Biology subjects

Wittler, L.

Publications and source records attributed to Wittler, L..

17 recordsLinked to original sources

bioRxiv2026

Comprehensive Lineage Tracing Maps the Landscape of Cell Fate Decisions in Mouse Embryogenesis

A comprehensive cell fate map of mammalian embryogenesis has remained out of reach due to the scale, cellular diversity, and non-deterministic nature of development in utero. Here, we use PEtracer to continuously install heritable genetic marks as cells divide, reconstructing lineage trees that resolve [~]75% of cell divisions across >1.5 million cells from 16 mouse embryos collected at half-day intervals from E7.5-E10.0. We pair these trees with deep transcriptional profiling to chart the landscape of cell fate decisions during gastrulation and early organogenesis. Using these data, we quantify cell fate biases, restriction timing, progenitor pool sizes, and lineage relationships across the embryo, revealing strikingly reproducible lineage architecture across replicate embryos despite the regulative flexibility of mammalian development. We further show how lineage, spatial position, and signaling jointly determine fate outcomes and timing, with their relative influence varying by tissue. This dataset provides a quantitative framework for understanding cell fate specification and a lineage-resolved reference for generating and contextualizing developmental hypotheses at organismal scale.

developmental biology↗

bioRxiv2025

Targeted sex ratio distortion in the mouse

In animal breeding, female or male progeny is preferred depending on the use of the animals. Especially in livestock, offspring with undesired sex is of low economic value and often culled. Here we achieve targeted sex ratio distortion in the mouse, leveraging components of a selfish supergene, the t-haplotype. Utilizing its central element, the t-responder, we generate a highly "selfless" genetic element disabling transgenic sperm. When integrated on the X or Y chromosome it results in a high prevalence of non-transgenic offspring of the desired sex. Our strategy overcomes key limitations of existing strategies, including compromised animal health, reduced fertility, and fully transgenic breeding stocks, which might foster acceptance of its application to livestock. The efficacy of our approach marks a groundbreaking advancement in animal breeding. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=130 HEIGHT=200 SRC="FIGDIR/small/660540v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1b11374org.highwire.dtl.DTLVardef@742ec5org.highwire.dtl.DTLVardef@1aaafc0org.highwire.dtl.DTLVardef@f87dac_HPS_FORMAT_FIGEXP M_FIG C_FIG Transgene integration on X or Y chromosome affects the motility of transgenic sperm resulting in a strong sex-bias towards non-transgenic/wildtype offspring. Application to livestock can reduce unwanted progeny and transform farm animal breeding.

genetics↗

bioRxiv2024

Enhancer adoption by an LTR retrotransposongenerates viral-like particles causingdevelopmental limb phenotypes

Mammalian genomes are scattered with transposable elements (TEs). TEs are epigenetically silenced to prevent harmful effects caused by either global activation leading to genome instability or insertional mutation disturbing gene transcription. However, whether the activation of a single element can contribute to pathological phenotypes without directly affecting gene expression is largely unknown. Here, we show that tissue-specific expression of a TE in the embryo leads to the production of viral-like particles (VLPs) which can affect organ formation. Failure to silence an LTR retrotransposon inserted upstream of the Fgf8 gene results in its co-expression with Fgf8 in the developing embryo. While local gene regulation is unaffected, the LTR retrotransposon participates in chromatin folding at the locus and adopts the expression of the regulatory domain it is located in. This drives the production of VLPs in the Fgf8-expressing cells of the developing limb, triggering apoptotic cell death at the time of digit outgrowth and resulting in a limb malformation resembling human ectrodactyly. This phenotype can be rescued by knock-out or knock-in of the retrotransposon causing mutations preventing its full retroviral cycle. Insertion of the same element at other developmental loci faithfully recapitulates expression according to the neighboring regulatory activity. Our findings provide a mechanism by which TE insertion is incorporated into the local genomic regulatory landscape and show how VLP production in post-implantation embryos can interfere with organ formation.

developmental biology↗

bioRxiv2024

Temporally restricted activities of En1 regulatory elements underlie distinct limb malformations

The precise spatiotemporal regulation of developmental genes is required for proper organogenesis. Engrailed-1 (En1) is essential for dorsal-ventral patterning during mouse limb development from embryonic day E9.5 to E11.5. Previously, we identified the long non-coding RNA locus Maenli, which drives limb-specific En1 expression at E9.5. In this study, we investigated the regulatory mechanisms sustaining En1 expression at later developmental stages when Maenli transcriptional activity is drastically reduced. Using in vivo CRISPR editing, we identified two intergenic enhancer elements, LSEE1 and LSEE2, that maintain En1 expression at E10.5 and E11.5. Mice lacking these enhancers exhibit only a subset of the limb malformations observed in En1 and Maenli mutants, indicating that the timing of En1 misexpression causes distinct phenotypes. These findings underscore the role of temporally restricted activities of cis-regulatory elements, including lncRNA loci and enhancers, in modulating gene expression and explaining subtle differences in complex disease phenotypes.

developmental biology↗

bioRxiv2023

Combinatorial microRNA activity is essential for the transition of pluripotent cells from proliferation into dormancy

Dormancy is a key feature of stem cell function in adult tissues as well as embryonic cells in the context of diapause. The establishment of dormancy is an active process that involves extensive transcriptional, epigenetic, and metabolic rewiring1,2. How these processes are coordinated to successfully transition cells to the resting dormant state remains unclear. Here we show that microRNA activity, which is otherwise dispensable for pre-implantation development, is essential for the adaptation of early mouse embryos to the dormant state of diapause. In particular, the pluripotent epiblast depends on miRNA activity, the absence of which results in loss of pluripotent cells. Through integration of high-sensitivity small RNA expression profiling of individual embryos and protein expression of miRNA targets with public data of protein-protein interactions, we constructed the miRNA-mediated regulatory network of mouse early embryos specific to diapause. We find that individual miRNAs contribute to the combinatorial regulation by the network and the perturbation of the network compromises embryo survival in diapause. Without miRNAs, nuclear and cytoplasmic bodies show aberrant expression, concurrent with splicing defects. We identified the nutrient-sensitive transcription factor TFE3 as an upstream regulator of diapause-specific miRNAs, linking cytoplasmic mTOR activity to nuclear miRNA biogenesis. Our results place miRNAs as a critical regulatory layer for the molecular rewiring of early embryos to establish dormancy.

molecular biology↗

bioRxiv2023

Genome-wide identification of notochord enhancers comprising the regulatory landscape of the Brachyury (T) locus in mouse

The node and notochord are important signaling centers organizing dorso-ventral patterning of cells arising from neuro-mesodermal progenitors forming the embryonic body anlage. Due to the scarcity of notochordal progenitors and notochord cells, a comprehensive identification of regulatory elements driving notochord-specific gene expression has been lacking. Here we have used ATAC-seq analysis of FACS-purified notochordal cells from TS12-13 mouse embryos to identify 8921 putative notochord enhancers. In addition, we established a new model for generating notochordal cells in culture, and found 3728 of these enhancers occupied by the essential notochordal regulators Brachyury (T) and/or Foxa2. We describe the regulatory landscape of the T locus comprising 10 putative enhancers occupied by these factors and confirmed the regulatory activity of 3 of these elements. Moreover, we characterized one new notochord enhancer, termed TNE2, in embryos. TNE2 complements the loss of TNE in the trunk notochord, and is essential for notochordal cell proliferation and differentiation in the tail. Our data demonstrate the essential role of Foxa2 in switching T expressing cells from a NMP/mesodermal trajectory to the notochordal fate. Summary statementCombining multi-omics assays of purified embryonic and in vitro generated cells we identified thousands of notochord enhancers comprising TNE2 essential for T expression and tail development of the mouse embryo.

developmental biology↗

bioRxiv2022

Co-option of the transcription factor SALL1 in mole ovotestis formation

Changes in gene expression represent an important source for phenotypical innovation. Yet, how such changes emerge and impact the evolution of traits remains elusive. Here, we explore the molecular mechanisms associated with the development of masculinizing ovotestes in female moles. By performing comparative analyses of epigenetic and transcriptional data in mole and mouse, we identified SALL1 as a co-opted gene for the formation of testicular tissue in mole ovotestes. Chromosome conformation capture analyses highlight a striking conservation of the 3D organization at the SALL1 locus, but a prominent evolutionary turnover of enhancer elements. Interspecies reporter assays support the capability of mole-specific enhancers to activate transcription in urogenital tissues. Through overexpression experiments in transgenic mice, we further demonstrate the capability of SALL1 to induce the ectopic gene expression programs that are a signature of mole ovotestes. Our results highlight the co-option of gene expression, through changes in enhancer activity, as a prominent mechanism for the evolution of traits.

evolutionary biology↗

bioRxiv2022

Single cell, whole embryo phenotyping of pleiotropic disorders of mammalian development

Mouse models are a critical tool for studying human diseases, particularly developmental disorders, as well as for advancing our general understanding of mammalian biology. However, it has long been suspected that conventional approaches for phenotyping are insufficiently sensitive to detect subtle defects throughout the developing mouse. Here we set out to establish single cell RNA sequencing (sc-RNA-seq) of the whole embryo as a scalable platform for the systematic molecular and cellular phenotyping of mouse genetic models. We applied combinatorial indexing-based sc-RNA-seq to profile 101 embryos of 26 genotypes at embryonic stage E13.5, altogether profiling gene expression in over 1.6M nuclei. The 26 genotypes include 22 mouse mutants representing a range of anticipated severities, from established multisystem disorders to deletions of individual enhancers, as well as the 4 wildtype backgrounds on which these mutants reside. We developed and applied several analytical frameworks for detecting differences in composition and/or gene expression across 52 cell types or trajectories. Some mutants exhibited changes in dozens of trajectories (e.g., the pleiotropic consequences of altering the Sox9 regulatory landscape) whereas others showed phenotypes affecting specific subsets of cells. We also identify differences between widely used wildtype strains, compare phenotyping of gain vs. loss of function mutants, and characterise deletions of topological associating domain (TAD) boundaries. Intriguingly, even among these 22 mutants, some changes are shared by heretofore unrelated models, suggesting that developmental pleiotropy might be "decomposable" through further scaling of this approach. Overall, our findings show how single cell profiling of whole embryos can enable the systematic molecular and cellular phenotypic characterization of mouse mutants with unprecedented breadth and resolution.

genomics↗

bioRxiv2022

GATA transcription factors drive initial Xist upregulation after fertilization through direct activation of a distal enhancer element

To ensure dosage compensation for X-linked genes between the sexes, one X chromosome is silenced during early embryonic development of female mammals. This process of X-chromosome inactivation (XCI) is initiated through upregulation of the RNA Xist from one X chromosome shortly after fertilization. Xist then mediates chromosome-wide gene silencing in cis and remains expressed in all cell types except the germ line and the pluripotent state, where XCI is reversed. The factors that drive Xist upregulation and thereby initiate XCI remain however unknown. We identify GATA transcription factors as potent Xist activators and demonstrate that they are essential for the activation of Xist in mice following fertilization. Through a pooled CRISPR activation screen we find that GATA1 can drive ectopic Xist expression in murine embryonic stem cells (mESCs). We demonstrate that all GATA factors can activate Xist directly via a GATA-responsive regulatory element (RE79) positioned 100 kb upstream of the Xist promoter. Additionally, GATA factors are essential for the induction of XCI in mouse preimplantation embryos, as simultaneous deletion of three members of the GATA family (GATA1/4/6) in mouse zygotes effectively prevents Xist upregulation. Thus, initiation of XCI and possibly its maintenance in distinct lineages of the preimplantation embryo is ensured by the combined activity of different GATA family members, and the absence of GATA factors in the pluripotent state likely contributes to X reactivation. We thus describe a form of regulation in which the combined action of numerous tissue-specific factors can achieve near-ubiquitous expression of a target gene.

genetics↗

bioRxiv2022

Combinatorial effects on gene expression at the Lbx1/Fgf8 locus resolve Split-Hand/Foot Malformation type 3

Split-Hand/Foot Malformation type 3 (SHFM3) is a congenital limb malformation associated with tandem duplications at the LBX1/FGF8 locus. Yet, the disease patho-mechanism remains unsolved. Here we investigated the functional consequences of SHFM3-associated rearrangements on chromatin conformation and gene expression in vivo in transgenic mice. We show that the Lbx1/Fgf8 locus consists of two separate, but interacting, regulatory domains. Re-engineering of a SHFM3-associated duplication and a newly reported inversion in mice resulted in restructuring of the chromatin architecture. This led to an ectopic activation of the Lbx1 and Btrc genes in the apical ectodermal ridge (AER) in an Fgf8-like pattern. Artificial repositioning of the AER-specific enhancers of Fgf8 was sufficient to induce misexpression of Lbx1 and Btrc. We provide evidence that the SHFM3 phenotype is the result of a combinatorial effect on gene misexpression and dosage in the developing limb. Our results reveal new insights into the molecular mechanism underlying SHFM3 and provide novel conceptual framework for how genomic rearrangements can cause gene misexpression and disease.

genetics↗

bioRxiv2021

Promoter repression and 3D-restructuring resolves divergent developmental gene expression in TADs

Cohesin loop extrusion facilitates precise gene expression by continuously driving promoters to sample all enhancers located within the same topologically-associated domain (TAD). However, many TADs contain multiple genes with divergent expression patterns, thereby indicating additional forces further refine how enhancer activities are utilised. Here, we unravel the mechanisms enabling a new gene, Rex1, to emerge with divergent expression within the ancient Fat1 TAD in placental mammals. We show that such divergent expression is not determined by a strict enhancer-promoter compatibility code, intra-TAD position or nuclear envelope-attachment. Instead, TAD-restructuring in embryonic stem cells (ESCs) separates Rex1 and Fat1 with distinct proximal enhancers that independently drive their expression. By contrast, in later embryonic tissues, DNA methylation renders the inactive Rex1 promoter profoundly unresponsive to Fat1 enhancers within the intact TAD. Combined, these features adapted an ancient regulatory landscape during evolution to support two entirely independent Rex1 and Fat1 expression programs. Thus, rather than operating only as rigid blocks of co-regulated genes, TAD-regulatory landscapes can orchestrate complex divergent expression patterns in evolution. HIGHLIGHTSO_LINew genes can emerge in evolution without taking on the expression pattern of their surrounding pre-existing TAD. C_LIO_LICompartmentalisation can restructure seemingly evolutionarily stable TADs to control a promoters access to enhancers. C_LIO_LILamina-associated domains neither prevent transcriptional activation nor enhancer-promoter communication. C_LIO_LIRepression rather than promoter-specificity refines when genes respond to promiscuous enhancer activities in specific tissues. C_LI

genetics↗

bioRxiv2021

In vivo dissection of a clustered-CTCF domain boundary reveals developmental principles of regulatory insulation

Vertebrate genomes organize into topologically associating domains (TADs), delimited by boundaries that insulate regulatory elements from non-target genes. However, how boundary function is established is not well understood. Here, we combine genome-wide analyses and transgenic mouse assays to dissect the regulatory logic of clustered-CTCF boundaries in vivo, interrogating their function at multiple levels: chromatin interactions, transcription and phenotypes. Individual CTCF binding sites (CBS) deletions revealed that the characteristics of specific sites can outweigh other factors like CBS number and orientation. Combined deletions demonstrated that CBS cooperate redundantly and provide boundary robustness. We show that divergent CBS signatures are not strictly required for effective insulation and that chromatin loops formed by non-convergently oriented sites could be mediated by a loop interference mechanism. Further, we observe that insulation strength constitutes a quantitative modulator of gene expression and phenotypes. Our results highlight the modular nature of boundaries and their control over developmental processes.

developmental biology↗

bioRxiv2020

A CRISPR-Cas9-engineered mouse model for GPI anchor deficiency mirrors human phenotype and shows hippocampal synaptic dysfunctions.

Pathogenic germline mutations in PIGV lead to glycosylphosphatidylinositol biosynthesis deficiency (GPIBD). Individuals with pathogenic biallelic mutations in genes of the glycosylphosphatidylinositol (GPI) anchor pathway exhibit cognitive impairments, motor delay, and often epilepsy. Thus far, the pathophysiology underlying the disease remains unclear, and suitable rodent models that mirror all symptoms observed in human patients have not been available. Therefore, we used CRISPR-Cas9 to introduce the most prevalent hypomorphic missense mutation in European patients, Pigv:c.1022C>A (p.A341E), at a site that is conserved in mice. Mirroring the human pathology, mutant Pigv341E mice exhibited deficits in motor coordination, cognitive impairments, and alterations in sociability and sleep patterns, as well as increased seizure susceptibility. Furthermore, immunohistochemistry revealed reduced synaptophysin immunoreactivity in Pigv341E mice, and electrophysiology recordings showed decreased hippocampal synaptic transmission that could underlie impaired memory formation. In single-cell RNA sequencing, Pigv341E-hippocampal cells exhibited changes in gene expression, most prominently in a subtype of microglia and subicular neurons. A significant reduction in Abl1 transcript levels in several cell clusters suggested a link to the signaling pathway of GPI-anchored ephrins. We also observed elevated levels of Hdc transcripts, which might affect histamine metabolism with consequences for circadian rhythm. This new mouse model will not only open the doors to further investigation into the pathophysiology of GPIBD, but will also deepen our understanding of the role of GPI-anchor-related pathways in brain development. Significance statementInherited GPI-anchor biosynthesis deficiencies (IGDs) explain many cases of syndromic intellectual disability. Although diagnostic methods are improving, the pathophysiology underlying the disease remains unclear. Furthermore, we lack rodent models suitable for characterizing cognitive and social disabilities. To address this issue, we generated the first viable mouse model for an IGD that mirrors the condition in human patients with a behavioral phenotype and susceptibility to epilepsy. Using the new model, we obtained neurological insights such as deficits in synaptic transmission that will facilitate understanding of the pathophysiology of IGDs.

neuroscience↗

bioRxiv2020

Mouse embryonic stem cells self-organize into trunk-like structures with neural tube and somites

Post-implantation embryogenesis is a highly dynamic process comprising multiple lineage decisions and morphogenetic changes inaccessible to deep analysis in vivo. Mouse embryonic stem cells (mESCs) can form aggregates reflecting the post-occipital embryo (gastruloids), but lacking proper morphogenesis. Here we show that embedding of aggregates derived from mESCs in an extracellular matrix compound results in Trunk-Like-Structures (TLS) with a high level of organization comprising a neural tube and somites. Comparative single-cell RNA-seq analysis demonstrates that TLS execute gene-regulatory programs in an embryo-like order, and generate primordial germ cell like cells (PGCLCs). TLS lacking Tbx6 form ectopic neural tubes, mirroring the embryonic mutant phenotype. ESC-derived trunk-like structures thus constitute a novel powerful in vitro platform for investigating lineage decisions and morphogenetic processes shaping the post-implantation embryo. One sentence summaryA platform for generating trunk-like-structures with precursors of spinal cord, bone and muscle from stem cells in a dish

developmental biology↗

bioRxiv2019

Emergence and patterning dynamics of mouse definitive endoderm

The segregation of definitive endoderm (DE) from mesendoderm progenitors leads to the formation of two distinct germ layers. Dissecting DE onset has been challenging as it occurs within a narrow spatio-temporal window in the embryo. Here we employ a dual Bra-GFP, Sox17-RFP reporter cell line to study DE onset dynamics. We find Sox17 starts in a few isolated cells in vivo. Using 2D and 3D in vitro models, we show that DE cells emerge from mesendoderm progenitors at a temporally regular, but spatially stochastic pattern, which is subsequently arranged by self-sorting of Sox17+ cells. Self-sorting coincides with up-regulation of E-cadherin but is not necessary for DE differentiation or proliferation. A subpopulation of Bra-high cells commits to a Sox17+ fate independent of external Wnt signal. Our in vivo and in vitro results highlight basic rules governing DE onset and patterning through the commonalities and differences between these systems.

developmental biology↗

bioRxiv2019

Functional dissection of TADs reveals non-essential and instructive roles in regulating gene expression

The genome is organized in megabase-sized three-dimensional units, called Topologically Associated Domains (TADs), that are separated by boundaries. TADs bring distant cis-regulatory elements into proximity, a process dependent on the cooperative action of cohesin and the DNA binding factor CTCF. Surprisingly, genome-wide depletion of CTCF has little effect on transcription, yet structural variations affecting TADs have been shown to cause gene misexpression and congenital disease. Here, we investigate TAD function in vivo in mice by systematically editing components of TAD organization at the Sox9/Kcnj locus. We find that TADs are formed by a redundant system of CTCF sites requiring the removal of all major sites within the TAD and at the boundary for two neighboring TADs to fuse. TAD fusion resulted in leakage of regulatory activity from the Sox9 to the Kcnj TAD, but no major changes in gene expression. This indicates that TAD structures provide robustness and precision, but are not essential for developmental gene regulation. Gene misexpression and resulting disease phenotypes, however, were attained by re-directing regulatory activity through inversions and/or the re-positioning of boundaries. Thus, efficient re-wiring of enhancer promoter interaction and aberrant disease causing gene activation is not induced by a mere loss of insulation but requires the re-direction of contacts.

genetics↗

bioRxiv2019

Preformed Chromatin Topology Assists TranscriptionalRobustness of Shh during Limb Development

Long-range gene regulation involves physical proximity between enhancers and promoters to generate precise patterns of gene expression in space and time. However, in some cases proximity coincides with gene activation, whereas in others preformed topologies already exist before activation. In this study, we investigate the preformed configuration underlying the regulation of the Shh gene by its unique limb enhancer, the ZRS, in vivo during mouse development. Abrogating the constitutive transcription covering the ZRS region led to a shift within the Shh-ZRS contacts and a moderate reduction in Shh transcription. Deletion of the CTCF binding sites around the ZRS resulted in a loss of the Shh-ZRS preformed interaction and a 50% decrease in Shh expression but no phenotype, suggesting an additional, CTCF-independent mechanism of promoter-enhancer communication. This residual activity, however, was diminished by combining the loss of CTCF binding with a hypomorphic ZRS allele resulting in severe Shh loss-of-function and digit agenesis. Our results indicate that the preformed chromatin structure of the Shh locus is sustained by multiple components and acts to reinforce enhancer-promoter communication for robust transcription.

developmental biology↗