MRC-Wellcome Trust Human Developmental Biology Research Articles

Expression patterns of ciliopathy genes ARL3 and CEP120 reveal roles in multisystem development

BackgroundJoubert syndrome and related disorders (JSRD) and Jeune syndrome are multisystem ciliopathy disorders with overlapping phenotypes. There are a growing number of genetic causes for these rare syndromes, including the recently described genes ARL3 and CEP120.MethodsWe sought to explore the developmental expression patterns of ARL3 and CEP120 in humans to gain additional understanding of these genetic conditions. We used an RNA in situ detection technique called RNAscope to characterise ARL3 and CEP120 expression patterns in human embryos and foetuses in collaboration with the MRC-Wellcome Trust Human Developmental Biology Resource.ResultsBoth ARL3 and CEP120 are expressed in early human brain development, including the cerebellum and in the developing retina and kidney, consistent with the clinical phenotypes seen with pathogenic variants in these genes.ConclusionsThis study provides insights into the potential pathogenesis of JSRD by uncovering the spatial expression of two JSRD-causative genes during normal human development.

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.

Neuronal Development and Onset of Electrical Activity in the Human Enteric Nervous System

The enteric nervous system (ENS) is the largest branch of the peripheral nervous system, comprising complex networks of neurons and glia, which are present throughout the gastrointestinal tract. Although development of a fully functional ENS is required for gastrointestinal motility, little is known about the ontogeny of ENS function in humans. We studied the development of neuronal subtypes and the emergence of evoked electrical activity in the developing human ENS. Human fetal gut samples (obtained via the MRC-Wellcome Trust Human Developmental Biology Resource-UK) were characterized by immunohistochemistry, calcium imaging, RNA sequencing, and quantitative real-time polymerase chain reaction analyses. Human fetal colon samples have dense neuronal networks at the level of the myenteric plexus by embryonic week (EW) 12, with expression of excitatory neurotransmitter and synaptic markers. By contrast, markers of inhibitory neurotransmitters were not observed until EW14. Electrical train stimulation of internodal strands did not evoke activity in the ENS of EW12 or EW14 tissues. However, compound calcium activation was observed at EW16, which was blocked by the addition of 1 μmol/L tetrodotoxin. Expression analyses showed that this activity was coincident with increases in expression of genes encoding proteins involved in neurotransmission and action potential generation. In analyses of human fetal intestinal samples, we followed development of neuronal diversity, electrical excitability, and network formation in the ENS. These processes are required to establish the functional enteric circuitry. Further studies could increase our understanding of the pathogenesis of a range of congenital enteric neuropathies.

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.

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.