Human Developmental Biology Resource Research Articles

Cardiac development demystified by use of the HDBR atlas.

Much has been learned over the last half century regarding the molecular and genetic changes that take place during cardiac development. As yet, however, these advances have not been translated into knowledge regarding the marked changes that take place in the anatomical arrangements of the different cardiac components. As such, therefore, many aspects of cardiac development are still described on the basis of speculation rather than evidence. In this review, we show how controversial aspects of development can readily be arbitrated by the interested spectator by taking advantage of the material now gathered together in the Human Developmental Biology Resource; HDBR. We use the material to demonstrate the changes taking place during the formation of the ventricular loop, the expansion of the atrioventricular canal, the incorporation of the systemic venous sinus, the formation of the pulmonary vein, the process of atrial septation, the remodelling of the pharyngeal arches, the major changes occurring during formation of the outflow tract, the closure of the embryonic interventricular communication, and the formation of the ventricular walls. We suggest that access to the resource makes it possible for the interested observer to arbitrate, for themselves, the ongoing controversies that continue to plague the understanding of cardiac development.

Expression of the schizophrenia associated gene FEZ1 in the early developing fetal human forebrain.

The protein fasciculation and elongation zeta-1 (FEZ1) is involved in axon outgrowth but potentially interacts with various proteins with roles ranging from intracellular transport to transcription regulation. Gene association and other studies have identified FEZ1 as being directly, or indirectly, implicated in schizophrenia susceptibility. To explore potential roles in normal early human forebrain neurodevelopment, we mapped FEZ1 expression by region and cell type. All tissues were provided with maternal consent and ethical approval by the Human Developmental Biology Resource. RNAseq data were obtained from previously published sources. Thin paraffin sections from 8 to 21 post-conceptional weeks (PCW) samples were used for RNAScope in situ hybridization and immunohistochemistry against FEZ1 mRNA and protein, and other marker proteins. Tissue RNAseq revealed that FEZ1 is highly expressed in the human cerebral cortex between 7.5-17 PCW and single cell RNAseq at 17-18 PCW confirmed its expression in all neuroectoderm derived cells. The highest levels were found in more mature glutamatergic neurons, the lowest in GABAergic neurons and dividing progenitors. In the thalamus, single cell RNAseq similarly confirmed expression in multiple cell types. In cerebral cortex sections at 8-10 PCW, strong expression of mRNA and protein appeared confined to post-mitotic neurons, with low expression seen in progenitor zones. Protein expression was observed in some axon tracts by 16-19 PCW. However, in sub-cortical regions, FEZ1 was highly expressed in progenitor zones at early developmental stages, showing lower expression in post-mitotic cells. FEZ1 has different expression patterns and potentially diverse functions in discrete forebrain regions during prenatal human development.

Sulfation Pathways During Neurodevelopment

Sulfate is an important nutrient that modulates a diverse range of molecular and cellular functions in mammalian physiology. Over the past 2 decades, animal studies have linked numerous sulfate maintenance genes with neurological phenotypes, including seizures, impaired neurodevelopment, and behavioral abnormalities. Despite sulfation pathways being highly conserved between humans and animals, less than one third of all known sulfate maintenance genes are clinically reportable. In this review, we curated the temporal and spatial expression of 91 sulfate maintenance genes in human fetal brain from 4 to 17 weeks post conception using the online Human Developmental Biology Resource Expression. In addition, we performed a systematic search of PubMed and Embase, identifying those sulfate maintenance genes linked to atypical neurological phenotypes in humans and animals. Those findings, together with a search of the Online Mendelian Inheritance in Man database, identified a total of 18 candidate neurological dysfunction genes that are not yet considered in clinical settings. Collectively, this article provides an overview of sulfate biology genes to inform future investigations of perturbed sulfate homeostasis associated with neurological conditions.

COVID-19 and Vertical Transmission: Assessing the Expression of ACE2 / TMPRSS2 in the Human Fetus and Placenta to Assess the Risk of SARS-CoV-2 Infection

Background: While pregnant women have been identified as a potentially at-risk group concerning COVID-19 infection, little is known regarding the susceptibility of the fetus to infection. Co-expression of ACE2 and TMPRSS2 has been identified as a pre-requisite for infection, and expression across different tissues is known to vary between children and adults. However, the expression of these proteins in the fetus is unknown.Methods: We performed a retrospective analysis of single cell data repositories. This data was then validated at both gene and protein level by performing qRT-PCR and two-colour immunohistochemistry on a library of second-trimester human fetal tissues.Findings: TMPRSS2 is present at both gene and protein level in the predominantly epithelial fetal tissues analysed. ACE2 is present at significant levels, only in the fetal intestine and kidney and is not expressed in the fetal lung. The placenta is also negative for the two proteins both during development and at term.Interpretation: This dataset indicates that the lungs are unlikely to be a viable route of SARS-CoV2 fetal infection. The fetal kidney, despite presenting both the proteins required for the infection, is anatomically protected from the exposure to the virus. However, the GI tract is likely to be susceptible to infection due to its high co-expression of both proteins, as well as its exposure to potentially infected amniotic fluid.Funding Information: This work was made possible by an MRC / UKRI COVID-19 Rapid response initiative grant (MR/V028480/1).Declaration of Interests: The authors declare no conflicts of interest related to this work or its developments. DC is founder, shareholder, and consultant of Next Generation Diagnostic SRL.Ethics Approval Statement: Human fetal tissues were obtained with consent through the Human Developmental Biology Resource (HDBR; REC 18/LO/0822 – IRAS 244325; Project ID 2000478). Placental samples were obtained at delivery of an uncomplicated, full-term pregnancy (median 39 weeks PCW [Range 38+1 -39+4] for 6 patients recruited through the EVERREST Prospective Study as normal controls (National Research Ethics reference 13/LO/1254), NCT02097667 registered 31st October 2013[10].

Abstract LB-220: Functional consequences of aberrant alternative splicing in embryonal tumor with multilayered rosettes

Abstract Embryonal tumor with multilayered rosettes (ETMR) is an aggressive and rare variant of embryonal brain tumors. Amplification of C19MC microRNA cluster and expression LIN28 are distinctive features of ETMR. Despite the increasing efforts to decipher ETMR, the biology remains poorly understood. To date, the role of aberrant alternative splicing in ETMR is not thoroughly investigated. In the current study a comprehensive analysis was performed on published unprocessed RNA-seq reads of tissue- matched ETMR (SRP032476), and fetal controls from the Human Developmental Biology Resource (HDBR) (E-MTAB-4840) datasets. Gene expression was quantified in samples using kallisto/sleuth pipeline. For the alternative splicing analysis, reads were mapped to the genome (hg38) using STAR. Identification and quantification of alternative splicing events (skipped exon, alternative 5′ and 3′splice sites, mutually exclusive exons, multi Exons Skip and retained intron) were carried out using SplAdder. Functional enrichment analysis for the differentially expressed and the differentially spliced genes was subsequently performed using Metascape. All statistical values reported were corrected for multiple hypothesis testing by Benjamini-Hochberg false discovery rate (q-values).The expression analysis revealed a total of 2205 differentially expressed genes (DEGs) between ETMR and fetal controls while 2446 showed differential alternative splicing patterns. DEGs with significant alternative splicing events were identified to be involved in ubiquitin mediated proteolysis, endocytosis, RNA transport, regulation of actin cytoskeleton pathways as well as histone modification. Interestingly, DEGs with intron retention events were involved in various signaling pathways including TGF-beta signaling pathway, MAPK signaling pathway and ErbB signaling pathway. These findings highlight the role of defective alternative splicing in shaping ETMR tumor landscape, and the identified pathways constitute potential therapeutic targets. Citation Format: Dina Hesham AbouRaya, Shahenda El-Naggar. Functional consequences of aberrant alternative splicing in embryonal tumor with multilayered rosettes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-220.

Embryonic and foetal expression patterns of the ciliopathy gene CEP164.

Nephronophthisis-related ciliopathies (NPHP-RC) are a group of inherited genetic disorders that share a defect in the formation, maintenance or functioning of the primary cilium complex, causing progressive cystic kidney disease and other clinical manifestations. Mutations in centrosomal protein 164 kDa (CEP164), also known as NPHP15, have been identified as a cause of NPHP-RC. Here we have utilised the MRC-Wellcome Trust Human Developmental Biology Resource (HDBR) to perform immunohistochemistry studies on human embryonic and foetal tissues to determine the expression patterns of CEP164 during development. Notably expression is widespread, yet defined, in multiple organs including the kidney, retina and cerebellum. Murine studies demonstrated an almost identical Cep164 expression pattern. Taken together, these data support a conserved role for CEP164 throughout the development of numerous organs, which, we suggest, accounts for the multi-system disease phenotype of CEP164-mediated NPHP-RC.

Trisomy 21 Driven Pro-Inflammatory Signalling in Fetal Bone Marrow May Play a Role in Perturbed B-Lymphopoiesis and Acute Lymphoblastic Leukemia of Down Syndrome

Introduction: Children with Down syndrome (DS) have a markedly increased risk of acute lymphoblastic leukemia (ALL), suggesting that trisomy 21 (T21) has specific effects on hematopoietic stem and progenitor cell (HSPC) biology in early life. Data from human fetal liver (FL) indicates that T21 alters fetal hematopoiesis, causing multiple defects in lympho-myelopoiesis. The impact of T21 on fetal B lymphopoiesis and how this may underpin the increase in ALL is not well known. We have recently found that fetal bone marrow (FBM) rather than FL is the main site of B lymphopoiesis; with a marked enrichment of fetal-specific progenitors (early lymphoid progenitors, ELP and PreProB progenitors) that lie upstream of adult type ProB progenitors (O'Byrne et al, Blood, in press). Previous preliminary data suggested that B progenitors were also reduced in T21 FBM (Roy et al, Blood. 124, 4331). Aim: To dissect putative molecular mechanisms responsible for the defects in T21 FBM B-lymphopoiesis and its association with childhood DS ALL. Methods: Second trimester human FBM and paediatric ALL samples were obtained from the Human Developmental Biology Resource and UK Childhood Leukaemia Cell Bank respectively. Multiparameter flow cytometry/sorting, transcriptome analysis by RNA-sequencing and microarray, and stromal co-culture assays were used to characterize HSPC and mesenchymal stromal cells (MSC) from normal (NM) disomic (n=21-35) and T21 (n=7-12) human FBM; RNASeq was performed on cytogenetically matched non-DS (n=13) and DS ALL (n=7). Results: In contrast to NM FBM, fetal specific progenitors were virtually absent (CD34+CD10-CD19-CD127+ ELP 2.8±0.4% vs. 0.8±0.4% of CD34+ cells) or very severely reduced (CD34+CD10-CD19+ PreProB 12.8±1 vs 2.6±0.7%) in T21 FBM. This was despite a >4-fold increase in the frequency of immunophenotypic HSC (4.2±1.2% vs 0.9±0.2% of CD34+ cells) and similar frequencies of MPP and LMPP in T21 FBM. As in adult BM, the vast majority of B progenitors in T21 FBM were CD34+CD10+CD19+ ProB progenitors with a frequency (28.8±8.3%) similar to NM FBM (30.3±2.3% of CD34+ cells). Thus, T21 causes a severe block in B-progenitor commitment at the LMPP stage, in tandem with a compensatory expansion of ProB progenitors. Consistent with this, T21 FBM HSC, MPP and LMPP had reduced B cell potential in vitro compared to NM FBM in MS5 co-cultures. RNAseq of NM (n=3) and T21 (n=3) FBM HSPC demonstrated global transcriptomic disruption by T21, with increased gene expression in HSC, MPP, LMPP and ProB progenitors. Cell cycle genes were enriched in T21 ProB progenitors. Despite these functional and global gene expression differences, expression of key B-lineage commitment genes was maintained suggesting the defect in B-lymphopoiesis may be secondary to lineage skewing of multipotent progenitors towards a non-B lymphoid fate and/or mediated by extrinsic factors. GSEA pointed to a role for multiple inflammatory pathways in T21 hematopoiesis with dysregulation of IFNα, IL6 and TGFβ signalling pathways in T21 HSC/LMPP. To investigate the role of the T21 microenvironment, we co-cultured NM HSC, MPP and LMPP with T21 or NM primary FBM MSC. T21 FBM MSC (n=3) had reduced capacity to support B cell differentiation in vitro consistent with perturbation of MSC function by T21. Similar to T21 FBM HSPC, transcriptomic analysis of T21 FBM MSC by microarray showed enrichment for IFNα signalling compared to NM; and T21 HSPC and MSC both showed increased gene expression for IFNα receptors IFNAR1 and IFNAR2, which are encoded on chromosome 21. Since IFNα was undetectable by ELISA of conditioned media from NM and T21 MSC, differences in secreted IFNα from MSC are unlikely to fully explain the increased IFN signalling in T21 HSPC and MSC. This suggests that T21 may drive autocrine rather than paracrine IFN signalling in FBM cells. Finally, RNASeq showed perturbed inflammatory signalling in DS ALL compared to non-DS ALL, suggesting a role for T21-driven inflammatory pathways in the biology of DS ALL. Conclusions: These data show that T21 severely impairs B lymphopoiesis in FBM and is associated with expression of proinflammatory gene expression programs in T21 FBM HSPC and MSC and DS ALL. The compensatory expansion of T21 FBM ProB progenitors, through self-renewal or via an alternative differentiation pathway; with concomitant T21-driven proinflammatory signalling may underpin the increased risk of B progenitor ALL in childhood. Disclosures No relevant conflicts of interest to declare.

HDBR Expression: A Unique Resource for Global and Individual Gene Expression Studies during Early Human Brain Development.

This paper describes a new resource, HDBR (Human Developmental Biology Resource) Expression, for studying prenatal human brain development. It is unique in the age range (4 post conception weeks [PCW] to 17PCW) and number of brains (172) studied, particularly those under 8PCW (33). The great majority of the samples are karyotyped. HDBR Expression is also unique in that both the large-scale data sets (RNA-seq data, SNP genotype data) and the corresponding RNA and DNA samples are available, the latter via the MRC-Wellcome Trust funded HDBR1(Gerrelli et al., 2015). There are 557 RNA-seq datasets from different brain regions, the majority between 4 and 12PCW. During this time the major brain regions are established and the early stages of cortex development occur (Bystron et al., 2008; O'Rahilly and Muller, 2008). In addition, there are 42 RNAseq data sets from spinal cord and 29 from cerebral choroid plexus. There are also 243 additional tissue specimens in paraffin wax blocks available for individual gene expression studies. For almost all of the brains and specimens in wax blocks there are corresponding SNP genotype data. Large-scale/high-throughput studies, such as next-generation sequencing, are providing raw material in a wide variety of research fields (for review of concepts and methodologies of RNA-seq, see Shin et al., 2014). Studies of human development are hampered by difficulties in obtaining tissue which means that publicly available large-scale data sets are particularly useful because data can be used and re-used (Kang et al., 2011; Zhang et al., 2011; Fietz et al., 2012; Miller et al., 2014; Darmanis et al., 2015).

Enabling research with human embryonic and fetal tissue resources.

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.

Investigating Embryonic Expression Patterns and Evolution of AHI1 and CEP290 Genes, Implicated in Joubert Syndrome

Joubert syndrome and related diseases (JSRD) are developmental cerebello-oculo-renal syndromes with phenotypes including cerebellar hypoplasia, retinal dystrophy and nephronophthisis (a cystic kidney disease). We have utilised the MRC-Wellcome Trust Human Developmental Biology Resource (HDBR), to perform in-situ hybridisation studies on embryonic tissues, revealing an early onset neuronal, retinal and renal expression pattern for AHI1. An almost identical pattern of expression is seen with CEP290 in human embryonic and fetal tissue. A novel finding is that both AHI1 and CEP290 demonstrate strong expression within the developing choroid plexus, a ciliated structure important for central nervous system development. To test if AHI1 and CEP290 may have co-evolved, we carried out a genomic survey of a large group of organisms across eukaryotic evolution. We found that, in animals, ahi1 and cep290 are almost always found together; however in other organisms either one may be found independent of the other. Finally, we tested in murine epithelial cells if Ahi1 was required for recruitment of Cep290 to the centrosome. We found no obvious differences in Cep290 localisation in the presence or absence of Ahi1, suggesting that, while Ahi1 and Cep290 may function together in the whole organism, they are not interdependent for localisation within a single cell. Taken together these data support a role for AHI1 and CEP290 in multiple organs throughout development and we suggest that this accounts for the wide phenotypic spectrum of AHI1 and CEP290 mutations in man.

Investigating gradients of gene expression involved in early human cortical development.

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.

06-P032 Human Developmental Biology Resource (HDBR): A unique resource for studying human embryo and fetal development

The Human Developmental Biology Resource (HDBR) is a unique national tissue bank resource, funded by the Wellcome Trust and Medical Research Council. It provides human embryonic and fetal tissue to the research community for gene expression studies. Ethical approval exists for the collection, storage and distribution of material between 4 and 12 weeks of development. This valuable material is intended for use primarily to study the expression of developmentally significant genes, particularly those implicated in birth defects and other inherited disorders. However, the HDBR will consider provision of material for any human gene expression study, provided the project is well justified. The HDBR can provide fresh, frozen or sectioned material. A significant proportion of the HDBR material is karyotyped and chromosomally normal material is routinely provided for research. In addition, karyotypically abnormal material can be provided on request. The HDBR offers an in-house service for the analysis of gene expression at the RNA or protein levels. There is a charge for this service, to cover staff time and consumable costs. The HDBR also provides electronic images for publication and advice on interpretation of results. All data generated from HDBR-supported projects will in future be deposited in a public database. This poster presents specimen data from recently completed HDBR studies.

MRC–Wellcome Trust Human Developmental Biology Resource: enabling studies of human developmental gene expression

A striking finding of the human and mouse genome sequencing projects is that, although there are many differences between the two species, they have similar numbers of genes. The differences arise during development and are driven, in part, by changes in gene expression. The MRC-Wellcome Trust Human Developmental Biology Resource (HDBR) is a unique resource that provides human embryonic and foetal tissues to the scientific community, enabling gene-expression studies at these crucial periods of development.