Probes for INS

Forward genetics in the mouse continues to be a useful and unbiased approach to identifying new genes and alleles with previously unappreciated roles in mammalian development and disease. Here, we report a new mouse allele of Cse1l that was recovered from an ENU mutagenesis screen. Embryos homozygous for the anteater allele of Cse1l display a number of variable phenotypes, with craniofacial and ocular malformations being the most obvious. We provide evidence that Cse1l is the causal gene through complementation with a novel null allele of Cse1l generated by CRISPR-Cas9 editing. While the variability in the anteater phenotype was high enough to preclude a detailed molecular analysis, we demonstrate a very penetrant reduction in Pax6 levels in the developing eye along with significant ocular developmental phenotypes. The eye gene discovery tool iSyTE shows Cse1l to be significantly expressed in the lens from early eye development stages in embryos through adulthood. Cse1l has not previously been shown to be required for organogenesis as homozygosity for a null allele results in very early lethality. Future detailed studies of Cse1l function in craniofacial and neural development will be best served with a conditional allele to circumvent the variable phenotypes we report here. We suggest that human next-generation (whole genome or exome) sequencing studies yielding variants of unknown significance in CSE1L could consider these findings as part of variant analysis.

Temporal transcription profiles of fetal testes with Sertoli cell ablation were examined in 4-day culture using a diphtheria toxin (DT)-dependent cell knockout system in AMH-TRECK transgenic (Tg) mice. RNA analysis revealed that ovarian-specific genes, including Foxl2, were ectopically expressed in DT-treated Tg testis explants initiated at embryonic days 12.5-13.5. FOXL2-positive cells were ectopically observed in two testicular regions-near the testicular surface epithelia and around its adjacent mesonephros. The surface FOXL2-positive cells, together with ectopic expression of Lgr5 and Gng13 (markers of ovarian cords), were derived from the testis epithelia/subepithelia, whereas another FOXL2-positive population was the 3βHSD-negative stroma near the mesonephros. In addition to high expression of Fgfr1/Fgfr2 and heparan sulfate proteoglycan (a reservoir for FGF ligand) in these two sites, exogenous FGF9 additives repressed DT-dependent Foxl2 upregulation in Tg testes. These findings imply retention of Foxl2 inducibility in the surface epithelia and peri-mesonephric stroma of the testicular parenchyma, in which certain paracrine signals, including FGF9 derived from fetal Sertoli cells, repress feminization in these two sites of the early fetal testis.

Open neural tube defects (NTDs) such as myelomeningocele (MMC) are debilitating and the most common congenital defects of the central nervous system. Despite their apparent clinical importance, the existing early prenatal diagnostic options for these defects remain limited. Using a well-accepted retinoic-acid-induced model of MMC established in fetal rats, we discovered that neurocan and phosphacan, the secreted chondroitin sulfate proteoglycans of the developing nervous system, are released into the amniotic fluid (AF) of fetal rats displaying spinal cord defects. In contrast to normal controls, elevated AF levels of neurocan and phosphacan were detected in MMC fetuses early in gestation and continued to increase during MMC progression, reaching the highest level in near-term fetuses. The molecular forms of neurocan and phosphacan identified in the AF of MMC fetuses and those found in MMC spinal cords were qualitatively similar. In summary, this is the first report demonstrating the presence of neurocan and phosphacan in the AF of MMC fetuses. The identification of elevated levels of neurocan and phosphacan in the AF of MMC fetuses provides two prospective biomarkers with the potential for early prenatal diagnosis of open NTDs.

The morphogenesis of the otic vesicle (OV) to form inner ear organs serves as an excellent model system to understand cell fate acquisition on a single cell level. Tbx2 and Tbx3 (Tbx2/3) encode closely related T-box transcription factors that are expressed widely in the mammalian OV. Inactivation of both genes in the OV (Tbx2/3cKO) results in failed morphogenesis into inner ear organs. To understand the basis of these defects, single cell RNA-sequencing (scRNA-seq) was performed on the OV lineage, in controls versus Tbx2/3cKO embryos. We identified a multipotent population termed otic progenitors in controls that are marked by expression of the known otic placode markers Eya1, Sox2, and Sox3 as well as new markers Fgf18, Cxcl12, and Pou3f3. The otic progenitor population was increased three-fold in Tbx2/3cKO embryos, concomitant with dysregulation of genes in these cells as well as reduced progression to more differentiated states of prosensory and nonsensory cells. An ectopic neural population of cells was detected in the posterior OV of Tbx2/3cKO embryos but had reduced maturation to delaminated neural cells. As all three cell fates were affected in Tbx2/3cKO embryos, we suggest that Tbx2/3 promotes progression of multipotent otic progenitors to more differentiated cell types in the OV.

Human genetic studies have linked rare coding variants in microglial genes, such as TREM2, and more recently PLCG2 to Alzheimer's disease (AD) pathology. The P522R variant in PLCG2 has been shown to confer protection for AD and to result in a subtle increase in enzymatic activity. PLCγ2 is a key component of intracellular signal transduction networks and induces Ca2+ signals downstream of many myeloid cell surface receptors, including TREM2. To explore the relationship between PLCγ2 and TREM2 and the role of PLCγ2 in regulating immune cell function, we generated human induced pluripotent stem cell (iPSC)- derived macrophages from isogenic lines with homozygous PLCG2 knockout (Ko). Stimulating TREM2 signalling using a polyclonal antibody revealed a complete lack of calcium flux and IP1 accumulation in PLCγ2 Ko cells, demonstrating a non-redundant role of PLCγ2 in calcium release downstream of TREM2. Loss of PLCγ2 led to broad changes in expression of several macrophage surface markers and phenotype, including reduced phagocytic activity and survival, while LPS-induced secretion of the inflammatory cytokines TNFα and IL-6 was unaffected. We identified additional deficits in PLCγ2- deficient cells that compromised cellular adhesion and migration. Thus, PLCγ2 is key in enabling divergent cellular functions and might be a promising target to increase beneficial microglial functions.

Fully grown mammalian oocytes store a large amount of RNA synthesized during the transcriptionally active growth stage. A large part of the stored RNA belongs to the long non-coding class which contain either transcriptional noise or important contributors to cellular physiology. Despite the expanding number of studies related to lncRNAs, their influence on oocyte physiology remains enigmatic. We found an oocyte specific antisense, long non-coding RNA, “Rose” (lncRNA in Oocyte Specifically Expressed) expressed in two variants containing two and three non-coding exons, respectively. Rose is localized in the nucleus of transcriptionally active oocyte and in embryo with polysomal occupancy in the cytoplasm. Experimental overexpression of Rose in fully grown oocyte did not show any differences in meiotic maturation. However, knocking down Rose resulted in abnormalities in oocyte cytokinesis and impaired preimplantation embryo development. In conclusion, we have identified an oocyte-specific maternal lncRNA that is essential for successful mammalian oocyte and embryo development.

Sensory neurons within DRGs are broadly divided into three types that transmit nociceptive, mechanical, and proprioceptive signals. These subtypes are established during in utero development when sensory neurons differentiate into distinct categories according to a complex developmental plan. Most of what we know about this developmental plan comes from studies in rodents and little is known about this process in humans. The present study documents the expression of key genes involved in human sensory neuron development during the late first and early second trimesters (9-16WG). We observed a decrease in the expression of SOX10 and BRN3A, factors associated with migration and proliferation of sensory neurons, towards the end of the first trimester. Small and large sensory neuron populations also emerged at the end of the first trimester, as well as the transcription factors responsible for defining distinct sensory neuron types. NTRK1, which is expressed in nociceptive neurons, emerged first at ~11 WG followed by NTRK2 in mechanoreceptors at ~12 WG, with NTRK3 for proprioceptors peaking at ~14 WG. These peaks were followed by increased expression of their respective neurotrophic factors. Our results show significant differences in the expression of key signalling molecules for human DRG development versus that of rodents, most notably the expression of neurotrophins that promote the survival of sensory neuron types. This highlights the importance of examining molecular changes in humans to better inform the application of data collected in pre-clinical models.

Transient receptor potential (TRP) channels play prominent roles in ion homeostasis by their ability to control cation influx. Mouse placentation is governed by the processes of trophoblast proliferation, invasion, differentiation, and fusion, all of which require calcium signaling. Although certain TRP channels have been shown to contribute to maternal-fetal transport of magnesium and calcium, a role for TRP channels in specific trophoblast functions has been disregarded. Using qRT-PCR and in situ hybridisation, the spatio-temporal expression pattern of TRP channels in the mouse placenta across gestation (E10.5-E18.5) was assessed. Prominent expression was observed for Trpv2, Trpm6, and Trpm7. Calcium microfluorimetry in primary trophoblast cells isolated at E14.5 of gestation further revealed the functional activity of TRPV2 and TRPM7. Finally, comparing TRP channels expression in mouse trophoblast stem cells (mTSCs) and mouse embryonic stem cells (mESC) confirmed the specific expression of TRPV2 during placental development. Moreover, TRP channel expression was similar in mTSCs compared to primary trophoblasts and validate mTSC as a model to study TRP channels in placental development. Collectivity, our results identify a specific spatio-temporal TRP channel expression pattern in trophoblasts, suggesting a possible involvement in regulating the process of placentation.

A precise sequence of axon guidance events is required for the development of the ocular motor system. Three cranial nerves grow towards, and connect with, six extraocular muscles in a stereotyped pattern, in order to control eye movements. The signalling protein alpha2-chimaerin (α2-CHN) plays a pivotal role in the formation of the ocular motor system; mutations in CHN1, encoding α2-CHN, cause the human eye movement disorder Duane Retraction Syndrome (DRS). Our research has demonstrated that manipulation of α2-chn signalling in the zebrafish embryo leads to ocular motor axon wiring defects, although the signalling cascades regulated by α2-chn remain poorly understood. Here, we demonstrate that several cytoskeletal regulatory proteins - collapsin response mediator protein 2 (CRMP2), (encoded by the gene dpysl2), stathmin1 and stathmin 2 - bind to α2-CHN. dpysl2, stathmin1 and especially stathmin2 are expressed by ocular motor neurons. We find that manipulation of dpysl2 and of stathmins in zebrafish larvae leads to defects in both the axon wiring of the ocular motor system and the optokinetic reflex, impairing horizontal eye movements. Knockdowns of these molecules in zebrafish larvae of either sex caused axon guidance phenotypes that included defasciculation and ectopic branching; in some cases these phenotypes were reminiscent of DRS. chn1 knockdown phenotypes were rescued by overexpression of CRMP2 and STMN1, suggesting that these proteins act in the same signalling pathway. These findings suggest that CRMP2 and stathmins signal downstream of α2-CHN to orchestrate ocular motor axon guidance, and to control eye movements.Significance statementThe precise control of eye movement is crucial for the life of vertebrate animals, including humans. In humans, this control depends on the arrangement of nerve wiring of the ocular motor system, composed of three nerves and six muscles, a system that is conserved across vertebrate phyla. Mutations in the protein alpha2-chimaerin have previously been shown to cause eye movement disorders (squint) and axon wiring defects in humans. Our recent work has unravelled how alpha2-chimaerin co-ordinates axon guidance of the ocular motor system in animal models. In this paper, we demonstrate key roles for the proteins CRMP2 and stathmin 1/2 in the signalling pathway orchestrated by alpha2-chimaerin, potentially giving insight into the aetiology of eye movement disorders in humans.

Background Orofacial clefts are the most common birth defects affecting 1 in 750 live births worldwide. Various genetic loci to be involved in nonsyndromic cleft lip and palate has been identified with a variation among populations. Wnt5a is expressed in the frontonasal prominence and maxillary process, which fuse to form the primary palate. Therefore, its dysregulation can lead to certain birth defects along with other diseases. Single nucleotide polymorphism (rs566926) in Wnt5A shows a significant association with nonsyndromic cleft lip and palate in Brazilian and European American populations. Objective The aim of the present study was to describe single nucleotide polymorphism (SNP; rs566926) in patients with nonsyndromic cleft lip and palate in a Pakistani population. Methods This study was conducted on 120 patients with nonsyndromic cleft lip and palate. Demographics and phenotypes were noted. Blood samples were collected in ethylenediaminetetraacetic acid vials. DNA was extracted followed by conventional polymerase chain reaction. SNP (566926) was determined by Sanger sequencing. Data were analyzed using NCBI Blast and SPSS (24.0). Results The mean age of n = 30 patients was 51.33 ± 61.33 months. Sixty percent were male and 40% were female. Regarding cleft types, 70% were both cleft lip and palate, 26% cleft lip only, and 3.3% cleft palate only. Heterozygous polymorphism (T/G) was seen in 33.3% of patients with both cleft lip and palate with bilateral involvement and heterozygous polymorphism (T) was seen in 16.6%. Conclusions SNP in the WNT5A gene is associated with cleft lip and palate, supporting its involvement in pathogenesis of cleft lip and palate. Further studies are recommended to determine the role of Wnt5a genes during craniofacial development.

GPR64/ADGRG2 is an orphan Adhesion G protein-coupled receptor (ADGR) known to be mainly expressed in the parathyroid gland and epididymis. This investigation aimed to delineate the cellular expression of GPR64 throughout the body with focus on the gastrointestinal (GI) tract. Transgenic Gpr64mCherry reporter mice were histologically examined throughout the body and reporter protein expression in intestinal tuft cells was confirmed by specific cell ablation. The GPCR repertoire of intestinal Gpr64mCherry-positive tuft cells was analyzed by quantitative RT-PCR analysis and in situ hybridization. The Gpr64mCherry was crossed into the general tuft cell reporter Trpm5GFP to generate small intestinal organoids for time-lapse imaging. Intestinal tuft cells were isolated from small intestine, FACS-purified and transcriptionally compared using RNA-seq analysis. Expression of the Gpr64mCherry reporter was identified in multiple organs and specifically in olfactory microvillous cells, enteric nerves, and importantly in respiratory and GI tuft cells. In the small intestine, cell ablation targeting Gpr64-expressing epithelial cells eliminated tuft cells. Transcriptional analysis of small intestinal Gpr64mCherry -positive tuft cells confirmed expression of Gpr64 and the chemo-sensors Sucnr1, Gprc5c, Drd3, and Gpr41/Ffar3. Time-lapse studies of organoids from Trpm5GFP:Gpr64mCherry mice revealed sequential expression of initially Trpm5GFP and subsequently also Gpr64mCherry in maturing intestinal tuft cells. RNA-seq analysis of small intestinal tuft cells based on these two markers demonstrated a dynamic change in expression of transcription factors and GPCRs from young to mature tuft cells. GPR64 is expressed in chemosensory epithelial cells across a broad range of tissues; however, in the GI tract, GPR64 is remarkably selectively expressed in mature versus young immunoregulatory tuft cells.

Bone marrow (BM) fibrosis was thought to be induced exclusively by mesenchymal stromal cells (MSCs). However, we and others found that neoplastic fibrocytes induce BM fibrosis in myelofibrosis (MF). Because glioma-associated oncogene-1 (GLI1), an effector of the Hedgehog pathway, plays a role in the induction of BM fibrosis, we wondered whether GLI1 affects fibrocyte-induced BM fibrosis in MF. Multiplexed fluorescence immunohistochemistry analysis of MF patients' BM detected high levels of GLI1 in MF fibrocytes compared to MSCs or normal fibrocytes. Immunostaining, RNA in situ hybridization, gene expression analysis, and western immunoblotting detected high levels of GLI1 and GLI1-induced matrix metalloproteases (MMP) 2 and 9 in MF patients BM-derived cultured fibrocytes. Similarly, MF patients' BM-derived GLI1+ fibrocytes were found in BMs and spleens of MF xenograft mice. GLI1 silencing reduced the levels of MMP2/9, phosphorylated SMAD2/3, and procollagen-I, and knockdown or inhibition of GLI1 decreased fibrocyte formation and induced apoptosis of both fibrocytes and fibrocyte progenitors. Because Janus kinase (JAK)2-induced STAT3 is constitutively activated in MF and because STAT3 induces GLI1 expression, we sought to determine whether STAT3 activates GLI1 in MF fibrocytes. Imaging analysis detected phosphotyrosine STAT3 in MF patients' BM fibrocytes, and transfection of fibrocytes with STAT3-siRNA or treatment with a JAK1/2 inhibitor ruxolitinib reduced GLI1 and MMP2/9 levels. Chromatin immunoprecipitation and a luciferase assay revealed that STAT3 induced the expression of the GLI1 gene in both MF BM fibrocytes and fibrocyte progenitors. Together, our data suggest that STAT3-activated GLI1 contributes to the induction of BM fibrosis in MF.