Biology subjects

Brivanlou, A. H.

Publications and source records attributed to Brivanlou, A. H..

11 recordsLinked to original sources

bioRxiv2023

HD mutation results in a dominant negative effect on HTT function

Huntingtons disease (HD) remains an incurable and fatal neurodegenerative disease long after CAG-expansion mutation in the huntingtin gene (HTT) was identified as the cause. The underlying pathological mechanism, whether HTT loss of function or gain of toxicity results from mutation, remains a matter of debate. In this study, we genetically modulated wild-type or mutant HTT expression levels in isogenic human embryonic stem cells to systematically investigate their contribution to HD-specific phenotypes. Using highly reproducible and quantifiable in vitro micropattern-based assays, we observed comparable phenotypes with HD mutation and HTT depletion. However, halving endogenous wild-type HTT levels did not strongly recapitulate the HD phenotypes, arguing against a classical loss of function mechanism. Remarkably, expression of CAG-expanded HTT in non-HD cells induced HD-like phenotypes akin to HTT depletion. By corollary, these results indicate a dominant negative effect of mutated HTT on its wild-type counterpart. Complementation with additional copies of wild-type HTT ameliorated the HD-associated phenotypes, strongly supporting a classical dominant negative mechanism. Understanding the molecular basis of this dominant negative effect will guide the development of efficient clinical strategies to counteract the deleterious impact of mutant HTT on the wild-type protein.

cell biology↗

bioRxiv2023

The emergence of human gastrulation upon in vitro attachment

While studied extensively in model systems, human gastrulation remains obscure. This process starts upon blastocyst implantation into the uterine wall, which is assumed to occur after 14 days post-fertilization. The scarcity and limited access to fetal biological material as well as ethical considerations limit our understanding of the cellular and molecular portrait of human gastrulation. In vitro culture of natural human blastocysts shed light on the second week of human development, unveiling an unexpected level of self-organization embedded in the pre-gastrulating embryo, yet whether they can undergo gastrulation upon in vitro attachment remains elusive. Blastocyst models called blastoids, which are derived from human pluripotent stem cells, provide the opportunity to reconstitute post-implantation human development in vitro with unlimited biological material. Here we show that human blastoids break symmetry and undergo gastrulation upon in vitro attachment. scRNA-seq of these models replicate the transcriptomic signature of the natural human gastrula, recapitulating aspects of the second to the third week of human development. Surprisingly, analysis of developmental timing reveals that in both blastoid models and natural in vitro attached human embryos, the onset of gastrulation as defined by molecular makers, can be traced to time scales equivalent to 12 days post-fertilization, which appeals for a reconsideration of the onset of human gastrulation upon extended in vitro culture.

developmental biology↗

bioRxiv2021

Self-organization of human dorsal-ventral forebrain structures by light induced SHH

Organizing centers secrete morphogens that specify the emergence of germ layers and the establishment of the bodys axes during embryogenesis. While traditional experimental embryology tools have been instrumental in dissecting the molecular aspects of organizers in model systems, they are impractical in human in-vitro model systems to quantitatively dissect the relationships between signaling and fate along embryonic coordinates. To systematically study human embryonic organizer centers, we devised a collection of optogenetic ePiggyBac vectors to express a photoactivatable Cre-loxP recombinase, that allows the systematic induction of organizer structures by shining blue-light on hESCs. We used a light stimulus to geometrically confine SHH expression in neuralizing hESCs. This led to the self-organization of mediolateral neural patterns from the organizer. scRNA-seq analysis established that these structures represent the dorsal-ventral forebrain, at the end of the first month of development. Here, we show that morphogen light-stimulation is a scalable tool that induces self-organizing centers.

developmental biology↗

bioRxiv2021

Role of YAP in early ectodermal specification anda Huntington's Disease model of human neurulation

The Hippo pathway, a highly conserved signaling cascade that functions as an integrator of molecular signals and biophysical states, ultimately impinges upon the transcription coactivator Yes-associated protein 1 (YAP). Hippo-YAP signaling has been shown to play key roles both at the early embryonic stages of implantation and gastrulation, and later during neurogenesis. To explore YAPs potential role in neurulation, we used self-organizing neuruloids grown from human embryonic stem cells on micropatterned substrates. We identified YAP activation as a key lineage determinant, first between neuronal ectoderm and non-neuronal ectoderm, and later between epidermis and neural crest, indicating that YAP activity can enhance the effect of BMP4 stimulation and therefore affect ectodermal specification at this developmental stage. Because aberrant Hippo-YAP signaling has been implicated in the pathology of Huntingtons Disease (HD), we used isogenic mutant neuruloids to explore the relationship between signaling and the disease. We found that HD neuruloids demonstrate ectopic activation of gene targets of YAP and that pharmacological reduction of YAPs transcriptional activity can partially rescue the HD phenotype.

developmental biology↗

bioRxiv2021

EOMES is responsible for WNT memory and can substitute for WNT in mesendoderm specification

Embryogenesis is guided by a limited set of signaling pathways dynamically expressed in different places. How a context dependent signaling response is generated has been a central question of developmental biology, which can now be addressed with in vitro models of human embryos that are derived from embryonic stem cells (hESCs) called gastruloids. Our previous work demonstrated that during early self-organization of gastruloids, cells chronicle signaling hierarchy. Only cells that have been exposed (primed) by WNT signaling can respond to subsequent Activin exposure and differentiate to mesendodermal (ME) fates. Here, we show that WNT priming does not alter SMAD2 binding nor its chromatin opening, but rather, acts by inducing the expression of the SMAD2 co-factor, EOMES. Expression of EOMES is sufficient to replace WNT upstream of Activin-mediated ME differentiation, thus unveiling the mechanistic basis for priming and cellular memory in early development.

developmental biology↗

bioRxiv2021

Huntingtin CAG expansion impairs germ layer patterning in synthetic human gastruloids through polarity defects.

Huntingtons disease (HD) is a fatal neurodegenerative disorder caused by an expansion of the CAG repeats in the Huntingtin gene (HTT). While HD has been shown to have a developmental component, how early during human embryogenesis the HTT-CAG expansion can cause embryonic defects remains unknown. Here, we demonstrate a specific and highly reproducible CAG length-dependent phenotypic signature in a synthetic model for human gastrulation derived from human embryonic stem cells (hESCs). Specifically, we observed a reduction in the extension of the ectodermal compartment that is associated with enhanced ACTIVIN signaling. Surprisingly, rather than a cell-autonomous effect, tracking the dynamics of TGF{beta} signaling demonstrated that HTT-CAG expansion perturbs the spatial restriction of ACTIVIN response. This is due to defects in the apicobasal polarization in the context of the polarized epithelium of the gastruloid, leading to ectopic subcellular localization of TGF{beta} receptors. This work refines the earliest developmental window for the prodromal phase of HD to the first two weeks of human development as modeled by our gastruloids.

developmental biology↗

bioRxiv2021

Self-organized stem cell-derived human lung buds with proximo-distal patterning and novel targets of SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the global COVID-19 pandemic and the lack of therapeutics hinders pandemic control1-2. Although lung disease is the primary clinical outcome in COVID-19 patients1-3, how SARS-CoV-2 induces tissue pathology in the lung remains elusive. Here we describe a high-throughput platform to generate tens of thousands of self-organizing, nearly identical, and genetically matched human lung buds derived from human pluripotent stem cells (hPSCs) cultured on micropatterned substrates. Strikingly, in vitro-derived human lung buds resemble fetal human lung tissue and display in vivo-like proximo-distal coordination of alveolar and airway tissue differentiation whose 3D epithelial self-organization is directed by the levels of KGF. Single-cell transcriptomics unveiled the cellular identities of airway and alveolar tissue and the differentiation of WNThi cycling alveolar stem cells, a human-specific lung cell type4. These synthetic human lung buds are susceptible to infection by SARS-CoV-2 and endemic coronaviruses and can be used to track cell type-dependent susceptibilities to infection, intercellular transmission and cytopathology in airway and alveolar tissue in individual lung buds. Interestingly, we detected an increased susceptibility to infection in alveolar cells and identified cycling alveolar stem cells as targets of SARS-CoV-2. We used this platform to test neutralizing antibodies isolated from convalescent plasma that efficiently blocked SARS-CoV-2 infection and intercellular transmission. Our platform offers unlimited, rapid and scalable access to disease-relevant lung tissue that recapitulate key hallmarks of human lung development and can be used to track SARS-CoV-2 infection and identify candidate therapeutics for COVID-19.

developmental biology↗

bioRxiv2020

Differential compartmentalization of BMP4/NOGGIN requires NOGGIN trans-epithelial transport

Using self-organizing human models of gastrulation, we previously showed that (i) BMP4 initiates the cascade of events leading to gastrulation; (ii) BMP4 signal-reception is restricted to the basolateral domain; and (iii) in a human-specific manner, BMP4 directly induces the expression of NOGGIN. Here, we report the surprising discovery that in human epiblasts, NOGGIN and BMP4 were secreted into opposite extracellular spaces. Interestingly, apically-presented NOGGIN could inhibit basally-delivered BMP4. Apically-imposed microfluidic flow demonstrated that NOGGIN traveled in the apical extracellular space. Our co-localization analysis detailed the endocytotic route that trafficked NOGGIN from the apical space to the basolateral intercellular space where BMP4 receptors were located. This apical-to-basal transcytosis was indispensable for NOGGIN inhibition. Taken together, the segregation of activator/inhibitor into distinct extracellular spaces challenges classical views of morphogen movement. We propose that the transport of morphogen inhibitors regulates the spatial availability of morphogens during embryogenesis.

developmental biology↗

bioRxiv2018

A wave of WNT signalling balanced by secreted inhibitors controls primitive streak formation in micropattern colonies of human embryonic stem cells

Long range signalling by morphogens and their inhibitors define embryonic patterning yet quantitative data and models are rare, especially in humans. Here we use a human embryonic stem cell \"gastruloid\" system to model formation of the primitive streak (PS) by WNT. In the pluripotent state E-CADHERIN (E-CAD) transduces boundary forces to focus WNT signalling to colony border. Following application of WNT ligand, E-CAD mediates a wave of epithelial-to-mesenchymal (EMT) conversion analogous to PS extension in an embryo. By knocking out the secreted WNT inhibitors active in our system, we show that DKK1 alone controls the extent and duration of patterning. The NODAL inhibitor CERBERUS1 acts downstream of WNT to refine the endoderm versus mesoderm fate choice. Our EMT wave is a generic property of a bistable system with diffusion and a single quantitative model describes both the wave and our knockout data.

developmental biology↗

bioRxiv2018

WNT signaling memory is required for ACTIVIN to function as a morphogen in human gastruloids

Self-organization of discrete fates in human gastruloids is mediated by a hierarchy of signaling pathways. How these pathways are integrated in time, and whether cells maintain a memory of their signaling history remains obscure. Here, we dissect the temporal integration of two key pathways, WNT and ACTIVIN, which along with BMP control gastrulation. CRISPR/Cas9 live reporters of SMAD1, 2 and 4 demonstrate that in contrast to the stable signaling by SMAD1, signaling and transcriptional response by SMAD2 is transient, and while necessary for pluripotency, it is insufficient for differentiation. Pre-exposure to WNT, however, endows cells with the competence to respond to graded levels of ACTIVIN, which induces differentiation without changing SMAD2 dynamics. This cellular memory of WNT signaling is necessary for ACTIVIN morphogen activity. A re-evaluation of the evidence gathered over decades in model systems, re-enforces our conclusions and points to an evolutionarily conserved mechanism.

developmental biology↗

bioRxiv2018

Molecular mechanism of symmetry breaking in a 3D model of a human epiblast

Breaking the anterior-posterior (AP) symmetry in mammals takes place at gastrulation. Much of the signaling network underlying this process has been elucidated in the mouse, however there is no direct molecular evidence of events driving axis formation in humans. Here, we use human embryonic stem cells to generate an in vitro 3D model of a human epiblast whose size, cell polarity, and gene expression are similar to a 10-day human epiblast. A defined dose of bone mor-phogenetic protein 4 (BMP4) spontaneously breaks axial symmetry, and induces markers of the primitive streak and epithelial to mesenchymal transition. By gene knockouts and live-cell imaging we show that, downstream of BMP4, WNT3 and its inhibitor DKK1 play key roles in this process. Our work demonstrates that a model human epiblast can break axial symmetry despite no asymmetry in the initial signal and in the absence of extraembryonic tissues or maternal cues. Our 3D model opens routes to capturing molecular events underlying axial symmetry breaking phenomena, which have largely been unexplored in model human systems.

developmental biology↗