ABSTRACT Gonadotropin‐releasing hormone (GnRH) neurons play a key role in the hypothalamic‐pituitary‐gonadal axis. We generated a new GnRH‐P2A‐iCreERT2‐P2A‐tdTomato (hereafter referred to as the GnRH‐CT‐tdT line) knock‐in mouse line utilizing CRISPR/Cas9 technology. Characterization analysis of this line revealed that tdTomato expression precisely recapitulates endogenous GnRH expression and iCreERT2 recombines in GnRH neurons with excellent specificity and sensitivity. Therefore, this newly constructed GnRH‐CT‐tdT mouse line provides a useful tool to perform genetic manipulation in GnRH neurons. As a proof‐of‐principle, we used this line to investigate the role of Chd7 (Chromodomain helicase DNA‐binding protein 7), a gene with its loss‐of‐function associated with isolated gonadotropin‐releasing hormone deficiency (IGD). Surprisingly, ablation of Chd7 in GnRH neurons does not affect the number of GnRH neurons, pubertal onset, and reproductive organ morphology. Therefore, Chd7 is dispensable in GnRH neurons after they have initiated GnRH expression. In summary, the GnRH‐CT‐tdT mouse line is a valuable tool to study gene function in GnRH neurons and to dissect the pathogenesis of IGD.
Transgenic reporters offer valuable opportunities to investigate the genetic and cellular basis of embryonic development in live organisms. While the specificity of transgenic reporters is essential for their effectiveness in developmental studies, many existing transgenic muscle reporters used for craniofacial muscle development in zebrafish also label trunk skeletal and cardiac muscles, as well as other head tissues. Here, we report the identification of a new transgenic reporter marking craniofacial muscles, Tg(nkx2.3:Alcama-GFP), in zebrafish. This transgenic reporter exhibits Alcama-GFP expression in growing muscle fibers at embryonic stages and in grown muscle fibers at larval stages. However, endogenous nkx2.3 transcripts are barely detected in growing craniofacial muscle fibers by in situ hybridization, and nkx2.3 mutants display fairly normal ventral jaw muscles adjacent to the lower jaw cartilages and extraocular muscles adjacent to the eye. Although an essential role for Nkx2.3 in craniofacial muscle formation remains to be determined, the Tg(nkx2.3:Alcama-GFP) craniofacial muscle reporter provides better opportunities for the precise analysis of craniofacial muscle development at the cellular and genomic levels.
Research into regeneration in sea cucumbers makes a significant contribution to understanding the fundamental mechanisms of this process. However, these studies focus only on the regenerating organs-the intestine and aquapharyngeal bulb. The source of cells for regeneration remains out of sight. The cells of the body wall, under the influence of various activating molecules (growth factors), dedifferentiate, determine the direction of migration, and move toward the injury. Therefore, the aim of this study was to identify the most active growth factor genes in the body wall during the regenerative process in the sea cucumber Eupentacta fraudatrix. We searched for and identified genes of the TGF-β, FGF, Neurotrophin, VEGF families. The sea cucumber Eupentacta fraudatrix possesses a set of growth factor genes characteristic of all echinoderms, with the exception of three orthologs of gdf2 genes. A significant increase in the expression of gdf2, bmp2/4, neurotrophin, and wnt5 genes was detected in the body wall as early as the first day of the regenerative process. A second increase in growth factor gene expression occurred on day 7, during the onset of the proliferation and growth phase. The results of this study identify the most promising genes for further investigation of their role in the signal transduction that triggers regeneration.
The uterus is an essential organ for fetal development in most mammals. Uterine glands, highly conserved structures in the mammalian uterus, play critical roles in the establishment and maintenance of pregnancy and have been implicated in the pathogenesis of uterine diseases, including endometrial cancer and endometriosis. Previous studies have shown that Retinol dehydrogenase 1 (Rdh1) is specifically expressed in the glandular epithelium (GE) from the onset of gland formation through adulthood. In this study, to develop a GE-specific Cre driver line, we generated Rdh1-iCre mice by introducing an improved Cre recombinase (iCre) into the Rdh1 locus using the CRISPR/Cas9 system. To evaluate the utility of this model, Rdh1-iCre mice were crossed with ROSA26-H2B-mCherry reporter mice, and Cre-dependent reporter expression was analyzed. Robust mCherry fluorescence was observed throughout the uterine glands at 2 weeks after birth, coinciding with the active elongation and branching of the GE. These results demonstrate that the Rdh1-iCre mouse line is a valuable and highly efficient tool for investigating the physiological roles of uterine glands during development and pregnancy, as well as their contribution to the progression of GE-derived uterine diseases.
Follicular thyroid cells in vertebrates are considered to have evolved from the endostyle of invertebrate chordates based on the shared features of the concentration of iodine and expression of thyroid-related genes, including Nkx2-1, FoxE, and TPO. We previously demonstrated using gene knockout (KO) experiments on the ascidian Ciona robusta that the thyroid-related transcription factors, Nkx2-1 and FoxE, were associated with endostyle development, and also that Nkx2-1 regulated the expression of TPO via FoxE, even though the expression of Pax2/5/8 homologs was not affected. In the present study, which involved detailed observations of Ciona Nkx2-1-KO and FoxE-KO specimens, we found that ectopic bulged structures neighboring the hypoplastic endostyle occasionally emerged. In situ hybridization revealed that the ectopic bulged structures expressed TPO and Pax2/5/8 homologs. Further KO experiments on Ciona Pax2/5/8 homologs revealed no effects on endostyle development or TPO expression. These results suggest that, in addition to the established "Nkx2-1 -> FoxE-FoxE -> TPO" pathway, the ventral pharynx offers an insight into a potential relationship between Pax2/5/8 homologs and TPO. Accordingly, ascidians may show that the ventral pharyngeal endoderm possessed multiple gene regulatory networks for TPO expression prior to the evolutionary divergence leading to the vertebrate follicular thyroid.
