PiggyBac Transposon-Mediated Mutagenesis in Rats Reveals a Crucial Role of Bbx in Growth and Male Fertility

Bobby sox homolog (Bbx) is an evolutionally conserved gene, but its biological function remains elusive. Here, we characterized defects of Bbx mutant rats that were created by PiggyBac-mediated insertional mutagenesis. Smaller body size and male infertility were the two major phenotypes of homozygous Bbx mutants. Bbx expression profile analysis showed that Bbx was more highly expressed in the testis and pituitary gland than in other organs. Histology and hormonal gene expression analysis of control and Bbx-null pituitary glands showed that loss of Bbx appeared to be dispensable for pituitary histogenesis and the expression of major hormones. BBX was localized in the nuclei of postmeiotic spermatids and Sertoli cells in wild-type testes, but absent in mutant testes. An increased presence of aberrant multinuclear giant cells and apoptotic cells was observed in mutant seminiferous tubules. TUNEL-positive cells costained with CREM (round spermatid marker), but not PLZF (spermatogonia marker), gammaH2Ax (meiotic spermatocyte marker), or GATA4 (Sertoli cell marker). Finally, there were drastically reduced numbers and motility of epididymal sperm from Bbx-null rats. These results suggest that loss of BBX induces apoptosis of postmeiotic spermatids and results in spermiogenesis defects and infertility

Live imaging of rat embryos with Doppler swept-source optical coherence tomography

The rat has long been considered an excellent system to study mammalian embryonic cardiovascular physiology, but has lacked the extensive genetic tools available in the mouse to be able to create single gene mutations. However, the recent establishment of rat embryonic stem cell lines facilitates the generation of new models in the rat embryo to link changes in physiology with altered gene function to define the underlying mechanisms behind congenital cardiovascular birth defects. Along with the ability to create new rat genotypes there is a strong need for tools to analyze phenotypes with high spatial and temporal resolution. Doppler OCT has been previously used for 3-D structural analysis and blood flow imaging in other model species. We use Doppler swept-source OCT for live imaging of early postimplantation rat embryos. Structural imaging is used for 3-D reconstruction of embryo morphology and dynamic imaging of the beating heart and vessels, while Doppler-mode imaging is used to visualize blood flow. We demonstrate that Doppler swept-source OCT can provide essential information about the dynamics of early rat embryos and serve as a basis for a wide range of studies on functional evaluation of rat embryo physiology

Trans-Activation between EphA and FGFR Regulates Self-Renewal and Differentiation of Mouse Embryonic Neural Stem/Progenitor Cells via Differential Activation of FRS2α.

Ephs and FGFRs belong to a superfamily of receptor tyrosine kinases, playing important roles in stem cell biology. We previously reported that EphA4 and FGFR form a heterodimer following stimulation with ligands, trans-activating each other and signaling through a docking protein, FRS2α, that binds to both receptors. Here, we investigated whether the interaction between EphA4 and FGFRs can be generalized to other Ephs and FGFRs, and, in addition, examined the downstream signal mediating their function in embryonic neural stem/progenitor cells. We revealed that various Ephs and FGFRs interact with each other through similar molecular domains. When neural stem/progenitor cells were stimulated with FGF2 and ephrin-A1, the signal transduced from the EphA4/FGFR/FRS2α complex enhanced self-renewal, while stimulation with ephrin-A1 alone induced neuronal differentiation. The downstream signal required for neuronal differentiation appears to be MAP kinase mainly linked to the Ras family of G proteins. MAP kinase activation was delayed and sustained, distinct from the transient activation induced by FGF2. Interestingly, this effect on neuronal differentiation required the presence of FGFRs. Specific FGFR inhibitor almost completely abolished the function of ephrin-A1 stimulation. These findings suggest that the ternary complex of EphA, FGFR and FRS2α formed by ligand stimulation regulates self-renewal and differentiation of mouse embryonic neural stem/progenitor cells by ligand-specific fine tuning of the downstream signal via FRS2α

Crosstalk of Humoral and Cell-Cell Contact-Mediated Signals in Postnatal Body Growth

The growth hormone (GH)–insulin-like growth factor 1 (IGF1) axis mediates postnatal body growth. The GH receptor has been regarded as the sole receptor that mediates the Janus kinase 2 (JAK2)/signal transducers and activators of the transcription 5B (STAT5B) signal toward IGF1 synthesis. Here, we report a signaling pathway that regulates postnatal body growth through EphA4, a member of the Eph family of receptor tyrosine kinases and a mediator of the cell-cell contact-mediated signaling. EphA4 forms a complex with the GH receptor, JAK2, and STAT5B and enhances Igf1 expression predominantly via the JAK2-dependent pathway, with some direct effect on STAT5B. Mice with a defective Epha4 gene have a gene dose-dependent short stature and low plasma IGF1 levels. Igf1 messenger RNA (mRNA) in the liver and many other tissues was also significantly reduced in Epha4-knockout mice, whereas pituitary Gh mRNA and plasma GH levels were not. These findings suggest that the local cell-cell contact-mediated ephrin/EphA4 signal is as important as the humoral GH signal in IGF1 synthesis and body size determination

EphA4 Regulates the Balance between Self-Renewal and Differentiation of Radial Glial Cells and Intermediate Neuronal Precursors in Cooperation with FGF Signaling.

In mouse cerebral corticogenesis, neurons are generated from radial glial cells (RGCs) or from their immediate progeny, intermediate neuronal precursors (INPs). The balance between self-renewal of these neuronal precursors and specification of cell fate is critical for proper cortical development, but the signaling mechanisms that regulate this progression are poorly understood. EphA4, a member of the receptor tyrosine kinase superfamily, is expressed in RGCs during embryogenesis. To illuminate the function of EphA4 in RGC cell fate determination during early corticogenesis, we deleted Epha4 in cortical cells at E11.5 or E13.5. Loss of EphA4 at both stages led to precocious in vivo RGC differentiation toward neurogenesis. Cortical cells isolated at E14.5 and E15.5 from both deletion mutants showed reduced capacity for neurosphere formation with greater differentiation toward neurons. They also exhibited lower phosphorylation of ERK and FRS2α in the presence of FGF. The size of the cerebral cortex at P0 was smaller than that of controls when Epha4 was deleted at E11.5 but not when it was deleted at E13.5, although the cortical layers were formed normally in both mutants. The number of PAX6-positive RGCs decreased at later developmental stages only in the E11.5 Epha4 deletion mutant. These results suggest that EphA4, in cooperation with an FGF signal, contributes to the maintenance of RGC self-renewal and repression of RGC differentiation through the neuronal lineage. This function of EphA4 is especially critical and uncompensated in early stages of corticogenesis, and thus deletion at E11.5 reduces the size of the neonatal cortex

Effect of stage-dependent EphA4 deletion on the cerebral cortex at P0.

<p><b>A–L</b>, Cresyl violet stain of coronal brain sections from control (A–D), <i>Nestin</i>;<i>Epha4</i>^<i>fx/fx</i> (E–H), and <i>GFAP</i>;<i>Epha4</i>^<i>fx/fx</i> (I–L) mice. Shown are the corresponding sections of these mice along the antero-posterior axis of the telencephalon. The <i>Nestin</i>;<i>Epha4</i>^<i>fx/fx</i> cortex appears smaller than the control cortex, while the <i>GFAP</i>;<i>Epha4</i>^<i>fx/fx</i> cortex is almost the same size as the control cortex. <b>M–P,</b> Quantification of cortical thickness (M, O) and proliferating zone (VZ+SVZ) width (N, P) in <i>Nestin;Epha4</i>^<i>fx/fx</i> (M, N) and <i>GFAP;Epha4</i>^<i>fx/fx</i> (O, P) mice. Solid lines in control animals (A to C) and corresponding lines in mutants (E–G, I–K) indicate the antero-posterior location where these thicknesses were measured. Enlargements of the measured areas in the anterior part (A, E, and I) are shown in the rightmost column (D, H, and L, respectively). Cortical thickness was reduced in all brain sections from <i>Nestin</i>;<i>Epha4</i>^<i>fx/fx</i> mice, and the proliferating zone width was thinner only in anterior sections. However, in <i>GFAP</i>;<i>Epha4</i>^<i>fx/fx</i> mice, cortical thickness and proliferating zone width were similar to controls. A, anterior; M, middle; P, posterior. N = 5 for each measurement, (*) P < 0.05. Error bars represent SD. Scale bar, 500 μm.</p

Expression and interaction of Ephs and FGFRs.

<p>(A) Expression of all Eph receptors, ephrin ligands, FGFRs and related molecules in mouse embryonic NSPCs. RT-PCR was performed with equal amounts of total RNA isolated from mouse NSPCs. Fragment lengths are indicated on the left in base pairs. (B) Co-expression of FGFRs, EphA4 and FRS2α (green, left panel), respectively, with the neural stem cell marker nestin (red, middle panel) in cultured neurospheres. Merged images are shown in right panels. Neurospheres were cultured on PLL-coated plates for a short time, fixed and immunostained. Immunofluorescent images were detected using a confocal microscopy with an appropriate optical filter. (C, D) Inhibition of FGFR-EphA binding with a dominant-negative EphA4 molecule, EphA4(ΔJM,KD), tagged with enhanced green fluorescence protein (ΔJM,KD-EGFP). FGFR1-HA (C) and FGFR3-HA (D) were co-expressed with Flag-tagged EphAs (EphA1, 2, 3, 5 and 7), respectively, and increasing doses of ΔJM,KD-EGFP in HEK293T cells. Binding of FGFR-HA with EphAs-Flag was examined with immunoprecipitation (IP) followed by SDS-PAGE and immunoblotting (IB) using the antibodies shown.</p