Glypicans and syndecans are heparan sulfate proteoglycans that are associated with the cell membrane. They regulate neural progenitor proliferation and differentiation, as well as neuronal migration, axon guidance, and synaptogenesis in the developing nervous system. The glypican family consists of 6 members encoded by the Gpc1 to Gpc6 genes, and the syndecan family consists of 4 members encoded by the Sdc1 to Sdc4 genes. To understand their roles, precise knowledge of their spatiotemporal gene expression is essential. In this study, their expression patterns in the mouse embryonic brain were examined by use of RT-PCR and in situ hybridization. RT-PCR revealed that expression of Gpc1, Gpc2, Gpc3, Gpc4, Gpc6, Sdc1, and Sdc3 was higher in the embryonic brain than in the adult brain, whereas expression of Gpc5 and Sdc4 was higher in the adult brain. In situ hybridization revealed that at embryonic days 13.5 (E13.5), E15.5, and E17.5, Gpc1, Gpc2, Gpc6, and Sdc3 were highly expressed throughout the brain, whereas Gpc3, Gpc4, Gpc5, and Sdc1 expression was more localized. According to their expression profiles, glypicans and syndecans can be divided into 5 groups: Gpc1, Gpc4, Gpc6, and Sdc1 showed high expression in the ventricular/subventricular zone and low expression in the adult brain. Gpc2 and Sdc3 showed high expression outside the ventricular/subventricular zone and lower expression in the adult brain. Gpc3 showed high expression in the meninges and choroid plexus of the embryonic brain and low expression in the adult brain. Gpc5 and Sdc4 showed low expression in the embryonic brain and high expression in the adult brain. Sdc2 showed low expression in both the embryonic and the adult brains. These expression profiles suggest that different groups of genes may play distinct roles in the developing brain.
Osteocytes are critical for bone development, and tightly regulate bone (re)modelling by directing the differentiation and activity of osteoblast and osteoclasts.Primary osteocyte isolation is challenging, therefore MLO-A5 and MLO-Y4 osteocyte-like cell lines have been instrumental in dissecting osteocytogenesis and bone mineralisation. Limited comparative analysis of these closely related in vitro models has been undertaken, therefore we used transcriptomic analysis to dissect their transcriptional similarities.Principal component analysis showed MLO-A5 and MLO-Y4 cells form distinct clusters, with 4,451 differentially expressed genes. Known osteoblast markers (Alpl and Col1a1) and early osteocyte markers (Gja1, Pdpn, and Tnfsf11) predominated in MLO-A5 and MLO-Y4 cells, respectively. MLO-A5 cells expressed genes associated with extracellular matrix (re)modelling and mineralisation pathways, whilst MLO-Y4 cells were enriched with genes associated with cell signalling, consistent with MLO-A5 and MLO-Y4 cells representing different stages in osteocyte development.Androgens have anabolic effects on the bone, so their impact on the osteocyte transcriptome and osteocyte function was investigated. Surprisingly, androgen (Ar) and oestrogen receptor (Esr2) expression was significantly lower in MLO-A5 and MLO-Y4 cells compared to mouse tibiae. Treatment with androgen receptor ligands (R1881 and enzalutamide) had minimal effect on MLO-A5 or MLO-Y4 gene transcription (<20 differentially expressed genes) and treated cells showed a striking lack of differentially expressed osteoblast and osteocyte regulatory genes.This work provides a comprehensive understanding of these important in vitro osteocyte models and highlights the limitations of using MLO-A5 or MLO-Y4 cells to investigate the impact of sex hormones on osteocyte function in vitro.
Pitt-Hopkins syndrome (PTHS) is a rare neurodevelopmental disorder caused by de novo mutations in the Transcription Factor 4 (TCF4) gene. Clinically, phenotypes of PTHS involve motor and cognitive domains as well as epilepsy and breathing abnormalities. These phenotypes exhibit significant overlap with another disorder, Rett syndrome (RTT), caused by mutations in the Methyl-CpG-Binding Protein 2 (MECP2) gene. We have shown that expression of metabotropic glutamate receptor 7 (mGlu7) is significantly decreased in brain samples of patients with RTT, and we showed therapeutic potential of increasing mGlu7 activity in RTT model mice using a small molecule positive allosteric modulator (PAM) with mGlu7 activity. A recent study mapped TCF4 binding sites throughout the human genome and identified a TCF4 binding site in the Metabotropic Glutamate Receptor 7 (GRM7) gene. Symptom overlap, as well as the potential regulation of GRM7 by TCF4, led us to profile mGlu7 expression in the brains of Tcf4+/− animals. These studies identified an unexpected upregulation of the receptor at both the mRNA and protein level in multiple brain areas, with brain region-specific differences in the regulation of the two main mGlu7 splice variants, suggesting that these two splice variants may be distinctly regulated in the context of Tcf4 heterozygosity.
Troponin I 1 (TNNI1) encodes the slow skeletal isoform of troponin I and is essential for the regulation of contraction in slow-twitch skeletal muscle. However, the developmental and tissue-specific expression of TNNI1-related genes across vertebrates remains incompletely characterized. In zebrafish, multiple tnni genes have been identified, including four tnni1-related paralogs (tnni1a, tnni1b, tnni1c, and tnni1d), which share high sequence similarity. While individual tnni genes have been partially characterized, a systematic comparison of the spatial and temporal expression patterns of these tnni1 paralogs during embryogenesis remains lacking. Here, we analyzed and compared the spatial and temporal expression patterns of the four tnni1 paralogs during zebrafish embryogenesis to assess their potential functional divergence. Multiple sequence alignment revealed that proteins encoded by zebrafish Tnni1 paralogs are highly conserved relative to mammalian TNNI1, with paralog-specific divergence primarily in the N-terminal region. Whole-mount in situ hybridization showed that tnni1a is first detected in the embryonic heart and was later expressed in cranial and hypaxial muscles. tnni1b was expressed in bilateral cardiac precursor cells, exhibited the strongest and most sustained cardiac expression among the paralogs, and was subsequently expressed in extraocular, craniofacial, hypaxial, and trunk muscles. In contrast, tnni1c and tnni1d were initially expressed in somites, displayed weaker or transient cardiac expression, and were later broadly expressed in extraocular, craniofacial, and hypaxial muscles. Taken together, these findings indicate that zebrafish tnni1 paralogs exhibit distinct yet partially overlapping spatiotemporal expression profiles, supporting partial subfunctionalization following gene duplication during cardiac and skeletal muscle development.
