The exon junction complex component EIF4A3 is essential for mouse and human cortical progenitor mitosis and neurogenesis.

Mutations in components of the exon junction complex (EJC) are associated with neurodevelopment and disease. In particular, reduced levels of the RNA helicase EIF4A3 cause Richieri-Costa-Pereira syndrome (RCPS) and copy number variations are linked to intellectual disability. Consistent with this, Eif4a3 haploinsufficient mice are microcephalic. Altogether, this implicates EIF4A3 in cortical development; however, the underlying mechanisms are poorly understood. Here, we use mouse and human models to demonstrate that EIF4A3 promotes cortical development by controlling progenitor mitosis, cell fate and survival. Eif4a3 haploinsufficiency in mice causes extensive cell death and impairs neurogenesis. Using Eif4a3;p53 compound mice, we show that apoptosis has the most impact on early neurogenesis, while additional p53-independent mechanisms contribute to later stages. Live imaging of mouse and human neural progenitors reveals that Eif4a3 controls mitosis length, which influences progeny fate and viability. These phenotypes are conserved, as cortical organoids derived from RCPS iPSCs exhibit aberrant neurogenesis. Finally, using rescue experiments we show that EIF4A3 controls neuron generation via the EJC. Altogether, our study demonstrates that EIF4A3 mediates neurogenesis by controlling mitosis duration and cell survival, implicating new mechanisms that underlie EJC-mediated disorders.

EIF4A3 deficient human iPSCs and mouse models demonstrate neural crest defects that underlie Richieri-Costa-Pereira syndrome.

Biallelic loss-of-function mutations in the RNA-binding protein EIF4A3 cause Richieri-Costa-Pereira syndrome (RCPS), an autosomal recessive condition mainly characterized by craniofacial and limb malformations. However, the pathogenic cellular mechanisms responsible for this syndrome are entirely unknown. Here, we used two complementary approaches, patient-derived induced pluripotent stem cells (iPSCs) and conditional Eif4a3 mouse models, to demonstrate that defective neural crest cell (NCC) development explains RCPS craniofacial abnormalities. RCPS iNCCs have decreased migratory capacity, a distinct phenotype relative to other craniofacial disorders. Eif4a3 haploinsufficient embryos presented altered mandibular process fusion and micrognathia, thus recapitulating the most penetrant phenotypes of the syndrome. These defects were evident in either ubiquitous or NCC-specific Eif4a3 haploinsufficient animals, demonstrating an autonomous requirement of Eif4a3 in NCCs. Notably, RCPS NCC-derived mesenchymal stem-like cells (nMSCs) showed premature bone differentiation, a phenotype paralleled by premature clavicle ossification in Eif4a3 haploinsufficient embryos. Likewise, nMSCs presented compromised in vitro chondrogenesis, and Meckel's cartilage was underdeveloped in vivo. These findings indicate novel and essential requirements of EIF4A3 for NCC migration and osteochondrogenic differentiation during craniofacial development. Altogether, complementary use of iPSCs and mouse models pinpoint unique cellular mechanisms by which EIF4A3 mutation causes RCPS, and provide a paradigm to study craniofacial disorders.

Santos syndrome is caused by mutation in the WNT7A gene.

We have recently described a family with a condition (Santos syndrome (SS; MIM 613005)) characterized by fibular agenesis/hypoplasia, hypoplastic femora and grossly malformed/deformed clubfeet with severe oligodactyly, ungual hypoplasia/anonychia, sometimes associated with mild brachydactyly and occasional pre-axial polydactyly. Autosomal dominant inheritance with incomplete penetrance was suggested, but autosomal recessive inheritance could not be ruled out, due to the high frequency of consanguineous matings in the region where the family lived. This report deals with linkage studies and exome sequencing, disclosing a novel variant in WNT7A, c.934G>A (p.Gly312Ser), as the cause of this syndrome. This variant was present in homozygous state in five individuals typically affected by the SS syndrome, and in heterozygous state in the son of one affected homozygous individual. The heterozygous boy presented only unilateral complex polysyndactyly and we hypothesize that he either presents a distinct defect or that his phenotype results from a rare, mild clinical manifestation of the variant in heterozygous state. Variants in WNT7A are known to cause at least two other limb defect disorders, the syndromes of Fuhrmann and Al-Awadi/Raas-Rothschild. Despite their variable degree of expressivity and overlap, the three related conditions can be differentiated phenotypically in most instances.

A human-specific enhancer fine-tunes radial glia potency and corticogenesis.

Humans evolved an extraordinarily expanded and complex cerebral cortex, associated with developmental and gene regulatory modifications 1-3 . Human accelerated regions (HARs) are highly conserved genomic sequences with human-specific nucleotide substitutions. Although there are thousands of annotated HARs, their functional contribution to human-specific cortical development is largely unknown 4,5 . HARE5 is a HAR transcriptional enhancer of the WNT signaling receptor Frizzled8 (FZD8) active during brain development 6 . Here, using genome-edited mouse and primate models, we demonstrate that human (Hs) HARE5 fine-tunes cortical development and connectivity by controlling the proliferative and neurogenic capacity of neural progenitor cells (NPCs). Hs-HARE5 knock-in mice have significantly enlarged neocortices containing more neurons. By measuring neural dynamics in vivo we show these anatomical features correlate with increased functional independence between cortical regions. To understand the underlying developmental mechanisms, we assess progenitor fate using live imaging, lineage analysis, and single-cell RNA sequencing. This reveals Hs-HARE5 modifies radial glial progenitor behavior, with increased self-renewal at early developmental stages followed by expanded neurogenic potential. We use genome-edited human and chimpanzee (Pt) NPCs and cortical organoids to assess the relative enhancer activity and function of Hs-HARE5 and Pt-HARE5. Using these orthogonal strategies we show four human-specific variants in HARE5 drive increased enhancer activity which promotes progenitor proliferation. These findings illustrate how small changes in regulatory DNA can directly impact critical signaling pathways and brain development. Our study uncovers new functions for HARs as key regulatory elements crucial for the expansion and complexity of the human cerebral cortex.

Subcellular mRNA localization and local translation of Arhgap11a in radial glial progenitors regulates cortical development.

mRNA localization and local translation enable exquisite spatial and temporal control of gene expression, particularly in polarized, elongated cells. These features are especially prominent in radial glial cells (RGCs), which are neural and glial precursors of the developing cerebral cortex and scaffolds for migrating neurons. Yet the mechanisms by which subcellular RGC compartments accomplish their diverse functions are poorly understood. Here, we demonstrate that mRNA localization and local translation of the RhoGAP ARHGAP11A in the basal endfeet of RGCs control their morphology and mediate neuronal positioning. Arhgap11a transcript and protein exhibit conserved localization to RGC basal structures in mice and humans, conferred by the 5' UTR. Proper RGC morphology relies upon active Arhgap11a mRNA transport and localization to the basal endfeet, where ARHGAP11A is locally synthesized. This translation is essential for positioning interneurons at the basement membrane. Thus, local translation spatially and acutely activates Rho signaling in RGCs to compartmentalize neural progenitor functions.

Craniofrontonasal Syndrome Caused by Introduction of a Novel uATG in the 5'UTR of EFNB1.

Craniofrontonasal syndrome (CFNS) is an X-linked disorder caused by EFNB1 mutations in which females are more severely affected than males. Severe male phenotypes are associated with mosaicism, supporting cellular interference for sex bias in this disease. Although many variants have been found in the coding region of EFNB1, only 2 pathogenic variants have been identified in the same nucleotide in 5'UTR, disrupting the stop codon of an upstream open reading frame (uORF). uORFs are known to be part of a wide range of post-transcriptional regulation processes, and just recently, their association with human diseases has come to light. In the present study, we analyzed EFNB1 in a female patient with typical features of CFNS. We identified a variant, located at c.-411, creating a new upstream ATG (uATG) in the 5'UTR of EFNB1, which is predicted to alter an existing uORF. Dual-luciferase reporter assays showed significant reduction in protein translation, but no difference in the mRNA levels. Our study demonstrates, for the first time, the regulatory impact of uATG formation on EFNB1 levels and suggests that this should be the target region in molecular diagnosis of CFNS cases without pathogenic variants in the coding and splice sites regions of EFNB1.

Down Syndrome iPSC-Derived Astrocytes Impair Neuronal Synaptogenesis and the mTOR Pathway In Vitro.

Several methods have been used to study the neuropathogenesis of Down syndrome (DS), such as mouse aneuploidies, post mortem human brains, and in vitro cell culture of neural progenitor cells. More recently, induced pluripotent stem cell (iPSC) technology has offered new approaches in investigation, providing a valuable tool for studying specific cell types affected by DS, especially neurons and astrocytes. Here, we investigated the role of astrocytes in DS developmental disease and the impact of the astrocyte secretome in neuron mTOR signaling and synapse formation using iPSC derived from DS and wild-type (WT) subjects. We demonstrated for the first time that DS neurons derived from hiPSC recapitulate the hyperactivation of the Akt/mTOR axis observed in DS brains and that DS astrocytes may play a key role in this dysfunction. Our results bear out that 21 trisomy in astrocytes contributes to neuronal abnormalities in addition to cell autonomous dysfunctions caused by 21 trisomy in neurons. Further research in this direction will likely yield additional insights, thereby improving our understanding of DS and potentially facilitating the development of new therapeutic approaches.

Complexity of the 5' Untranslated Region of EIF4A3, a Critical Factor for Craniofacial and Neural Development.

Repeats in coding and non-coding regions have increasingly been associated with many human genetic disorders, such as Richieri-Costa-Pereira syndrome (RCPS). RCPS, mostly characterized by midline cleft mandible, Robin sequence and limb defects, is an autosomal-recessive acrofacial dysostosis mainly reported in Brazilian patients. This disorder is caused by decreased levels of EIF4A3, mostly due to an increased number of repeats at the EIF4A3 5'UTR. EIF4A3 5'UTR alleles are CG-rich and vary in size and organization of three types of motifs. An exclusive allelic pattern was identified among affected individuals, in which the CGCA-motif is the most prevalent, herein referred as "disease-associated CGCA-20nt motif." The origin of the pathogenic alleles containing the disease-associated motif, as well as the functional effects of the 5'UTR motifs on EIF4A3 expression, to date, are entirely unknown. Here, we characterized 43 different EIF4A3 5'UTR alleles in a cohort of 380 unaffected individuals. We identified eight heterozygous unaffected individuals harboring the disease-associated CGCA-20nt motif and our haplotype analyses indicate that there are more than one haplotype associated with RCPS. The combined analysis of number, motif organization and haplotypic diversity, as well as the observation of two apparently distinct haplotypes associated with the disease-associated CGCA-20nt motif, suggest that the RCPS alleles might have arisen from independent unequal crossing-over events between ancient alleles at least twice. Moreover, we have shown that the number and sequence of motifs in the 5'UTR region is associated with EIF4A3 repression, which is not mediated by CpG methylation. In conclusion, this study has shown that the large number of repeats in EIF4A3 does not represent a dynamic mutation and RCPS can arise in any population harboring alleles with the CGCA-20nt motif. We also provided further evidence that EIF4A3 5'UTR is a regulatory region and the size and sequence type of the repeats at 5'UTR may contribute to clinical variability in RCPS.