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Funder: NIHR Cambridge Biomedical Research centre, Background: Holoprosencephaly is a spectrum of developmental disorder of the embryonic forebrain in which there is failed or incomplete separation of the prosencephalon into two cerebral hemispheres. To date, dominant mutations in sonic hedgehog (SHH) pathway genes are the predominant Mendelian causes, and have marked interfamilial and intrafamilial phenotypical variabilities. Methods: We describe two families in which offspring had holoprosencephaly spectrum and homozygous predicted-deleterious variants in phospholipase C eta-1 (PLCH1). Immunocytochemistry was used to examine the expression pattern of PLCH1 in human embryos. We used SHH as a marker of developmental stage and of early embryonic anatomy. Results: In the first family, two siblings had congenital hydrocephalus, significant developmental delay and a monoventricle or fused thalami with a homozygous PLCH1 c.2065C>T, p.(Arg689*) variant. In the second family, two siblings had alobar holoprosencephaly and cyclopia with a homozygous PLCH1 c.4235delA, p.(Cys1079ValfsTer16) variant. All parents were healthy carriers, with no holoprosencephaly spectrum features. We found that the subcellular localisation of PLCH1 is cytoplasmic, but the p.(Cys1079ValfsTer16) variant was predominantly nuclear. Human embryo immunohistochemistry showed PLCH1 to be expressed in the notorcord, developing spinal cord (in a ventral to dorsal gradient), dorsal root ganglia, cerebellum and dermatomyosome, all tissues producing or responding to SHH. Furthermore, the embryonic subcellular localisation of PLCH1 was exclusively cytoplasmic, supporting protein mislocalisation contributing to the pathogenicity of the p.(Cys1079ValfsTer16) variant. Conclusion: Our data support the contention that PLCH1 has a role in prenatal mammalian neurodevelopment, and deleterious variants cause a clinically variable holoprosencephaly spectrum phenotype.

Holoprosencephaly is a spectrum of developmental disorder of the embryonic forebrain in which there is failed or incomplete separation of the prosencephalon into two cerebral hemispheres. To date, dominant mutations in sonic hedgehog (SHH) pathway genes are the predominant Mendelian causes, and have marked interfamilial and intrafamilial phenotypical variabilities.We describe two families in which offspring had holoprosencephaly spectrum and homozygous predicted-deleterious variants in phospholipase C eta-1 (In the first family, two siblings had congenital hydrocephalus, significant developmental delay and a monoventricle or fused thalami with a homozygousOur data support the contention that PLCH1 has a role in prenatal mammalian neurodevelopment, and deleterious variants cause a clinically variable holoprosencephaly spectrum phenotype.

The evolution of gene expression in mammalian organ development remains largely uncharacterized. Here we report the transcriptomes of seven organs (cerebrum, cerebellum, heart, kidney, liver, ovary and testis) across developmental time points from early organogenesis to adulthood for human, rhesus macaque, mouse, rat, rabbit, opossum and chicken. Comparisons of gene expression patterns identified correspondences of developmental stages across species, and differences in the timing of key events during the development of the gonads. We found that the breadth of gene expression and the extent of purifying selection gradually decrease during development, whereas the amount of positive selection and expression of new genes increase. We identified differences in the temporal trajectories of expression of individual genes across species, with brain tissues showing the smallest percentage of trajectory changes, and the liver and testis showing the largest. Our work provides a resource of developmental transcriptomes of seven organs across seven species, and comparative analyses that characterize the development and evolution of mammalian organs.

International audience; Definitive haematopoiesis in the fetal liver supports self-renewal anddifferentiation of haematopoietic stem cells and multipotent progenitors(HSC/MPPs) but remains poorly defined in humans. Here, using single-celltranscriptome profiling of approximately 140,000 liver and 74,000 skin, kidneyand yolk sac cells, we identify the repertoire of human blood and immune cellsduring development. We infer differentiation trajectories from HSC/MPPs andevaluate the influence of the tissue microenvironment on blood and immune celldevelopment. We reveal physiological erythropoiesis in fetal skin and thepresence of mast cells, natural killer and innate lymphoid cell precursors in theyolk sac. We demonstrate a shift in the haemopoietic composition of fetal liverduring gestation away from being predominantly erythroid, accompanied by aparallel change in differentiation potential of HSC/MPPs, which we functionallyvalidate. Our integrated map of fetal liver haematopoiesis provides a blueprintfor the study of paediatric blood and immune disorders, and a reference forharnessing the therapeutic potential of HSC/MPPs.

Combat-related blasts have caused the majority of injuries in Operation Iraqi Freedom with traumatic brain injury (TBI) emerging as a preeminent injury. The purpose of this dissertation is to describe the scope of combat-related blast injuries and the career performance outcomes of service personnel injured in blasts. Factors associated with career performance outcomes in service personnel injured in blasts and specifically in those with mild TBI were also explored. The Combat Trauma Registry Expeditionary Medical Encounter Database (CTR-EMED) is a collection of datasets from frontline medical care in Iraq and subsequent hospital care. The CTR-EMED provided demographics of injured service personnel and injury episode information. Outcome variables were ascertained from the Career History Archival Medical and Personnel System, which is a database of career and medical information for service personnel. In the 4623 blast episodes examined, mild TBI was the most frequent single injury type (10.8%) and with extremity injuries combined, the extremities was the area most commonly injured. Surface wounds of the extremities comprised 27.6% of all injuries. When examining service discharge in 4255 personnel injured in combat-related blasts, 37.8% experienced normal attrition and 8.3% early attrition. In service personnel with a discharge code, 29.8% had a disability-related discharge. Both early attrition and disability discharge proportions were higher in those with PTSD. In those without PTSD, a dose response relationship existed between injury severity and both disability discharge and early attrition. In those with a PTSD diagnosis, injury severity was associated with these adverse outcomes but the relationship was more complex. In the 790 service personnel diagnosed with mild TBI after a combat-related blast, 24% experienced a disability discharge. The most common acute TBI symptoms documented were headache (63.0%), loss of consciousness (37.0%) and tinnitus (31.0%). In those without PTSD, disability discharge was associated with age, total injury severity, time to discharge and post-concussive syndrome, while disability discharge was only associated with post- concussive syndrome in those with PTSD. These results provide important information for acute and rehabilitative care providers. The impact of PTSD on recovery after combat-related blast injuries should be explored to maximize functional outcome in these service personnel

SummaryDefinitive haematopoiesis in the fetal liver supports self-renewal and differentiation of haematopoietic stem cells/multipotent progenitors (HSC/MPPs), yet remains poorly defined in humans. Using single cell transcriptome profiling of ~133,000 fetal liver and ~65,000 fetal skin and kidney cells, we identify the repertoire of blood and immune cells in first and early second trimesters of development. From this data, we infer differentiation trajectories from HSC/MPPs, and evaluate the impact of tissue microenvironment on blood and immune cell development. We predict coupling of mast cell differentiation with erythro-megakaryopoiesis and identify physiological erythropoiesis in fetal skin. We demonstrate a shift in fetal liver haematopoietic composition during gestation away from being erythroid-predominant, accompanied by a parallel change in HSC/MPP differentiation potential, which we functionally validate. Our integrated map of fetal liver haematopoiesis provides a blueprint for the study of paediatric blood and immune disorders, and a valuable reference for understanding and harnessing the therapeutic potential of HSC/MPPs.

Close-up of human cerebellar development Early on, cerebellar development shares similarities across humans, nonhuman primates, and even mice. But differences emerge while development progresses, as cellular and molecular analyses by Haldipur et al. now reveal. The rhombic lip persists longer during cerebellar development in humans than in either the mouse or the macaque and generates a pool of neuroprogenitor cells. Similarly, the ventricular zone of the human cerebellum goes a step further than that of the mouse in developing an additional proliferative layer with outer radial glia cells. Transcriptome analysis revealed detailed similarities and differences between progenitor cells of the developing human cerebellum and neocortex. Science , this issue p. 454

Neurexins (NRXNs) are presynaptic terminal proteins and candidate neurodevelopmental disorder susceptibility genes; mutations presumably upset synaptic stabilization and function. However, analysis of human cortical tissue samples by RNAseq and quantitative real-time PCR at 8-12 postconceptional weeks, prior to extensive synapse formation, showed expression of all three NRXNs as well as several potential binding partners. However, the levels of expression were not identical; NRXN1 increased with age and NRXN2 levels were consistently higher than for NRXN3. Immunohistochemistry for each NRXN also revealed different expression patterns at this stage of development. NRXN1 and NRXN3 immunoreactivity was generally strongest in the cortical plate and increased in the ventricular zone with age, but was weak in the synaptogenic presubplate (pSP) and marginal zone. On the other hand, NRXN2 colocalized with synaptophysin in neurites of the pSP, but especially with GAP43 and CASK in growing axons of the intermediate zone. Alternative splicing modifies the role of NRXNs and we found evidence by RNAseq for exon skipping at splice site 4 and concomitant expression of KHDBRS proteins which control this splicing. NRXN2 may play a part in early cortical synaptogenesis, but NRXNs could have diverse roles in development including axon guidance, and intercellular communication between proliferating cells and/or migrating neurons.

In 2017, the Law Commission of Ontario (LCO) initiated an independent study to consider Ontario’s experience with class actions since the enactment of the Class Proceedings Act, 1992 (CPA). During this period, class actions have grown significantly in volume, complexity, and impact in Ontario and across Canada. Class actions also have systemic implications for access to justice, court procedures and efficiency, and government and corporate liability. Finally, this form of litigation has had major financial, policy and even cultural implications across the country. Following an intensive 18-month research project that included over 100 interviews with stakeholders and an empirical examination of Ontario cases, the LCO published its final report, and made over 40 recommendations for amendment and reform of the CPA. It is the first review of the CPA since the Act's inception, and the most detailed examination of class actions in Canada in three decades.

This article describes the DRiVe (demonstrate, replicate, investigate, variate, evaluate) Inquiry Framework, which provides teachers with specific detailed strategies and graphic organizers to support them in developing their science inquiry practices and in shifting to more open-ended science inquiry. Designed by teachers for teachers, the DRiVe Inquiry Framework has been implemented extensively in classrooms across Canada. This article takes readers through the details of using the framework in a Grade 7/8 combined class in which students tested pop bottle water filters. The part of the activity this article focuses on used the following science practices: asking questions; planning and carrying out investigations; analyzing and interpreting data; using mathematics and computational thinking; constructing explanations; engaging in argument from evidence; and obtaining, evaluating and communicating information.

Pediatric and adult kidney tumors differUnderstanding tumor origins and the similarities and differences between organ-specific cancers is important for determining treatment options. Younget al.generated more than 72,000 single-cell transcriptomes from healthy and cancerous human kidneys. From these data, they determined that Wilms tumor, a pediatric kidney cancer, originates from aberrant fetal cells, whereas adult kidney cancers are likely derived from a specific subtype of proximal convoluted tubular cell.Science, this issue p.594

Laminins are heterotrimeric glycoproteins of the extracellular matrix. Eleven different laminin chains have been identified in vertebrates. They are ubiquitously expressed in the human body, with a distinct tissue distribution. Laminin expression in neural retina and their functional role during human retinogenesis is still unknown. This study investigated the laminin expression in human developing and adult retina, showing laminin α1, α5, β1, β2 and γ1 to be predominantly expressed in Bruch’s membrane and the inner limiting membrane. Laminin-332 and laminin γ3 expression were mainly observed in the neural retina during retinal histogenesis. These expression patterns were largely conserved in pluripotent stem cell-derived retinal organoids. Blocking of laminin γ3 function in retinal organoids resulted in the disruption of laminar organisation and synapse formation, the loss of photoreceptor organisation and retinal ganglion cells. Our data demonstrate a unique temporal and spatial expression for laminins and reveal a novel role for laminin γ3 during human retinogenesis.

Immune landscape of the human kidney Single-cell RNA sequencing has begun to shed light on the full cellular diversity of specific organs. However, these studies rarely examine organ-specific immune cells. Stewart et al. sequenced healthy adult and fetal kidney samples at a single-cell level to define the heterogeneity in epithelial, myeloid, and lymphoid cells. From this dataset, they identified zonation of cells, with relevance to disease and the varied perturbations that occur in different tumor settings. This profiling of the human kidney generates a comprehensive census of existing cell populations that will help inform the diagnosis and treatment of kidney-related diseases. Science , this issue p. 1461

Human development is regulated by spatiotemporally restricted molecular programmes and is pertinent to many areas of basic biology and human medicine, such as stem cell biology, reproductive medicine and childhood cancer. Mapping human development has presented significant technological, logistical and ethical challenges. The availability of established human developmental biorepositories and the advent of cutting-edge single-cell technologies provide new opportunities to study human development. Here, we present a working framework for the establishment of a human developmental cell atlas exploiting single-cell genomics and spatial analysis. We discuss how the development atlas will benefit the scientific and clinical communities to advance our understanding of basic biology, health and disease.

Mutations in pre-mRNA processing factors (PRPFs) cause 40% of autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed PRPFs cause retinal disease. To understand the molecular basis of this phenotype, we have generated RP type 11 (PRPF31-mutated) patient-specific retinal organoids and retinal pigment epithelium (RPE) from induced pluripotent stem cells (iPSC). Impaired alternative splicing of genes encoding pre-mRNA splicing proteins occurred in patient-specific retinal cells and Prpf31 /- mouse retinae, but not fibroblasts and iPSCs, providing mechanistic insights into retinal-specific phenotypes of PRPFs. RPE was the most affected, characterised by loss of apical-basal polarity, reduced trans-epithelial resistance, phagocytic capacity, microvilli, and cilia length and incidence. Disrupted cilia morphology was observed in patient-derived-photoreceptors that displayed progressive features associated with degeneration and cell stress. In situ gene-editing of a pathogenic mutation rescued key structural and functional phenotypes in RPE and photoreceptors, providing proof-of-concept for future therapeutic strategies.

The extracellular matrix (ECM) plays an important role in numerous processes including cellular proliferation, differentiation, migration, maturation, adhesion guidance and axonal growth. To date, there has been no detailed analysis of the ECM distribution during retinal ontogenesis in humans and the functional importance of many ECM components is poorly understood. In this study, the expression of key ECM components in adult mouse and monkey retina, developing and adult human retina and retinal organoids derived from human pluripotent stem cells was studied. Our data indicate that basement membrane ECMs (Fibronectin and Collagen IV) were expressed in Bruch's membrane and the inner limiting membrane of the developing human retina, whilst the hyalectins (Versican and Brevican), cluster of differentiation 44 (CD44), photoreceptor-specific ECMs Interphotoreceptor Matrix Proteoglycan 1 (IMPG1) and Interphotoreceptor Matrix Proteoglycan 2 (IMPG2) were detected in the developing interphotoreceptor matrix (IPM). The expression of IMPG1, Versican and Brevican in the developing IPM was conserved between human developing retina and human pluripotent stem cell-derived retinal organoids. Blocking the action of CD44 and IMPG1 in pluripotent stem cell derived retinal organoids affected the development of photoreceptors, their inner/outer segments and connecting cilia and disrupted IPM formation, with IMPG1 having an earlier and more significant impact. Together, our data suggest an important role for IMPG1 and CD44 in the development of photoreceptors and IPM formation during human retinogenesis.The expression and the role of many extracellular matrix (ECM) components during human retinal development is not fully understood. In this study, expression of key ECM components (Collagen IV, Fibronectin, Brevican, Versican, IMPG1 and IMPG2) was investigated during human retinal ontogenesis. Collagen IV and Fibronectin were expressed in Bruch's membrane; whereas Brevican, Versican, IMPG1IMPG2 in the developing interphotoreceptor matrix (IPM). Retinal organoids were successfully generated from pluripotent stem cells. The expression of ECM components was examined in the retinal organoids and found to recapitulate human retinal development in vivo. Using functional blocking experiments, we were able to highlight an important role for IMPG1 and CD44 in the development of photoreceptors and IPM formation.

SummaryMutations in pre-mRNA processing factors (PRPFs) cause 40% of autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed PRPFs cause retinal disease. To understand the molecular basis of this phenotype, we have generated RP type 11 (PRPF31-mutated) patient-specific retinal organoids and retinal pigment epithelium (RPE) from induced pluripotent stem cells (iPSC). Impaired alternative splicing of genes encoding pre-mRNA splicing proteins occurred in patient-specific retinal cells and Prpf31+/− mouse retinae, but not fibroblasts and iPSCs, providing mechanistic insights into retinal-specific phenotypes of PRPFs. RPE was the most affected, characterised by loss of apical-basal polarity, reduced trans-epithelial resistance, phagocytic capacity, microvilli, and cilia length and incidence. Disrupted cilia morphology was observed in patient-derived-photoreceptors that displayed progressive features associated with degeneration and cell stress. In situ gene-editing of a pathogenic mutation rescued key structural and functional phenotypes in RPE and photoreceptors, providing proof-of-concept for future therapeutic strategies.eTOCPRPF31 is a ubiquitously expressed pre-mRNA processing factor that when mutated causes autosomal dominant RP. Using a patient-specific iPSC approach, Buskin and Zhu et al. show that retinal-specific defects result from altered splicing of genes involved in the splicing process itself, leading to impaired splicing, loss of RPE polarity and diminished phagocytic ability as well as reduced cilia incidence and length in both photoreceptors and RPE.HighlightsSuccessful generation of iPSC-derived RPE and photoreceptors from four RP type 11 patientsRPE cells express the mutant PRPF31 protein and show the lowest expression of wildtype proteinPRPF31 mutations result in altered splicing of genes involved in pre-mRNA splicing in RPE and retinal organoidsPrpf31 haploinsufficiency results in altered splicing of genes involved in pre-mRNA splicing in mouse retinaRPE cells display loss of polarity, reduced barrier function and phagocytosisPhotoreceptors display shorter and fewer cilia and degenerative featuresRPE cells display most abnormalities suggesting they might be the primary site of pathogenesisIn situ gene editing corrects the mutation and rescues key phenotypes

The cerebral cortex is divided stereotypically into a number of functionally distinct areas. According to the protomap hypothesis formulated by Rakic neural progenitors in the ventricular zone form a mosaic of proliferative units that provide a primordial species-specific cortical map. Positional information of newborn neurons is maintained during their migration to the overlying cortical plate. Much evidence has been found to support this hypothesis from studies of primary cortical areas in mouse models in particular. Differential expansion of cortical areas and the introduction of new functional modules during evolution might be the result of changes in the progenitor cells. The human cerebral cortex shows a wide divergence from the mouse containing a much higher proportion of association cortex and a more complicated regionalised repertoire of neuron sub-types. To what extent does the protomap hypothesis hold true for the primate brain? This review summarises a growing number of studies exploring arealised gene expression in the early developing human telencephalon. The evidence so far is that the human and mouse brain do share fundamental mechanisms of areal specification, however there are subtle differences which could lead us to a better understanding of cortical evolution and the origins of neurodevelopmental diseases.

Inherited microcephaly exposes Zika culprit Microcephaly has been the terrifying hallmark of the recent outbreak of Zika virus (ZIKV) in the Americas. How the virus damages brain development in the fetus is enigmatic. Chavali et al. found that in congenital microcephaly, mutations in a neural precursor protein, Musashi-1 (MSI1), impede RNA binding to neural stem cell targets, resulting in abnormal brain development (see the Perspective by Griffin). MSI1 also binds ZIKV RNA to amplify viral replication in cells. This interaction could put a pregnant woman at risk of giving birth to a microcephalic child. Furthermore, MSI1 is expressed at high levels in the mouse testis, which may explain the sexual transmission of this virus. Science , this issue p. 83 ; see also p. 33

GABAergic interneurons are crucial to controlling the excitability and responsiveness of cortical circuitry. Their developmental origin may differ between rodents and human. We have demonstrated the expression of 12 GABAergic interneuron-associated genes in samples from human neocortex by quantitative rtPCR from 8 to 12 postconceptional weeks (PCW) and shown a significant anterior to posterior expression gradient, confirmed by in situ hybridization or immunohistochemistry for GAD1 and 2, DLX1, 2, and 5, ASCL1, OLIG2, and CALB2. Following cortical plate (CP) formation from 8 to 9 PCW, a proportion of cells were strongly stained for all these markers in the CP and presubplate. ASCL1 and DLX2 maintained high expression in the proliferative zones and showed extensive immunofluorescent double-labeling with the cell division marker Ki-67. CALB2-positive cells increased steadily in the SVZ/VZ from 10 PCW but were not double-labeled with Ki-67. Expression of GABAergic genes was generally higher in the dorsal pallium than in the ganglionic eminences, with lower expression in the intervening ventral pallium. It is widely accepted that the cortical proliferative zones may generate CALB2-positive interneurons from mid-gestation; we now show that the anterior neocortical proliferative layers especially may be a rich source of interneurons in the early neocortex.

A five generation family with an X linked ocular disorder has been investigated. The major clinical features were reduced visual acuity, nystagmus, and myopia. Although impaired night vision was not a symptom, using psychophysical and electrophysiological testing both rod and cone function were found to be abnormal in all affected males. No abnormality was detected in carrier females. Gene location studies showed X linked transmission of a gene that maps to proximal Xp11. The findings observed in this cohort are similar to those previously reported in both congenital stationary night blindness type 2 (CSNB2) and Aland Island eye disease (AIED). This study addresses whether CSNB2 and AIED are a single entity or whether the latter is a subset of the former.

Hypertrophic cardiomyopathy (HCM) is caused by mutations in sarcomeric proteins, the commonest being MYBPC3 encoding myosin-binding protein C. It is characterised by left ventricular hypertrophy but there is an important pre-hypertrophic phenotype with features including crypts, abnormal mitral leaflets and trabeculae. We investigated these during mouse cardiac development using high-resolution episcopic microscopy. In embryonic hearts from wildtype, homozygous (HO) and heterozygous (HET) Mybpc3-targeted knock-out (KO) mice we show that crypts (one or two) are a normal part of wildtype development but they almost all resolve by birth. By contrast, HO and HET embryos had increased crypt presence, abnormal mitral valve formation and alterations in the compaction process. In scarce normal human embryos, crypts were sometimes present. This study shows that features of the human pre-hypertrophic HCM phenotype occur in the mouse. In an animal model we demonstrate that there is an embryological HCM phenotype. Crypts are a normal part of cardiac development but, along with the mitral valve and trabeculae, their developmental trajectory is altered by the presence of HCM truncating Mybpc3 gene mutation.

Neurogenesis of excitatory neurons in the developing human cerebral neocortex is a complex and dynamic set of processes and the exact mechanisms controlling the specification of human neocortical neuron subtypes are poorly understood due to lack of relevant cell models available. It has been shown that the transcription factors Pax6, Tbr2 and Tbr1, which are sequentially expressed in the rodent neocortex, regulate and define corticogenesis of glutamatergic neocortical neurons. In humans the homologues of these genes are generally expressed in a similar pattern, but with some differences. In this study, we used purified human umbilical cord blood stem cells, expressing pluripotency marker genes (OCT4, SOX2 and NANOG), to model human neocortical neurogenesis in vitro. We analyzed the expression patterns of PAX6, TBR2 and TBR1, at both protein and mRNA levels, throughout the 24 days of a sequential neuronal induction protocol. Their expression patterns correlated with those found in the developing human neocortex where they define different developmental stages of neocortical neurons. The derived cord blood neuron-like cells expressed a number of neuronal markers. They also expressed components of glutamatergic neurotransmission including glutamate receptor subunits and transporters, and generated calcium influxes upon stimulation with glutamate. Thus we have demonstrated that it is possible to model neocortical neurogenesis using cord blood stem cells in vitro. This may allow detailed analysis of the molecular mechanisms regulating neocortical neuronal specification, thus aiding the development of potential therapeutic tools for diseases and injuries of the cerebral cortex.

Developing neocortical progenitors express transcription factors in gradients that induce programs of region-specific gene expression. Our previous work identified anteriorly upregulated expression gradients of a number of corticofugal neuron-associated gene probe sets along the anterior–posterior axis of the human neocortex (8-12 postconceptional weeks [PCW]). Here, we demonstrate by real-time polymerase chain reaction, in situ hybridization and immunohistochemistry that 3 such genes, ROBO1, SRGAP1, and CTIP2 are highly expressed anteriorly between 8-12 PCW, in comparison with other genes (FEZF2, SOX5) expressed by Layer V, VI, and subplate neurons. All 3 were prominently expressed by early postmitotic neurons in the subventricular zone, intermediate zone, and cortical plate (CP) from 8 to 10 PCW. Between 12 and 15 PCW expression patterns for ER81 and SATB2 (Layer V), TBR1 (Layer V/VI) and NURR1 (Layer VI) revealed Layer V forming. By 15 PCW, ROBO1 and SRGAP1 expression was confined to Layer V, whereas CTIP2 was expressed throughout the CP anteriorly. We observed ROBO1 and SRGAP1 immunoreactivity in medullary corticospinal axons from 11 PCW onward. Thus, we propose that the coexpression of these 3 markers in the anterior neocortex may mark the early location of the human motor cortex, including its corticospinal projection neurons, allowing further study of their early differentiation.

Mutations in PLA2G6, which encodes calcium-independent phospholipase A2 group VIA (iPLA2-VIA), underlie the autosomal recessive disorder infantile neuroaxonal dystrophy (INAD). INAD typically presents in the first year of life, and leads to optic atrophy and psychomotor regression. We have examined PLA2G6 expression in early human embryonic development by in situ hybridization. At Carnegie Stage (CS) 19 (approximately 7 post conception weeks [PCW]), strong expression is evident in the ventricular zone (VZ) of midbrain and forebrain suggestive of expression in neural stem and progenitor cells. At CS23 (8 PCW) expression is also detectable in the VZ of the hindbrain and the subventricular zone (SVZ) of the developing neocortex, ganglionic eminences and diencephalon. By 9 PCW strong expression in the post-mitotic cells of the cortical plate can be seen in the developing neocortex. In the eye, expression is seen in the lens and retina at all stages examined. PLA2G6 expression is also evident in the alar plate of the spinal cord, dorsal root ganglia, the retina and lens in the eye and and several non-neuronal tissues, including developing bones, lung, kidney and gut. These findings suggest a role for PLA2G6 in neuronal proliferation throughout the developing brain and in maturing neurons in the cortical plate and hindbrain. Although widespread PLA2G6 expression is detected in neuronal tissues, the pattern shows dynamic changes with time and indicates that INAD pathogenesis may begin prior to birth.

Autosomal recessive primary microcephaly (MCPH) is a disorder of neurodevelopment resulting in a small brain1,2. We identified WDR62 as the second most common cause of MCPH after finding homozygous missense and frame-shifting mutations in seven MCPH families. In human cell lines, we found that WDR62 is a spindle pole protein, as are ASPM and STIL, the MCPH7 and MCHP7 proteins3–5. Mutant WDR62 proteins failed to localize to the mitotic spindle pole. In human and mouse embryonic brain, we found that WDR62 expression was restricted to neural precursors undergoing mitosis. These data lend support to the hypothesis that the exquisite control of the cleavage furrow orientation in mammalian neural precursor cell mitosis, controlled in great part by the centrosomes and spindle poles, is critical both in causing MCPH when perturbed and, when modulated, generating the evolutionarily enlarged human brain6–9.

We are developing a three-dimensional (3D) atlas of the human embryonic brain using anatomical landmarks and gene expression data to define major subdivisions through 12 stages of development [Carnegie Stages (CS) 12-23; approximately 26-56 days post conception (dpc)]. Virtual 3D anatomical models are generated from intact specimens using optical projection tomography (OPT). Using MAPAINT software, selected gene expression data, gathered using standard methods of in situ hybridization and immunohistochemistry, are mapped to a representative 3D model for each chosen Carnegie stage. In these models, anatomical domains, defined on the basis of morphological landmarks and comparative knowledge of expression patterns in vertebrates, are linked to a developmental neuroanatomic ontology. Human gene expression patterns for genes with characteristic expression in different vertebrates (e.g. PAX6, GAD65 and OLIG2) are being used to confirm and/or refine the human anatomical domain boundaries. We have also developed interpolation software that digitally generates a full domain from partial data. Currently, the 3D models and a preliminary set of anatomical domains and ontology are available on the atlas pages along with gene expression data from approximately 100 genes in the HUDSEN Human Spatial Gene Expression Database (http://www.hudsen.org). The aim is that full 3D data will be generated from expression data used to define a more detailed set of anatomical domains linked to a more advanced anatomy ontology and all of these will be available online, contributing to the long-term goal of the atlas, which is to help maximize the effective use and dissemination of data wherever it is generated.

The division of the neocortex into functional areas (the cortical map) differs little between individuals, although brain lesions in development can lead to substantial re-organization of regional identity. We are studying how the cortical map is established in the human brain as a first step towards understanding this plasticity. Previous work on rodent development has identified certain transcription factors (e.g. Pax6, Emx2) expressed in gradients across the neocortex that appear to control regional expression of cell adhesion molecules and organization of area-specific thalamocortical afferent projections. Although mechanisms may be shared, the human neocortex is composed of different and more complex local area identities. Using Affymetrix gene chips of human foetal brain tissue from 8 to 12.5 post-conceptional weeks [PCW, equivalent to Carnegie stage (CS) 23, to Foetal stage (F) 4], human material obtained from the MRC-Wellcome Trust Human Developmental Biology Resource (http://www.hdbr.org), we have identified a number of genes that exhibit gradients along the anterior-posterior axis of the neocortex. Gene probe sets that were found to be upregulated posteriorally compared to anteriorally, included EMX2, COUPTFI and FGF receptor 3, and those upregulated anteriorally included cell adhesion molecules such as cadherins and protocadherins, as well as potential motor cortex markers and frontal markers (e.g. CNTNAP2, PCDH17, ROBO1, and CTIP2). Confirmation of graded expression for a subset of these genes was carried out using real-time PCR. Furthermore, we have established a dissociation cell culture model utilizing tissue dissected from anteriorally or posteriorally derived developing human neocortex that exhibits similar gradients of expression of these genes for at least 72 h in culture.

The subplate is a largely transient zone containing precocious neurons involved in several key steps of cortical development. The majority of subplate neurons form a compact layer in mouse, but are dispersed throughout a much larger zone in the human. In rodent, subplate neurons are among the earliest born neocortical cells, whereas in primate, neurons are added to the subplate throughout cortical neurogenesis. Magnetic resonance imaging and histochemical studies show that the human subplate grows in size until the end of the second trimester. Previous microarray experiments in mice have shown several genes that are specifically expressed in the subplate layer of the rodent dorsal cortex. Here we examined the human subplate for some of these markers. In the human dorsal cortex, connective tissue growth factor-positive neurons can be seen in the ventricular zone at 15–22 postconceptional weeks (PCW) (most at 17 PCW) and are present in the subplate at 22 PCW. The nuclear receptor-related 1 protein is mostly expressed in the subplate in the dorsal cortex, but also in lower layer 6 in the lateral and perirhinal cortex, and can be detected from 12 PCW. Our results suggest that connective tissue growth factor- and nuclear receptor-related 1-positive cells are two distinct cell populations of the human subplate. Furthermore, our microarray analysis in rodent suggested that subplate neurons produce plasma proteins. Here we demonstrate that the human subplate also expresses α2zinc-binding globulin and Alpha-2-Heremans-Schmid glycoprotein/human fetuin. In addition, the established subplate neuron marker neuropeptide Y is expressed superficially, whereas potassium/chloride co-transporter (KCC2)-positive neurons are localized in the deep subplate at 16 PCW. These observations imply that the human subplate shares gene expression patterns with rodent, but is more compartmentalized into superficial and deep sublayers. This increased complexity of the human subplate may contribute to differential vulnerability in response to hypoxia/ischaemia across the depth of the cortex. Combining knowledge of cell-type specific subplate gene expression with modern imaging methods will enable a better understanding of neuropathologies involving the subplate.

Visualisation and interpretation of gene expression data have been crucial to advances in our understanding of mechanisms underlying early brain development. As most developmental processes involve complex changes in size, shape and structure, spatial-data can most readily provide information at multiple levels (cell type, cell location in relation to tissue organisation or body axes, etc.), that can be related to these complex changes. Although three-dimensional (3D) spatial-data are ideal, the restricted availability of suitable tissues makes it difficult to generate these for genes expressed at early human fetal stages. Mapping gene expression data to representative 3D models facilitates combinatorial analysis of multiple expression patterns but does not overcome the problems of sparsely sampled data in time and space. Here we describe software that allows 3D domains to be reconstructed by interpolating between sparse 2D gene expression patterns that have been mapped to 3D representative models of corresponding human developmental stages. A set of procedures are proposed to infer expression domains in these gaps. The procedures, which are connected in a serial way, include components clustering, components tracking, shape matching and points interpolation. Each procedure consists of a graphical user interface and a set of algorithms. Results on exemplar gene data are provided.

The transcription factors Emx2 and Pax6 are expressed in the proliferating zones of the developing rodent neocortex, and gradients of expression interact in specifying caudal and rostral identities. Pax6 is also involved in corticoneurogenesis, being expressed by radial glial progenitors that give rise to cells that also sequentially express Tbr2, NeuroD and Tbr1, genes temporally downstream of Pax6. In this study, using in situ hybridization, we analysed the expression of EMX2, PAX6, TBR2, NEUROD and TBR1 mRNA in the developing human cortex between 8 and 12 postconceptional weeks (PCW). EMX2 mRNA was expressed in the ventricular (VZ) and subventricular zones (SVZ), but also in the cortical plate, unlike in the rodent. However, gradients of expression were similar to that of the rodent at all ages studied. PAX6 mRNA expression was limited to the VZ and SVZ. At 8 PCW, PAX6 was highly expressed rostrally but less so caudally, as has been seen in the rodent, however this gradient disappeared early in corticogenesis, by 9 PCW. There was less restricted compartment-specific expression of TBR2, NEUROD and TBR1 mRNA than in the rodent, where the gradients of expression were similar to that of PAX6 prior to 9 PCW. The gradient disappeared for TBR2 by 10 PCW, and for NEUROD and TBR1 by 12 PCW. These data support recent reports that EMX2 but not PAX6 is more directly involved in arealization, highlighting that analysis of human development allows better spatio-temporal resolution than studies in rodents.

Duane's retraction syndrome (DRS) is a complex congenital eye movement disorder caused by aberrant innervation of the extraocular muscles by axons of brainstem motor neurons. Studying families with a variant form of the disorder (DURS2-DRS), we have identified causative heterozygous missense mutations in CHN1 , a gene on chromosome 2q31 that encodes α2-chimaerin, a Rac guanosine triphosphatase–activating protein (RacGAP) signaling protein previously implicated in the pathfinding of corticospinal axons in mice. We found that these are gain-of-function mutations that increase α2-chimaerin RacGAP activity in vitro. Several of the mutations appeared to enhance α2-chimaerin translocation to the cell membrane or enhance its ability to self-associate. Expression of mutant α2-chimaerin constructs in chick embryos resulted in failure of oculomotor axons to innervate their target extraocular muscles. We conclude that α2-chimaerin has a critical developmental function in ocular motor axon pathfinding.

We have employed immunohistochemistry for multiple markers to investigate the structure and possible function of the different compartments of human cerebral wall from the formation of cortical plate at 8 postconceptional weeks (PCW) to the arrival of thalamocortical afferents at 17 PCW. New observations include the subplate emerging as a discrete differentiated layer by 10 PCW, characterized by synaptophysin and vesicular gamma-aminobutyric acid transporter expression also seen in the marginal zone, suggesting that these compartments may maintain a spontaneously active synaptic network even before the arrival of thalamocortical afferents. The subplate expanded from 13 to 17 PCW, becoming the largest compartment and differentiated further, with NPY neurons located in the outer subplate and KCC2 neurons in the inner subplate. Glutamate decarboxylase and calretinin-positive inhibitory neurons migrated tangentially and radially from 11.5 PCW, appearing in larger numbers toward the rostral pole. The proliferative zones, marked by Ki67 expression, developed a complicated structure by 12.5 PCW reflected in transcription factor expression patterns, including TBR2 confined to the inner subventricular and outer ventricular zones and TBR1 weakly expressed in the subventricular zone (SVZ). PAX6 was extensively expressed in the proliferative zones such that the human outer SVZ contained a large reservoir of PAX6-positive potential progenitor cells.

During vertebrate central nervous system development, the apical neuroepithelium is bathed with embryonic Cerebrospinal Fluid (e-CSF) which plays regulatory roles in cortical cell proliferation and maintenance. Here, we report the first proteomic analysis of human e-CSF and compare it to an extensive proteomic analysis of rat e-CSF. As expected, we identified a large collection of protease inhibitors, extracellular matrix proteins, and transport proteins in CSF. However, we also found a surprising suite of signaling and intracellular proteins not predicted by previous proteomic analysis. Some of the intracellular proteins are likely to represent the contents of microvesicles recently described within the CSF (Marzesco, A. M., et al. J. Cell Sci. 2005, 118 (Pt. 13), 2849-2858). Defining the rich composition of e-CSF will enable a greater understanding of its concerted actions during critical stages of brain development.

Congenital anomalies are a significant burden on human health. Understanding the developmental origins of such anomalies is key to developing potential therapies. The Human Developmental Biology Resource (HDBR), based in London and Newcastle, UK, was established to provide embryonic and fetal material for a variety of human studies ranging from single gene expression analysis to large-scale genomic/transcriptomic studies. Increasingly, HDBR material is enabling the derivation of stem cell lines and contributing towards developments in tissue engineering. Use of the HDBR and other fetal tissue resources discussed here will contribute to the long-term aims of understanding the causation and pathogenesis of congenital anomalies, and developing new methods for their treatment and prevention.

Murine embryonic stem (ES) cells can replicate indefinitely in culture and can give rise to all tissues, including the germline, when reimplanted into a murine blastocyst. ES cells can also be differentiated in vitro into a wide range of cell types. We have utilized a liver-specific marker to demonstrate that murine ES cells can differentiate into hepatocytes in vitro. We have used ES cells carrying a gene trap vector insertion (I.114) into an ankyrin repeat-containing gene (Gtar) that we have previously shown provides an exclusive beta-galactosidase marker for the early differentiation of hepatocytes in vivo. beta-Galactosidase-positive cells were differentiated from I.114 ES cells in vitro. The identity of these cells was confirmed by the expression of the proteins alpha-fetoprotein, albumin, and transferrin and by the fact that they have an ultrastructural appearance consistent with that of embryonic hepatocytes. We propose that this model system of hepatic differentiation in vitro could be used to define factors that are involved in specification of the hepatocyte lineage. In addition, human ES cells have recently been derived and it has been proposed that they may provide a source of differentiated cell types for cell replacement therapies in the treatment of a variety of diseases.

The first haematopoietic stem cells in mammalian and non-mammalian vertebrates are derived from mesoderm, therefore genes that are important in mesoderm patterning and formation might also play an essential role in haematopoietic stem cell commitment and differentiation. Several members of the Wnt gene family are expressed in very specific patterns in embryonic mesoderm and have previously been shown to act as haematopoietic growth factors. In order to investigate in detail the role that such secreted proteins play in the biology of early haematopoietic commitment we have used in vitro differentiation of murine embryonal stem (ES) as a model system. Using reverse-transcriptase polymerase chain reaction analysis we identified several candidate Wnt genes whose expression pattern was consistent with a role in generation, maintenance and/or differentiation of early haematopoietic progenitor cells including three genes previously shown to have a role in haematopoiesis (Wnt5a, Wnt2b and Wnt10b). The most interesting candidate was Wnt3, because of its strong and regulated expression during in vitro differentiation of murine ES cells as well as its early embryonic expression in mesoderm. Overexpression of Wnt3 was sufficient to cause a consistent increase in the number of embryoid bodies committing to haematopoiesis further strengthening the evidence that this protein can enhance haematopoietic commitment during in vitro differentiation of ES cells. In addition, overexpression of Wnt3 caused a marked upregulation of Brachyury expression, thus providing some evidence that Brachyury may be one of the target genes for the Wnt3 signalling pathway.

SRY, SOX9, and DAX1 are key genes in human sex determination, by virtue of their associated male-to-female sex reversal phenotypes when mutated (SRY, SOX9) or over-expressed (DAX1). During human sex determination, SRY is expressed in 46,XY gonads coincident with sex cord formation, but also persists as nuclear protein within Sertoli cells at 18 weeks gestation. High-level SOX9 expression in the sex cords of the testis parallels that seen during mouse development, however in humans, SOX9 transcripts also are detected in the developing ovary. Low-level DAX1 expression predates peak SRY expression by at least 10 days, and persists in Sertoli cells throughout the entire sex determination period. In Dosage Sensitive Sex reversal, the anti-testis properties of DAX1 over-expression could act prior to the peak effects of SRY and continue during the period of SOX9 expression. These findings highlight expression differences for the SRY, SOX9, and DAX1 genes during sex determination in humans and mice. These results provide a direct framework for future investigation into the mechanisms underlying normal and abnormal human sex determination.

The transcription factors SF-1 and WT1 play pivotal roles in mammalian gonadal development and sexual differentiation. In human embryos, both SF-1 and WT1 are expressed when the indifferent gonadal ridge first forms at 32 days post-ovulation. As the sex cords develop - providing morphological evidence of testis differentiation - SF-1 localises predominantly to developing Sertoli cells in the sex cords, whereas WT1 retains a broader pattern of expression. Later, SF-1 localises predominantly to steroidogenic Leydig cells, and WT1 localises to the sex cords. In the ovary, SF-1 and WT1 transcripts persist in the gonadal ridge from the earliest developmental stages throughout the critical period of sex determination. These studies, which delineate for the first time the sequential expression profiles of SF-1 and WT1 during human gonadal development, provide a framework for understanding human sex reversal phenotypes associated with their mutations.

The objective of this article is to measure risk-adjusted outcomes following the introduction of a pediatric practice guideline for the care of asthma. This includes a measurement of outcome variables that were identified during design of a pediatric practice guideline (PPG) for the treatment of asthma in hospitalized pediatric patients: one historical control group off the PPG (PPGOff/h, n = 116), and two concurrent groups of patients (PPGOn/c, n = 100, and PPGOff/c, n = 66) treated at Texas Children's Hospital, a stand-alone, medical school-based teaching hospital. This includes the use of a physician-coordinated practice guideline with multidisciplinary input that is part of the medical record, and functions as an order set to guide care by both physicians and support staff. The main outcome is the effect of a pediatrie practice guideline upon risk-adjusted outcome variables measuring quality of care, health status, and utilization of resources. The practice guideline was used in 63.7% of the concurren...