoietic system, particularly regarding traffic of progenitors between different compartments. In contrast, experimental manipulation of the developing mouse in utero is possible only during the second half of gestation, that is, the fetal period. This approach has been very useful in understanding how the immune system learns to distinguish self from nonself.

[Neural crest and vertebrate evolution]. / Crête neurale et évolution des vertébrés.

The neural crest (NC) is a remarkable structure of the Vertebrate embryo, which forms from the lateral borders of the neural plate (designated as neural folds) during neural tube closure. As soon as the NC is formed, its constitutive cells detach and migrate away from the neural primordium along definite pathways and at precise periods of time according to a rostro-caudal progression. The NC cells aggregate in definite places in the developing embryo, where they differentiate into a large variety of cell types including the neurons and glial cells of the peripheral nervous system, the pigment cells dispersed throughout the body and endocrine cells such as the adrenal medulla and the calcitonin producing cells. At the cephalic level only, in higher Vertebrates (but along the whole neural axis in Fishes and Amphibians), the NC is also at the origin of mesenchymal cells differentiating into connective tissue chondrogenic and osteogenic cells. Vertebrates belong to the larger group of Cordates which includes also the Protocordates (Cephalocordates and the Urocordates). All Cordates are characterized by the same body plan with a dorsal neural tube and a notochord which, in Vertebrates, exists only at embryonic stages. The main difference between Protocordates and Vertebrates is the very rudimentary development of cephalic structures in the former. As a result, the process of cephalization is one of the most obvious characteristics of Vertebrates. It was accompanied by the apparition of the NC which can therefore be considered as an innovation of Vertebrates during evolution. The application of a cell marking technique which consists in constructing chimeric embryos between two species of birds, the quail and the chicken, has led to show that the vertebrate head is mainly formed by cells originating from the NC, meaning that this structure was an important asset in Vertebrate evolution. Recent studies, described in this article, have strengthened this view by showing that the NC does not only provide the cells that build up the facial skeleton and most of the skull but plays a major role in early brain neurogenesis. It was shown that the cephalic NC cells produce signaling molecules able to regulate the activity of the two secondary organizing centers previously identified in the developing brain: the anterior neural ridge and the midbrain-hindbrain junction, which secrete Fgf8, a potent stimulator of early brain neurogenesis.