A simple smiFISH pipeline to quantify mRNA at the single-cell level in 3D.

Techniques allowing the precise quantification of mRNA at the cellular level are essential for understanding biological processes. Here, we present a semi-automated smiFISH (single-molecule inexpensive FISH) pipeline enabling quantification of mRNA in a small number of cells (∼40) in fixed whole mount tissue. We describe steps for sample preparation, hybridization, image acquisition, cell segmentation, and mRNA quantification. Although the protocol was developed in Drosophila, it can be optimized for use in other organisms. For complete details on the use and execution of this protocol, please refer to Guan et al.1.

Post-transcriptional regulation of transcription factor codes in immature neurons drives neuronal diversity.

How the vast array of neuronal diversity is generated remains an unsolved problem. Here, we investigate how 29 morphologically distinct leg motoneurons are generated from a single stem cell in Drosophila. We identify 19 transcription factor (TF) codes expressed in immature motoneurons just before their morphological differentiation. Using genetic manipulations and a computational tool, we demonstrate that the TF codes are progressively established in immature motoneurons according to their birth order. Comparing RNA and protein expression patterns of multiple TFs reveals that post-transcriptional regulation plays an essential role in shaping these TF codes. Two RNA-binding proteins, Imp and Syp, expressed in opposing gradients in immature motoneurons, control the translation of multiple TFs. The varying sensitivity of TF mRNAs to the opposing gradients of Imp and Syp in immature motoneurons decrypts these gradients into distinct TF codes, establishing the connectome between motoneuron axons and their target muscles.

Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle.

The majority of work on the neuronal specification has been carried out in genetically and physiologically tractable models such as C. elegans, Drosophila larvae, and fish, which all engage in undulatory movements (like crawling or swimming) as their primary mode of locomotion. However, a more sophisticated understanding of the individual motor neuron (MN) specification-at least in terms of informing disease therapies-demands an equally tractable system that better models the complex appendage-based locomotion schemes of vertebrates. The adult Drosophila locomotor system in charge of walking meets all of these criteria with ease, since in this model it is possible to study the specification of a small number of easily distinguished leg MNs (approximately 50 MNs per leg) both using a vast array of powerful genetic tools, and in the physiological context of an appendage-based locomotion scheme. Here we describe a protocol to visualize the leg muscle innervation in an adult fly.

Two Protocols to Study the Interactions of Thyroid Hormone Receptors with Other Proteins and Chromatin.

Understanding the transcriptional function of thyroid hormone receptors implies a precise analysis of their interactions with chromatin and other protein components of the cells. We present here two protocols that are routinely used in our laboratory. The first co-immunoprecipitation procedure allows addressing the capacity of proteins to form stable multiprotein complexes with TRs in cells. The chromatin affinity purification enables us to define the sites occupied by TRs on chromatin. In this case the lack of high quality antibodies is circumvented by introducing an N-terminal tag in TR, with unspecific affinity for immunoglobulins.

CRISPR/Cas9 Editing of the Mouse Thra Gene Produces Models with Variable Resistance to Thyroid Hormone.

BACKGROUND: Resistance to thyroid hormone due to THRA mutations (RTHα) is a recently discovered genetic disease, displaying important variability in its clinical presentation. The mutations alter the function of TRα1, one of the two nuclear receptors for thyroid hormone. METHODS: The aim of this study was to understand the relationship between specific THRA mutations and phenotype. CRISPR/Cas9 genome editing was used to generate five new mouse models of RTHα, with frameshift or missense mutations. RESULTS: Like human patients, mutant mice displayed a hypothyroid-like phenotype, with altered development. Phenotype severity varied between the different mouse models, mainly depending on the ability of the mutant receptor to interact with transcription corepressor in the presence of thyroid hormone. CONCLUSION: The present mutant mice represent highly relevant models for the human genetic disease which will be useful for future investigations.

Methylcytosine dioxygenase TET3 interacts with thyroid hormone nuclear receptors and stabilizes their association to chromatin.

Thyroid hormone receptors (TRs) are members of the nuclear hormone receptor superfamily that act as ligand-dependent transcription factors. Here we identified the ten-eleven translocation protein 3 (TET3) as a TR interacting protein increasing cell sensitivity to T3. The interaction between TET3 and TRs is independent of TET3 catalytic activity and specifically allows the stabilization of TRs on chromatin. We provide evidence that TET3 is required for TR stability, efficient binding of target genes, and transcriptional activation. Interestingly, the differential ability of different TRα1 mutants to interact with TET3 might explain their differential dominant activity in patients carrying TR germline mutations. So this study evidences a mode of action for TET3 as a nonclassical coregulator of TRs, modulating its stability and access to chromatin, rather than its intrinsic transcriptional activity. This regulatory function might be more general toward nuclear receptors. Indeed, TET3 interacts with different members of the superfamily and also enhances their association to chromatin.

Characteristics of patients with late manifestation of resistance thyroid hormone syndrome: a single-center experience.

Resistance to thyroid hormone (RTH) is a rare genetic disease caused by reduced tissue sensitivity to thyroid hormone. The hallmark of RTH is elevated serum levels of thyroid hormone with unsuppressed thyrotropin (TSH). However, the most common form of RTH results from minor defects in the ligand-binding domain or hinge domain of the TRß gene, resulting in impaired T3-induced transcriptional activity, often showing mild presentation. Early diagnosis can be challenging. The objective of the current study was to characterize this specific group of RTH patients. This was a retrospective study. Patients diagnosed as RTH with TRß mutations were enrolled in a single institute between 2004 and 2014. A total of 14 patients were diagnosed as RTH with mutation in THß gene. The median age at diagnosis was 22.5 (IQR: 13.25-32.75). Goiter was the most common clinical finding. TSH was significantly elevated after TRH injection (median peak was 21.83 µIU/l, IQR: 13.59-31.48), 9.2-fold compared to the basal level. We found 10 mutations in TRß gene, all located in the last four exons, and including one novel mutation, H271D. In vitro study found that H271D mutation reduced TR affinity to T3. Four patients with intact thyroid were diagnosed after 16 years old, defined as late manifestation. Compared to those diagnosed before 10 years old, patients with late manifestation presented with normal growth and mental development. Interestingly, three of them carried R438H mutation. We identified a novel p.H271D mutation in TRß associated with RTH. Endocrinologists should be alert that RTH is frequently found in euthyroid patients with mild symptoms and often leads to misleading diagnosis as well as inappropriate treatment.