Biology subjects

Opitz, R.

Publications and source records attributed to Opitz, R..

8 recordsLinked to original sources

bioRxiv2026

Quantification of the effects of single nucleotide variants in NKX2.1 transcription factor binding sites

Transcription factors recognise and bind specific DNA sequence patterns in promoters and enhancers thereby regulating gene expression. Variations in the DNA sequence of transcription factor binding sites (TFBSs) can alter gene regulation and may disrupt development. The transcription factor NKX2.1 is a crucial regulator of thyroid, lung, and neural development. Mutations in its coding gene NKX2-1 may cause choreoathetosis and congenital hypothyroidism with or without pulmonary dysfunction (CAHTP, OMIM #610978). Most genetically solved patients carry mutations in the coding regions of NKX2-1 that affect DNA binding, while the majority of patients with CAHTP-like symptoms do not carry mutations in the NKX2-1 coding sequence. We hypothesise that variations in the DNA-sequence at promoter or enhancer sites to which the transcription factor NKX2.1 binds could cause disease as well. We employed EMSA-seq to quantify the effects of genetic variation on NKX2.1 binding strength and used this data to train neural network models to forecast the influence of DNA variation on NKX2.1 binding. We validated our models using microscale thermophoresis, X-ray crystallography, and publicly available ChIP-seq data sets. The neural networks were able to detect TFBSs in ChIP-seq data and can thus be used to evaluate whole genome sequencing data of CAHTP-patients in order to prioritise potential disease-causing variants in regulatory elements. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/708450v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@167cedeorg.highwire.dtl.DTLVardef@3e5291org.highwire.dtl.DTLVardef@19eb7f9org.highwire.dtl.DTLVardef@1404057_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Central T3 deprivation disturbs cortical cilia formation, oligodendrocyte lineage and neuronal cell-cell-communication in a MCT8/OATP1C1 deficient Allan-Herndon-Dudley Syndrome mouse model

BackgroundThe Allan-Herndon-Dudley syndrome (AHDS) is a rare, X-linked human genetic disorder caused by mutations in the monocarboxylate transporter 8 (MCT8), essential for thyroid hormone (TH) transport across the blood-brain-barrier. The resulting central TH deprivation disrupts brain maturation and function, leading to intellectual disability and movement disorders. Cortical development, highly dependent on TH, is particularly affected and contributes significantly to AHDS pathologies. MethodsTo elucidate disrupted cortical processes, we conducted single nucleus RNA sequencing in a mouse model engineered to mimic the central TH deficiency characteristic of human AHDS. This murine AHDS model features the concomitant deletion of both MCT8 and OATP1C1, a T4 transporter largely absent in human brain capillaries that in mice plays a role in TH transport. The phenotype of dKO mice bears striking resemblance to the pathologies observed in human AHDS patients. ResultsSingle nuclei were isolated from the cortex and attached striatum of 21-day old WT and MCT8/OATP1C1 dKO mice and sequenced using the 10x Genomics workflow. Cell proportion analyses on the resulting 48 clusters suggested elevated numbers of GABAergic striatal D1 and D2 neurons in the dKO mice. Diminished levels of mature oligodendrocytes coincided with a bifurcation within the oligodendrocyte lineage trajectory, leading to distinct subpopulations of WT and dKO oligodendrocytes. Differentially expressed gene (DEG) patterns align poorly with Slc16a2 and Slco1c1 mRNA levels in the respective clusters, but closely with prior published cortical bulk RNAseq data of mice with systemic hypothyroidism or MCT8/OATP1C1 deficiency. These parallels confirm the reliability of our data and provide new insights by pinpointing TH-responsive DEGs to specific cellular clusters. Moreover, inferred cell-cell communication using NeuronChat suggested a disbalance in GABAergic versus glutamatergic signaling. We further uncovered perturbed primary cilia formation in several GABAergic and glutamatergic clusters of the dKO cortex. DiscussionMolecular signatures and perturbations uncovered by our snRNAseq study reveal new molecular characteristics of the AHDS. The imbalance in GABAergic versus glutamatergic cell-cell-communication, perturbed primary cilia formation, and bifurcation of the oligodendrocyte lineage align with pathologies observed in AHDS patients and highlight the role of TH signaling in maintaining neuronal network homeostasis in the cortex.

neuroscience↗

bioRxiv2023

RNA-sequencing reveals strong predominance of THRA splicing isoform 2 in the developing and adult human brain

Thyroid hormone receptor alpha (THR) is a nuclear hormone receptor that binds triiodothyronine (T3) and acts as an important transcription factor in development, metabolism and reproduction. THR has in mammals two major splicing isoforms, THR1 and THR2. The better characterized isoform, THR1, is a transcriptional stimulator of genes involved in cell metabolism and growth. The less well characterized isoform, THR2, lacks the Ligand Binding Domain (LBD) and is thought to act as an inhibitor of THR1 action. The ratio of THR1 to THR2 splicing isoforms is therefore critical for transcriptional regulation in different tissues and during development. However, the expression patterns of both isoforms have not been studied in healthy human tissues or in the developing brain. Given the lack of commercially available isoform-specific antibodies, we addressed this question by analyzing four bulk RNA-sequencing datasets and two scRNA-sequencing datasets to determine the RNA expression levels of human THRA1 and THRA2 transcripts in healthy adult tissues and in the developing brain. We demonstrate how 10X Chromium scRNA-seq datasets can be used to perform splicing-sensitive analyses of isoforms that differ at the 3-end. In all datasets, we discovered a strong predominance of THRA2 transcripts at all investigated stages of human brain development and in the central nervous system from healthy human adults.

genetics↗

bioRxiv2023

Partial resistance to thyroid hormone-induced tachycardia and cardiac hypertrophy in mice lacking thyroid hormone receptor beta

BackgroundThyroid hormones regulate cardiac functions mainly via direct actions in the heart and binding to the thyroid hormone receptor (TR) isoforms 1 and {beta}. While the role of the most abundantly expressed isoform, TR1, is widely studied and well characterized, the role of TR{beta} in regulating heart functions is still poorly understood, primarily due to the accompanying elevation of circulating thyroid hormone in mice lacking TR{beta} (TR{beta}-KO). However, their hyperthyroidism is ameliorated at thermoneutrality, which allows studying the role of TR{beta} without this confounding factor. MethodsHere we non-invasively monitored heart rate in TR{beta}-KO mice over several days using radiotelemetry at different housing temperatures (22{degrees}C and 30{degrees}C), and upon T3 administration in comparison to wildtype animals. ResultsTR{beta}-KO mice displayed normal average heart rate at both 22{degrees}C and 30{degrees}C with only minor changes in heart rate frequency distribution, which was confirmed by independent electrocardiogram recordings in freely-moving conscious mice. Parasympathetic nerve activity was, however, impaired in TR{beta}-KO mice at 22{degrees}C, and only partly rescued at 30{degrees}C. As expected, oral treatment with pharmacological doses of T3 at 30{degrees}C led to tachycardia in wildtypes, accompanied by broader heart rate frequency distribution and increased heart weight, while TR{beta}-KO mice showed blunted tachycardia, as well as resistance to changes in heart rate frequency distribution and heart weight. At the molecular level, these observations were paralleled by a blunted cardiac mRNA induction of several important genes, including the pacemaker channels Hcn2 and Hcn4, as well as Kcna7. ConclusionsThe phenotyping of TR{beta}-KO mice conducted at thermoneutrality allows novel insights on the role of TR{beta} in cardiac functions in absence of the usual confounding hyperthyroidism. Even though TR{beta} is expressed at lower levels than TR1 in the heart, our findings demonstrate an important role for this isoform in the cardiac response to thyroid hormones.

physiology↗

bioRxiv2023

Spatiotemporal expression of thyroid hormone transporter MCT8 and THRA mRNA in human cerebral organoids recapitulating first trimester cortex development

Thyroid hormones (TH) play critical roles during nervous system development and patients carrying coding variants of MCT8 (Monocarboxylate transporter 8) or THRA (Thyroid hormone receptor alpha) present a spectrum of neurological phenotypes resulting from perturbed local TH action during early brain development. Recently, human cerebral organoids (hCOs) emerged as powerful three-dimensional in vitro tools for disease modelling recapitulating key aspects of early human cerebral cortex development. To begin exploring prospects of this model for thyroid research, we performed a detailed characterization of the spatiotemporal expression of the TH transporter MCT8 and TH receptor THRA in developing hCOs. Immunostaining of hCOs showed MCT8 membrane expression in neuronal progenitor cell types including early neuroepithelial cells, SOX2+/Nestin+ radial glia cells (RGCs), TBR2+ intermediate progenitors and HOPX+ outer RGCs. In addition, we detected robust MCT8 protein expression in CTIP2+ deep layer neurons and at later developmental stages in SATB2+ upper layer neurons. Spatiotemporal SLC16A2 mRNA expression, detected by fluorescent in situ hybridization (FISH), was highly concordant with MCT8 protein expression across cortical cell layers. FISH detected THRA mRNA expression already in neuroepithelium before the onset of neurogenesis and THRA expression was maintained in RGCs of the ventricular zone. Increased THRA expression was later detected in the subventricular zone whereas highest THRA expression was observed in excitatory neurons in peripheral hCO regions. In combination with strong up-regulation of known T3 response genes following short-term T3 treatment of hCOs, these observations show that hCOs provide a promising and experimentally tractable model to probe local TH action during human cortical neurogenesis and eventually to model the consequences of impaired TH function for early cortex development.

neuroscience↗

bioRxiv2020

Timed mesodermal FGF and BMP govern the multi-step thyroid specification

Thyroid tissue is the site for de novo synthesis of thyroid hormones which are essential for vertebrate development and growth. Defects in embryonic thyroid morphogenesis are a predominant cause for congenital thyroid diseases but the molecular pathomechanisms are incompletely understood. The first molecularly recognizable step of thyroid development is the specification of thyroid precursors at a defined position in the anterior foregut endoderm. While recent studies identified FGF and BMP pathways as critical signaling factors for thyroid specification, the interplay between extrinsic signaling cues and thyroid transcription factor expression remained elusive. Here, we used zebrafish embryos to decipher the dynamics of thyroid transcription factor induction in relation to FGF and BMP signaling activities in pharyngeal endoderm. We first identified a previously unrecognized endodermal thyroid progenitor cell population expressing Pax2a but not Nkx2.4b. This cell population is characterized by enhanced FGF signaling but initially lacks detectable BMP signaling. A subpopulation of Pax2a-expressing progenitors differentiates subsequently into thyroid lineage-committed precursor cells co-expressing Pax2a and Nkx2.4b. We next combined pharmacological approaches with genetic models permitting inhibition or ectopic overactivation of signaling pathways to timely manipulate FGF and BMP activities. These experiments support a model where FGF signaling primarily regulates Pax2a expression whereas BMP signaling has dual functions in regulation of both Pax2a and Nkx2.4b expression. Collectively, our data allow us to formulate a refined model of thyroid cell specification from foregut endoderm.

developmental biology↗

bioRxiv2019

Enhanced canonical Wnt signaling during early zebrafish development perturbs the interaction of cardiac mesoderm and pharyngeal endoderm and causes thyroid specification defects

BackgroundCongenital hypothyroidism (CH) due to thyroid dysgenesis is a frequent congenital endocrine disorder for which the molecular mechanisms remain unresolved in the far majority of cases. This situation reflects in part our still limited knowledge about the mechanisms involved in the early steps of thyroid specification from the endoderm, in particular the extrinsic signaling cues that regulate foregut endoderm patterning. In this study, we used small molecules and genetic zebrafish models to characterize the role of various signaling pathways in thyroid specification. MethodsWe treated zebrafish embryos during different developmental periods with small molecule compounds known to modulate the activity of Wnt signaling pathway and observed effects in thyroid, endoderm and cardiovascular development using whole mount in situ hybridization and transgenic fluorescent reporter models. We used an antisense morpholino to create a zebrafish acardiac model. For thyroid rescue experiments, BMP pathway induction in zebrafish embryos was obtained by using heatshock inducible transgenic lines. ResultsInterestingly, combined analyses of thyroid and cardiovascular development revealed that overactivation of Wnt signaling during early development leads to impaired thyroid specification concurrent with severe defects in the cardiac specification. When using a model of morpholino-induced blockage of cardiomyocyte differentiation, a similar correlation was observed, suggesting that defective signaling between cardiac mesoderm and endodermal thyroid precursors contributes to thyroid specification impairment. Rescue experiments through transient overactivation of BMP signaling could partially restore thyroid specification in models with defective cardiac development. ConclusionCollectively, our results indicate that BMP signaling is critically required for thyroid cell specification and identify cardiac mesoderm as a likely source of BMP signals.

developmental biology↗

bioRxiv2019

Small Molecule Screening in Zebrafish Embryos Identifies Signaling Pathways Regulating Early Thyroid Development

BackgroundDefects in embryonic development of the thyroid gland are a major cause for congenital hypothyroidism in human newborns but the underlying molecular mechanisms are still poorly understood. Organ development relies on a tightly regulated interplay between extrinsic signaling cues and cell intrinsic factors. At present, however, there is limited knowledge about the specific extrinsic signaling cues that regulate foregut endoderm patterning, thyroid cell specification and subsequent morphogenetic processes in thyroid development. MethodsTo begin to address this problem in a systematic way, we used zebrafish embryos to perform a series of in vivo phenotype-driven chemical genetic screens to identify signaling cues regulating early thyroid development. For this purpose, we treated zebrafish embryos during different developmental periods with a panel of small molecule compounds known to manipulate the activity of major signaling pathways and scored phenotypic deviations in thyroid, endoderm and cardiovascular development using whole mount in situ hybridization and transgenic fluorescent reporter models. ResultsSystematic assessment of drugged embryos recovered a range of thyroid phenotypes including expansion, reduction or lack of the early thyroid anlage, defective thyroid budding as well as hypoplastic, enlarged or overtly disorganized presentation of the thyroid primordium after budding. Our pharmacological screening identified BMP and FGF signaling as key factors for thyroid specification and early thyroid organogenesis, highlight the importance of low Wnt activities during early development for thyroid specification and implicate drug-induced cardiac and vascular anomalies as likely indirect mechanisms causing various forms of thyroid dysgenesis. ConclusionsBy integrating the outcome of our screening efforts with previously available information from other model organisms including Xenopus, chicken and mouse, we conclude that signaling cues regulating thyroid development appear broadly conserved across vertebrates. We therefore expect that observations made in zebrafish can inform mammalian models of thyroid organogenesis to further our understanding of the molecular mechanisms of congenital thyroid diseases.

developmental biology↗