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Although serine-arginine rich (SR) proteins have often been implicated in the positive regulation of splicing, recent studies have shown that one unusual SR protein, SRp38, serves, contrastingly, as a splicing repressor during mitosis and stress response. We have identified a novel developmental role for SRp38 in the regulation of neural differentiation. SRp38 is expressed in the neural plate during embryogenesis and is transcriptionally induced by the neurogenic bHLH protein neuroD. Overexpression of SRp38 inhibits primary neuronal differentiation at a step between neurogenin and neuroD activity. This repression of neuronal differentiation requires activation of the Notch pathway. Conversely, depletion of SRp38 activity results in a dysregulation of neurogenesis. Finally, SRp38 can interact with the peptidyltransferase center of 28S rRNA, suggesting that SRp38 activity may act, in part, via regulation of ribosome biogenesis or function. Strikingly, recent studies of several cell cycle regulators during primary neurogenesis have also revealed a crucial control step between neurogenin and neuroD. SRp38 may mediate one component of this control by maintaining splicing and translational silencing in undifferentiated neural cells.
Fig. 1. Sequence and expression pattern of SRp38. (A) Schematic diagram of SRp38. SRp38 is a 238 amino acid protein with an N-terminal RNA recognition motif (RRM) containing two conserved ribonucleoprotein (RNP) domains and a C-terminal serine-arginine rich (SR) domain. RNPs are underlined in red and SR domain is underlined in blue. (B) The RRM of SRp38 is 87% identical to that of the mouse SRp38 isoforms (TASR1 and TASR2). The SR domain is low complexity and the homology drops considerably in this region. (C-F) Expression pattern of SRp38. (C,E) Dorsal view. (D,F) Lateral views. (C,D) At stage 15, SRp38 is expressed diffusely throughout the neural plate. (E,F) At later stages, SRp38 is expressed in the anterior of the embryo, throughout the neural tube, eyes and branchial arches, and surrounding the otic vesicle.
Fig. 2. SRp38 inhibits neural development but not initial induction of neural tissues. (A) In situ hybridization for neural specific nrp1, stage 21. Control embryo shows staining in the neural tube and eyes. Embryo injected in one cell at the two-cell stage with 500 pg SRp38 (right). Nrp-1 expression is lost at the site of injection (red arrowhead). (B) Simplified schematic of neural induction. Inhibition of bone morphogenetic protein (BMP) signaling by BMP antagonists, such as noggin, allows expression of Sox genes (Sox2 and Sox3) which then induce neural fates. (C) SRp38 does not block expression of Sox3 or Sox2 in whole embryos. Dorsal view of control embryos (left column) expressing Sox2 and Sox3 in the neural plate. Embryos injected in one cell at the two cell stage with 500 pg SRp38 (right column) also express Sox2 and Sox3 normally in the neural plate. Injected sides marked with red arrowhead. Lineage tracer in pink. (D) Ectodermal explant RT-PCR, stage 10.5. Noggin mRNA injection induces robust expression of Sox3 (lane 4). SRp38 co-expression is unable to block Sox3 expression (lane 6). (E) Ectodermal explant RT-PCR, stage 10.5. SRp38 mRNA injection does not induce expression of the BMP target Vex1. Embryos were injected with 500 pg (lane 4), 250 pg (lane 5) or 125 pg (lane 6). EF1α is a loading control and Xbra controls for mesodermal contamination. (F) In situ hybridization for nrp1 (neural). Noggin injected animal caps express nrp1 and co-injection of SRp38 prevents nrp1 expression.
Fig. 3. SRp38 can inhibit mesodermal differentiation. Control embryos on left. SRp38 injection (250 pg each) on right. Some embryos are also stained for lacZ activity (in red) as a lineage tracer. All injection sites are marked with black arrowhead. (A-C) Early mesoderm injections were targeted toward ventral domains. In situ hybridization for Bix1, stage 9.5 (A); brachyury, stage 11 (B); and wnt8, stage 11 (C). SRp38 injection (250 pg each, right) does not inhibit expression of Bix1 but does inhibit expression of Brachyury and wnt8. (D) In situ hybridization for myoD, stage 25. Dorsal injection of SRp38 (250 pg each, right) inhibits expression of myoD at the site of injection (marked by red tracer). (E) In situ hybridization for α-globin, stage 25. Ventral injection of SRp38 (250 pg each, right) inhibits expression of α-globin at the site of injection. (F) Tor70 antibody reveals disrupted notochord development in embryos injected dorsally with SRp38 (two sections on right, notochord indicated by red arrowhead).
Fig. 4. SRp38 inhibits the activity of neurogenin but not that of neuroD. (A) SRp38 blocks neurogenin induction of neuroD. Stage 15 lateral view, neuroD in situ hybridization. Control embryo: normal expression pattern of neuroD (in trigeminal ganglia indicated by red arrowhead). Expression of 500 pg SRp38 inhibits neuroD expression. However, expression of 250 pg of neurogenin induces ectopic expression of neuroD, while co-expression of SRp38 with neurogenin inhibits induction of neuroD (right embryo). (B) Ectodermal explant RT-PCR. SRp38 blocks neurogenin induction of NCAM, neuronal β-tubulin, synaptobrevin II and neuroD. Analysis of animal caps expressing either 500 pg of SRp38 and/or 250 pg neurogenin as indicated. EF1α is a loading control and muscle actin (mActin) controls for mesodermal contamination. (C) SRp38 does not block neuroD induction of synaptobrevin II (sybII). Stage 25 embryos stained for sybII. Control embryo: normal expression pattern of sybII in trigeminal ganglia and neurons. Expression of 500 pg SRp38 inhibits sybII expression. Ectopic expression of 250pg of neuroD induces ectopic expression of for sybII, while co-expression of SRp38 with neuroD does not inhibit neuroD induction of sybII. (D) Simplified schematic of neurogenic cascade. In the neural plate, neurogenin activates transcription of several downstream genes, including neuroD. neuroD then induces genes characteristic of neuronal differentiation such as neuronal β-tubulin and synaptobrevin. Notch signaling can inhibit the neurogenic effects of neurogenin but not neuroD.
Fig. 5. SRp38 inhibits neurogenesis and induces Delta/Id3. (A) In situ hybridization for non-specific β-tubulin stains the ciliated epidermis and neurons. Left, control embryo. Middle, injection of 500 pg SRp38 targeted to the neural plate results in a loss of neuronalβ -tubulin. Right, overexpression of 500 pg SRp38 in the epidermis leads to a decrease in ciliated epidermal cells. Both of these phenotypes are symptomatic of Notch activation. Injection sites marked by red arrowhead. (B) Schematic model of SRp38 inhibition of neurogenesis. SRp38 inhibition of neurogenin activity may act via Delta and Id3. (C) Lateral views of stage 18 embryos stained for Delta. Left: control embryo. Right: injection of 500 pg of SRp38 induces robust expression of Delta (red arrowhead). (D) RT-PCR analysis of animal caps expressing 500 pg SRp38 (lane 4). (Uninjected control: C, lane 3.) SRp38 induces ectopic expression of Delta, lane 4. EF1α is a loading control and muscle actin (MA) controls for mesodermal contamination. (E) Lateral views of stage 18 embryos stained for Id3. Left: control embryo. Right: injection of 500 pg of SRp38 induces expression of Id3 (red arrowhead). (F) RT-PCR analysis of Id3 splicing. Primers were designed to span exons 1-2 or exons 2-3. Embryos treated with decreasing doses of SRp38 (500 pg to 100 pg) were analyzed for changes in the amount of spliced products. No discernible changes were found.
Fig. 6. SRp38 is required for regulated neurogenesis. (A) Dorsal views, in situ hybridization for neuronal specific β-tubulin. Top, left to right: control embryos show expression of β-tubulin in neurons and trigeminal ganglia; SRp38-injected embryos show a loss ofβ -tubulin-expressing cells; injection of antisense morpholino oligonucleotides (AMOs) does not perturb expression ofβ -tubulin. Bottom, left to right: inhibitory Delta (Deltastu) injection results in increased and disorganized expression of β-tubulin; co-expression of Deltastu and SRp38 results in almost normal embryos (compare with +SRp38 above); co-injection of Deltastu and AMOs results in increased expression of β-tubulin (compare with +AMO embryo above and to controls). (B) In situ hybridization for nrp1, dorsoanterior view, stage 17. Left: control embryos show nrp1 staining in the neural plate and eyeprimordia. Middle: SRp38 overexpression inhibits expression of nrp1, red arrows. Right: co-injection of SRp38 with the function-blocking sequence C3 rescues expression of nrp1, red arrows. (C) RT-PCR analysis on animal cap ectodermal explants. Expression of 100 pg of neurogenin in the animal cap results in expression of neuronal β-tubulin and a mild decrease in Id3 (lane 3). Co-expression of neurogenin and 5ng of C3 results in a greater increase of neuronalβ -tubulin and concomitant decrease in Id3 (compare control lane 5 with lane 3). C3 alone (lane 6, 5 ng) results in complete loss of Delta and no effect on Id3 expression. CyclinD1 and p27xic1 expression are unchanged in neuralized explants upon addition of C3 (compare lane 5 to lane 4). (D) TUNEL staining indicates apoptotic cells. Dorsal views of stage 15 embryos. Control embryos show very few TUNEL-positive cells (black arrowhead), while embryos injected with 250 pg of SRp38 RNA in one cell at the two-cell stage show a variable, though clear increase, in the number of TUNEL-positive cells (red arrowheads). (E) Primary neurogenesis is increased in the absence of SRp38 and Delta function.
Fig. 7. SRp38 is induced by neuroD. (A) Dorsal views of SRp38 expression are pictured at stage 21. Left: control. Right: injection of 250 pg of neuroD into one cell at two cell-stage induces robust expression of SRp38. (B) RT-PCR on ectodermal explants. Injection of 250 pg of neuroD (+N) induces expression of SRp38. EF1α is a loading control and muscle actin (MA) controls for mesodermal contamination. (C) SRp38 northern blot on total RNA in embryos overexpressing increasing doses of neuroD (125 pg to 500 pg) results in increasing amounts of SRp38 RNA. (D) SRp38 is itself regulated by neuroD, suggesting it is a component of a neuroD-induced mechanism serving to limit neurogenesis.
vamp2 (vesicle-associated membrane protein 2 (synaptobrevin 2))gene expression in Xenopus laevis embryos, NF stage 25, as assayed by in situ hybridization. Lateral view: anteriorleft, dorsal up.
