Neural Patterning Research Articles

Hand2 Functions to Synergistically Activate Gli Target Genes in Mandibular Neural Crest Cells

The Hedgehog (Hh) pathway has been well‐studied and implicated in neural crest cell (NCC) survival and facial patterning; however, the mechanism(s) by which the transcription factors (TFs) of the pathway, the Gli TFs, orchestrate Hh signal transduction remains elusive. For example, Gli ChIP‐seq data from NCCs in the developing mandibular prominence (MNP) reveal that the presence of a peak is not sufficient to predict a Gli “target.” In light of these data and previous studies suggesting Gli proteins interact with co‐factors, we performed genome‐wide analyses to identify motifs that frequently appear near Gli binding regions (GBRs). These analyses revealed that E‐box motifs (bHLH TF binding sites) were significantly enriched (p=0.001) within Gli ChIP‐seq peaks compared to random sequences. Additionally, ChIP‐seq peaks for Hand2, a bHLH TF known to be necessary for MNP development, were 111‐fold enriched within Gli peaks. A comparison of Hand2f/f;Wnt1‐Cre and Gli2f/f;Gli3f/f;Wnt1‐Cre MNP phenotypes shows striking similarities, which in conjunction with the significant overlap of Gli and Hand2 co‐occupied genomic regions suggests these two proteins may work in tandem to influence Gli target gene expression. Interrogation of DNA motifs within genomic locations occupied by Gli and Hand2 together versus those occupied by Gli alone revealed the existence of two distinct types of GBRs: a ‘canonical’ GBR defined by the presence of a medially located A (GACCACCC), and a ‘deviant’ GBR defined by a T in the medial position (GACCTCCC). Interestingly, the ‘canonical’ GBR is 19‐fold enriched (p<10−136) in Gli3 peaks without a Hand2 peak, whereas it is only 3‐fold enriched (p<10−47) in Gli3 peaks that contain a Hand2 peak. To test if these distinct GBRs convey biological relevance, we identified canonical and deviant GBRs within Gli and Hand2 ChIP‐seq peaks of Gli targets important for mandibular development and linked them to reporter constructs transfected into 09–1 cells. We found that Hand2 and Gli utilize a deviant GBR to synergistically activate Gli targets including Foxd1 and Foxd2. In sum, our work establishes insight into the existence of a sequence‐dependent mechanism for Gli target gene regulation in which molecular accomplices may be necessary for optimal transcription of Gli targets in NCCs of the developing MNP.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Hox-TALE regulatory circuit for neural crest patterning is conserved across vertebrates

In jawed vertebrates (gnathostomes), Hox genes play an important role in patterning head and jaw formation, but mechanisms coupling Hox genes to neural crest (NC) are unknown. Here we use cross-species regulatory comparisons between gnathostomes and lamprey, a jawless extant vertebrate, to investigate conserved ancestral mechanisms regulating Hox2 genes in NC. Gnathostome Hoxa2 and Hoxb2 NC enhancers mediate equivalent NC expression in lamprey and gnathostomes, revealing ancient conservation of Hox upstream regulatory components in NC. In characterizing a lamprey hoxα2 NC/hindbrain enhancer, we identify essential Meis, Pbx, and Hox binding sites that are functionally conserved within Hoxa2/Hoxb2 NC enhancers. This suggests that the lamprey hoxα2 enhancer retains ancestral activity and that Hoxa2/Hoxb2 NC enhancers are ancient paralogues, which diverged in hindbrain and NC activities. This identifies an ancestral mechanism for Hox2 NC regulation involving a Hox-TALE regulatory circuit, potentiated by inputs from Meis and Pbx proteins and Hox auto-/cross-regulatory interactions.

Distinct Activities of Gli1 and Gli2 in the Absence of Ift88 and the Primary Cilia.

The primary cilia play essential roles in Hh-dependent Gli2 activation and Gli3 proteolytic processing in mammals. However, the roles of the cilia in Gli1 activation remain unresolved due to the loss of Gli1 transcription in cilia mutant embryos, and the inability to address this question by overexpression in cultured cells. Here, we address the roles of the cilia in Gli1 activation by expressing Gli1 from the Gli2 locus in mouse embryos. We find that the maximal activation of Gli1 depends on the cilia, but partial activation of Gli1 by Smo-mediated Hh signaling exists in the absence of the cilia. Combined with reduced Gli3 repressors, this partial activation of Gli1 leads to dorsal expansion of V3 interneuron and motor neuron domains in the absence of the cilia. Moreover, expressing Gli1 from the Gli2 locus in the presence of reduced Sufu has no recognizable impact on neural tube patterning, suggesting an imbalance between the dosages of Gli and Sufu does not explain the extra Gli1 activity. Finally, a non-ciliary Gli2 variant present at a higher level than Gli1 when expressed from the Gli2 locus fails to activate Hh pathway ectopically in the absence of the cilia, suggesting that increased protein level is unlikely the major factor underlying the ectopic activation of Hh signaling by Gli1 in the absence of the cilia.

Arx Expression Suppresses Ventralization of the Developing Dorsal Forebrain

Early brain development requires a tight orchestration between neural tube patterning and growth. How pattern formation and brain growth are coordinated is incompletely understood. Previously we showed that aristaless-related homeobox (ARX), a paired-like transcription factor, regulates cortical progenitor pool expansion by repressing an inhibitor of cell cycle progression. Here we show that ARX participates in establishing dorsoventral identity in the mouse forebrain. In Arx mutant mice, ventral genes, including Olig2, are ectopically expressed dorsally. Furthermore, Gli1 is upregulated, suggesting an ectopic activation of SHH signaling. We show that the ectopic Olig2 expression can be repressed by blocking SHH signaling, implicating a role for SHH signaling in Olig2 induction. We further demonstrate that the ectopic Olig2 accounts for the reduced Pax6 and Tbr2 expression, both dorsal specific genes essential for cortical progenitor cell proliferation. These data suggest a link between the control of dorsoventral identity of progenitor cells and the control of their proliferation. In summary, our data demonstrate that ARX functions in a gene regulatory network integrating normal forebrain patterning and growth, providing important insight into how mutations in ARX can disrupt multiple aspects of brain development and thus generate a wide spectrum of neurodevelopmental phenotypes observed in human patients.

Xenopus slc7a5 is essential for notochord function and eye development

slc7a5 (also known as LAT1), largely accepted as an amino acid transporter, has been shown to play important roles in cancer and developmental processes. Because knockout mice of Slc7a5 are embryonically lethal due to placental defects, it is difficult to evaluate its role in early development. In this study, expression and function of slc7a5 were evaluated in Xenopus laevis embryos that develop without a placenta. Expression of slc7a5 was detected in the notochord and in the eye and it was not co-localized with slc3a2, which helps slc7a5 to localize at the plasma membrane, before the late neurula stage. Loss-of-function experiment with a morpholino antisense oligonucleotide led to defect of neural and non-neural patterning, inhibition of primary neurogenesis, and disruption of eye development. Disruption of neural development and primary neurogenesis was likely due to impaired notochord development as sonic hedgehog (shh) signaling pathway was compromised in slc7a5-inhibited embryos. These results suggest that slc7a5 is required for notochord development and subsequent primary neurogenesis via shh/gli signaling and for eye development. These novel developmental roles of slc7a5 appeared to be independent of transport function at least before the late neurula stage.

Potential therapeutic roles of retinoids for prevention of neuroinflammation and neurodegeneration in Alzheimer's disease.

All retinoids, which can be natural and synthetic, are chemically related to vitamin A. Both natural and synthetic retinoids use specific nuclear receptors such as retinoic acid receptors and retinoid X receptors to activate specific signaling pathways in the cells. Retinoic acid signaling is extremely important in the central nervous system. Impairment of retinoic acid signaling pathways causes severe pathological processes in the central nervous system, especially in the adult brain. Retinoids have major roles in neural patterning, differentiation, axon outgrowth in normal development, and function of the brain. Impaired retinoic acid signaling results in neuroinflammation, oxidative stress, mitochondrial malfunction, and neurodegeneration leading to progressive Alzheimer’s disease, which is pathologically characterized by extra-neuronal accumulation of amyloid plaques (aggregated amyloid-beta) and intra-neurofibrillary tangles (hyperphosphorylated tau protein) in the temporal lobe of the brain. Alzheimer’s disease is the most common cause of dementia and loss of memory in old adults. Inactive cholinergic neurotransmission is responsible for cognitive deficits in Alzheimer’s disease patients. Deficiency or deprivation of retinoic acid in mice is associated with loss of spatial learning and memory. Retinoids inhibit expression of chemokines and neuroinflammatory cytokines in microglia and astrocytes, which are activated in Alzheimer’s disease. Stimulation of retinoic acid receptors and retinoid X receptors slows down accumulation of amyloids, reduces neurodegeneration, and thereby prevents pathogenesis of Alzheimer’s disease in mice. In this review, we described chemistry and biochemistry of some natural and synthetic retinoids and potentials of retinoids for prevention of neuroinflammation and neurodegeneration in Alzheimer’s disease.

Transcriptional regulation of the Ciona Gsx gene in the neural plate

The ascidian neural plate consists of a defined number of identifiable cells organized in a grid of rows and columns, representing a useful model to investigate the molecular mechanisms controlling neural patterning in chordates. Distinct anterior brain lineages are specified via unique combinatorial inputs of signalling pathways with Nodal and Delta-Notch signals patterning along the medial-lateral axis and FGF/MEK/ERK signals patterning along the anterior-posterior axis of the neural plate. The Ciona Gsx gene is specifically expressed in the a9.33 cells in the row III/column 2 position of anterior brain lineages, characterised by a combinatorial input of Nodal-OFF, Notch-ON and FGF-ON. Here, we identify the minimal cis-regulatory element (CRE) of 376 bp, which can recapitulate the early activation of Gsx. We show that this minimal CRE responds in the same way as the endogenous Gsx gene to manipulation of FGF- and Notch-signalling pathways and to overexpression of Snail, a mediator of Nodal signals, and Six3/6, which is required to demarcate the anterior boundary of Gsx expression at the late neurula stage. We reveal that sequences proximal to the transcription start site include a temporal regulatory element required for the precise transcriptional onset of gene expression. We conclude that sufficient spatial and temporal information for Gsx expression is integrated in 376 bp of non-coding cis-regulatory sequences.

GLIS3 Transcriptionally Activates WNT Genes to Promote Differentiation of Human Embryonic Stem Cells into Posterior Neural Progenitors.

Anterior-posterior (A-P) specification of the neural tube involves initial acquisition of anterior fate followed by the induction of posterior characteristics in the primitive anterior neuroectoderm. Several morphogens have been implicated in the regulation of A-P neural patterning; however, our understanding of the upstream regulators of these morphogens remains incomplete. Here, we show that the Krüppel-like zinc finger transcription factor GLI-Similar 3 (GLIS3) can direct differentiation of human embryonic stem cells (hESCs) into posterior neural progenitor cells in lieu of the default anterior pathway. Transcriptomic analyses reveal that this switch in cell fate is due to rapid activation of Wingless/Integrated (WNT) signaling pathway. Mechanistically, through genome-wide RNA-Seq, ChIP-Seq, and functional analyses, we show that GLIS3 binds to and directly regulates the transcription of several WNT genes, including the strong posteriorizing factor WNT3A, and that inhibition of WNT signaling is sufficient to abrogate GLIS3-induced posterior specification. Our findings suggest a potential role for GLIS3 in the regulation of A-P specification through direct transcriptional activation of WNT genes. Stem Cells 2018 Stem Cells 2019;37:202-215.

Human Cortical Neuron Generation Using Cell Reprogramming: A Review of Recent Advances.

The study and treatment of neurological disorders have been hampered by a lack of access to live, healthy, or disease-affected human neurons. The recent advances in the field of cell reprogramming offer exciting new possibilities for disease modeling, drug development, and cell-based therapies. Since the derivation of human embryonic stem cells (hESCs) and their differentiation into neurons, cell reprogramming technologies have built on these protocols to generate mature human neurons of disease-associated phenotypes from somatic cells. Mechanistic knowledge of neural patterning and neurogenesis has been essential for the establishment of reprogramming strategies that employ a combination of transcription factors and small molecules selected due to their critical role in brain development. The generation of reprogrammed human neurons has the potential to further enhance our knowledge of pathways underlying the developmental process of the human brain, the current knowledge of which has predominantly come from animal studies, postmortem tissue, and most recently hESCs. Somatic cell reprogramming began in 2006 with the first report of induced pluripotent stem cell (iPSC) derivation from mouse fibroblasts. This has now expanded to direct-to-induced neuron and direct-to-induced neural stem or precursor reprogramming using a variety of viral and nonviral delivery methods. Most recently, iPSC technology has been extended to the development of three-dimensional brain structures referred to as brain spheroids or organoids. This review will discuss the reprogramming strategies that have been formulated to generate cortical neurons that are associated with many diseases, including autism spectrum disorders and schizophrenia.

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence.

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

Differential Effects of Heparin and Hyaluronic Acid on Neural Patterning of Human Induced Pluripotent Stem Cells.

A lack of well-established animal models that can efficiently represent human brain pathology has led to the development of human induced pluripotent stem cell (hiPSC)-derived brain tissues. Brain organoids have enhanced our ability to understand the developing human brain and brain disorders (e.g., Schizophrenia, microcephaly), but the organoids still do not accurately recapitulate the anatomical organization of the human brain. Therefore, it is important to evaluate and optimize induction and signaling factors in order to engineer the next generation of brain organoids. In this study, the impact of hyaluronic acid (HA), a major brain extracellular matrix (ECM) component that interacts with cells through ligand-binding receptors, on the patterning of brain organoids from hiPSCs was evaluated. To mediate HA- binding capacity of signaling molecules, heparin was added in addition to HA or conjugated to HA to form hydrogels (with two different moduli). The neural cortical spheroids derived from hiPSCs were treated with either HA or heparin plus HA (Hep- HA) and were analyzed for ECM impacts on neural patterning. The results indicate that Hep-HA has a caudalizing effect on hiPSC-derived neural spheroids, in particular for stiff Hep-HA hydrogels. Wnt and Hippo/Yes-associated protein (YAP) signaling was modulated (using Wnt inhibitor IWP4 or actin disruption agent Cytochalasin D respectively) to understand the underlying mechanism. IWP4 and cytochalasin D promote forebrain identity. The results from this study should enhance the understanding of influence of biomimetic ECM factors for brain organoid generation.

FoxO6 regulates Hippo signaling and growth of the craniofacial complex.

The mechanisms that regulate post-natal growth of the craniofacial complex and that ultimately determine the size and shape of our faces are not well understood. Hippo signaling is a general mechanism to control tissue growth and organ size, and although it is known that Hippo signaling functions in neural crest specification and patterning during embryogenesis and before birth, its specific role in postnatal craniofacial growth remains elusive. We have identified the transcription factor FoxO6 as an activator of Hippo signaling regulating neonatal growth of the face. During late stages of mouse development, FoxO6 is expressed specifically in craniofacial tissues and FoxO6-/- mice undergo expansion of the face, frontal cortex, olfactory component and skull. Enlargement of the mandible and maxilla and lengthening of the incisors in FoxO6-/- mice are associated with increases in cell proliferation. In vitro and in vivo studies demonstrated that FoxO6 activates Lats1 expression, thereby increasing Yap phosphorylation and activation of Hippo signaling. FoxO6-/- mice have significantly reduced Hippo Signaling caused by a decrease in Lats1 expression and decreases in Shh and Runx2 expression, suggesting that Shh and Runx2 are also linked to Hippo signaling. In vitro, FoxO6 activates Hippo reporter constructs and regulates cell proliferation. Furthermore PITX2, a regulator of Hippo signaling is associated with Axenfeld-Rieger Syndrome causing a flattened midface and we show that PITX2 activates FoxO6 expression. Craniofacial specific expression of FoxO6 postnatally regulates Hippo signaling and cell proliferation. Together, these results identify a FoxO6-Hippo regulatory pathway that controls skull growth, odontogenesis and face morphology.

Evolution of Locomotor Rhythms

Nervous systems control locomotion using rhythmically active networks that orchestrate motor neuron firing patterns. Whether animals use common or distinct genetic programs to encode motor rhythmicity remains unclear. Cross-species comparisons have revealed remarkably conserved neural patterning systems but have also unveiled divergent circuit architectures that can generate similar locomotor behaviors.

Expression of NK cluster genes in the onychophoran Euperipatoides rowelli: implications for the evolution of NK family genes in nephrozoans

BackgroundUnderstanding the evolution and development of morphological traits of the last common bilaterian ancestor is a major goal of the evo-devo discipline. The reconstruction of this “urbilaterian” is mainly based on comparative studies of common molecular patterning mechanisms in recent model organisms. The NK homeobox genes are key players in many of these molecular pathways, including processes regulating mesoderm, heart and neural development. Shared features seen in the expression patterns of NK genes have been used to determine the ancestral bilaterian characters. However, the commonly used model organisms provide only a limited view on the evolution of these molecular pathways. To further investigate the ancestral roles of NK cluster genes, we analyzed their expression patterns in the onychophoran Euperipatoides rowelli.ResultsWe identified nine transcripts of NK cluster genes in E. rowelli, including single copies of NK1, NK3, NK4, NK5, Msx, Lbx and Tlx, and two copies of NK6. All of these genes except for NK6.1 and NK6.2 are expressed in different mesodermal organs and tissues in embryos of E. rowelli, including the anlagen of somatic musculature and the heart. Furthermore, we found distinct expression patterns of NK3, NK5, NK6, Lbx and Msx in the developing nervous system. The same holds true for the NKL gene NK2.2, which does not belong to the NK cluster but is a related gene playing a role in neural patterning. Surprisingly, NK1, Msx and Lbx are additionally expressed in a segment polarity-like pattern early in development—a feature that has been otherwise reported only from annelids.ConclusionOur results indicate that the NK cluster genes were involved in mesoderm and neural development in the last common ancestor of bilaterians or at least nephrozoans (i.e., bilaterians to the exclusion of xenacoelomorphs). By comparing our data from an onychophoran to those from other bilaterians, we critically review the hypothesis of a complex “urbilaterian” with a segmented body, a pulsatile organ or heart, and a condensed mediolaterally patterned nerve cord.

Serine Threonine Kinase Receptor-Associated Protein Deficiency Impairs Mouse Embryonic Stem Cells Lineage Commitment Through CYP26A1-Mediated Retinoic Acid Homeostasis.

Retinoic acid (RA) signaling is essential for the differentiation of embryonic stem cells (ESCs) and vertebrate development. RA biosynthesis and metabolism are controlled by a series of enzymes, but the molecular regulators of these enzymes remain largely obscure. In this study, we investigated the functional role of the WD-domain protein STRAP (serine threonine kinase receptor-associated protein) in the pluripotency and lineage commitment of murine ESCs. We generated Strap knockout (KO) mouse ESCs and subjected them to spontaneous differentiation. We observed that, despite the unchanged characteristics of ESCs, Strap KO ESCs exhibited defects for lineage differentiation. Signature gene expression analyses revealed that Strap deletion attenuated intracellular RA signaling in embryoid bodies (EBs), and exogenous RA significantly rescued this deficiency. Moreover, loss of Strap selectively induced Cyp26A1 expression in mouse EBs, suggesting a potential role of STRAP in RA signaling. Mechanistically, we identified putative Krüppel-like factor 9 (KLF9) binding motifs to be critical in the enhancement of non-canonical RA-induced transactivation of Cyp26A1. Increased KLF9 expression in the absence of STRAP is partially responsible for Cyp26A1 induction. Interestingly, STRAP knockdown in Xenopus embryos influenced anterior-posterior neural patterning and impaired the body axis and eye development during early Xenopus embryogenesis. Taken together, our study reveals an intrinsic role for STRAP in the regulation of RA signaling and provides new molecular insights for ESC fate determination. Stem Cells 2018;36:1368-1379.

An ancestral role for Semaphorin3F-Neuropilin signaling in patterning neural crest within the new vertebrate head

The origin of the vertebrate head is one of the great unresolved issues in vertebrate evolutionary developmental biology. Although many of the novelties in the vertebrate head and pharynx derive from the neural crest, it is still unknown how early vertebrates patterned the neural crest within the ancestral body plan they inherited from invertebrate chordates. Here, using a basal vertebrate, the sea lamprey, we show that homologs of Semaphorin3F (Sema3F) ligand and its Neuropilin (Nrp) receptors show complementary and dynamic patterns of expression that correlate with key periods of neural crest development (migration and patterning of cranial neural crest-derived structures). Using CRISPR/Cas9-mediated mutagenesis, we demonstrate that lamprey Sema3F is essential for patterning of neural crest-derived melanocytes, cranial ganglia and the head skeleton, but is not required for neural crest migration or patterning of trunk neural crest derivatives. Based on comparisons with jawed vertebrates, our results suggest that the deployment of Nrp-Sema3F signaling, along with other intercellular guidance cues, was pivotal in allowing early vertebrates to organize and pattern cranial neural crest cells into many of the hallmark structures that define the vertebrate head.

The development and metamorphosis of the indirect developing acorn worm Schizocardium californicum (Enteropneusta: Spengelidae)

BackgroundEnteropneusts are benthic marine invertebrates that belong to the deuterostome phylum Hemichordata. The two main clades of enteropneusts are defined by differences in early life history strategies. In the Spengelidae and Ptychoderidae, development is indirect via a planktotrophic tornaria larva. In contrast, development in the Harrimanidae is direct without an intervening larval life history stage. Most molecular studies in the development and evolution of the enteropneust adult body plan have been carried out in the harrimanid Saccoglossus kowalevskii. In order to compare these two developmental strategies, we have selected the spengelid enteropneust Schizocardium californicum as a suitable indirect developing species for molecular developmental studies. Here we describe the methods for adult collecting, spawning and larval rearing in Schizocardium californicum, and describe embryogenesis, larval development, and metamorphosis, using light microscopy, immunocytochemistry and confocal microscopy.ResultsAdult reproductive individuals can be collected intertidally and almost year-round. Spawning can be triggered by heat shock and large numbers of larvae can be reared through metamorphosis under laboratory conditions. Gastrulation begins at 17 h post-fertilization (hpf) and embryos hatch at 26 hpf as ciliated gastrulae. At 3 days post-fertilization (dpf), the tornaria has a circumoral ciliary band, mouth, tripartite digestive tract, protocoel, larval muscles and a simple serotonergic nervous system. The telotroch develops at 5 dpf. In the course of 60 days, the serotonergic nervous system becomes more elaborate, the posterior coeloms develop, and the length of the circumoral ciliary band increases. At the end of the larval stage, larval muscles disappear, gill slits form, and adult muscles develop. Metamorphosis occurs spontaneously when the larva reaches its maximal size (ca. 3 mm), and involves loss and reorganization of larval structures (muscles, nervous system, digestive tract), as well as development of adult structures (adult muscles, tripartite body organization).ConclusionsThis study will enable future research in S. californicum to address long standing questions related to the evolution of axial patterning mechanisms, germ layer induction, neurogenesis and neural patterning, the mechanisms of metamorphosis, the relationships between larval and adult body plans, and the evolution of metazoan larval forms.

Generation of Neural Organoids from Human Embryonic Stem Cells Using the Rotary Cell Culture System: Effects of Microgravity on Neural Progenitor Cell Fate.

Progress in aeronautics and spaceflight technologies requires in parallel further research on how microgravity may affect human tissue. To date, little is known about the effects of microgravity on human development. In this study we used the rotary cell culture system to investigate whether microgravity supports the generation and maintenance of neural organoids derived from human embryonic stem cells (hESCs) as a model of human brain development. Our results show that although neural organoids could be generated and maintained in microgravity conditions, there were changes in expression of rostral–caudal neural patterning genes and cortical markers compared to organoids generated in standard conditions. This phenomenon was also observed in hESC-derived cortical organoids exposed to microgravity for relatively shorter periods. These results are one of the first for analyzing human neurogenesis in a microgravity environment.

Neuropathology of holoprosencephaly.

Holoprosencephaly (HPE) is a primary disorder of neural induction and patterning of the rostral neural tube resulting in noncleavage of the forebrain with failure to form two separate distinct hemispheres. The spectrum of HPE is very broad and encompasses various neuropathological phenotypes of different severity. The recent literature has demonstrated that the phenotypic variability of HPE ranges from aprosencephaly-atelencephaly, at the most severe end, to milder forms such as the "middle interhemispheric variant" of HPE at the less severe end of the spectrum. Between them, different intermediate forms demonstrate a continuum in a wide phenotypic spectrum rather than well-defined categories. Although the term "HPE" suggests a disorder affecting only the prosencephalon, other brain structures are involved, underlining the complexity of the malformation. Because of close spatiotemporal interactions and common signaling pathways contributing to the development of both brain and face, concomitant facial and ocular anomalies are associated with brain malformation. In this review, the characteristic neuropathological features of the various forms of HPE are described as well as their associated brain, face, and ocular malformations, to delineate the different phenotypes.