The N terminus of Ascl1 underlies differing proneural activity of mouse and Xenopus Ascl1 proteins

The proneural basic-helix-loop-helix (bHLH) transcription factor Ascl1 is a master regulator of neurogenesis in both central and peripheral nervous systems in vivo, and is a central driver of neuronal reprogramming in vitro. Over the last three decades, assaying primary neuron formation in Xenopus embryos in response to transcription factor overexpression has contributed to our understanding of the roles and regulation of proneural proteins like Ascl1, with homologues from different species usually exhibiting similar functional effects. Here we demonstrate that the mouse Ascl1 protein is twice as active as the Xenopus protein in inducing neural-β-tubulin expression in Xenopus embryos, despite there being little difference in protein accumulation or ability to undergo phosphorylation, two properties known to influence Ascl1 function. This superior activity of the mouse compared to the Xenopus protein is dependent on the presence of the non-conserved N terminal region of the protein, and indicates species-specific regulation that may necessitate care when interpreting results in cross-species experiments.


Introduction
Proneural basic-helix-loop-helix (bHLH) transcription factors have conserved roles from Drosophila to vertebrates, acting in cascades to drive and coordinate the multiple stages of neurogenesis 1 . Vertebrate homologues of the Drosophila achaetescute complex include mammalian Ascl1 (mash1/mAscl1) 2 , and Xenopus Ascl1 (Xash1/xAscl1) 3 . In mammals, Ascl1 is involved in development of GABAergic neurons in the ventral telencephalon and dorsal spine 4 , mesencephalic dopaminergic neurons 5 , rhombencephalic serotonergic neurons 6 and central and peripheral noradrenergic neurons 7 . In parallel, Xenopus Ascl1 is expressed pre and post-metamorphically in the ventricular zone of the pros-, mes-, and rhombencephalon 3 , with a distinct role in GABAergic fate in the retina 8 , and in noradrenergic identity in antero-ventral neural precursors 9 .
Development of Xenopus primary neurons expressing neuralβ-tubulin has provided a simple assay for investigating proneural protein activity and regulation in vivo, for example 10-12 . Proneural gene homologues from different species are often used interchangeably due to the striking conservation of expression and function as outlined above. Ascl1 is also a key cellular reprogramming factor in the regenerative medicine field 13 and its molecular mechanisms of action are a current area of study where species-specific phenomena may be critical for selection of appropriate model systems. We present a short study to compare the activity of mouse and Xenopus Ascl1 proteins in driving neurogenesis in Xenopus embryos. Surprisingly, we observe a marked difference in potency between the mouse and Xenopus Ascl1 homologues, and show that the differences in potency map to the non-conserved residues in the N terminus of the protein.

Animal care
All work has been carried out under UK Home Office Licence and has passed an Institutional ethical review committee assessment at the University of Cambridge.

Plasmids and constructs
The coding regions of mouse Ascl1 (Genbank accession number NM008553) and Xenopus Ascl1 (Genbank accession number NM001085778) were cloned into pCS2+ vectors between EcoR1 and Xho1 or BamH1 and Xho1 sites respectively, adding a single HA tag at 3' end. The chimeric N-m/bHC-xAscl1 construct was generated by double digest of both plasmids with BamH1 and Bgl1. The N terminal fragment of the mouse gene was purified as the insert and the bHLH and C terminus of the Xenopus gene in pCS2+ was used as the vector for ligation. The NTdelxAscl1 construct was generated by PCR amplification of the conserved N terminal region, bHLH and C terminus of the Xenopus gene, (therefore removing the non-conserved 5'region of the N terminus) using primers Thus, X. laevis eggs were obtained by standard hormone methods of induction, and subsequently fertilised in vitro. pCS2+ constructs were linearised and capped mRNA was transcribed in vitro using the SP6 mMessage mMachine ® kit (Ambion). Embryos were injected unilaterally at the two cell stage with mRNA as indicated in the text, with GFP (for qPCR) or β-gal (ISH) as lineage tracers. Embryos were cultured at 18°C in Ficol solution and staged according to 15 . At stage 18, embryos were either snap-frozen for qPCR analysis or fixed in MEMFA for 90 minutes, washed twice in PBS, followed by PBS supplemented with 2mM MgCl 2 and embryos stained in 1 mg/ml X-gal in X-gal mixer. For recipes see 12 . Embryos were again washed twice in PBS before dehydration and storage in methanol.

Whole mount in situ hybridisation (ISH)
Dig-oxigenin-labelled anti-sense probes were synthesised from the following plasmids: X. laevis neural-β-tubulin 16 and X. laevis xMyt1 17 . Whole mount ISH was performed as described in 12 and embryos were scored for the extent and pattern of marker expression as described in data analysis.

Western blotting
For western blot analysis, 12 embryos were snap frozen at stage 12.5 and whole embryo protein was extracted as described in 12. For detection of phospho-status, samples were incubated in the presence or absence of lambda protein phosphatase (NEB) for 1 hour at 30°C. 60µg total protein was loaded on to pre-cast BioRad Criterion ™ TGX 18% gels in Tris-Glycine buffer. Primary antibodies were used at 1:2000 dilution for at least 1 hour at room temperature (tubulin) or at 4°C overnight (HA): Rat HRP-conjugated anti-HA clone 3F-10 antibody (Roche; 12013819001) and mouse anti-α-tubulin clone B-5-1-2 antibody (Sigma; T5618). Anti-tubulin antibody was detected with a sheep HRP-conjugated anti-mouse antibody at 1:10000 dilution (GE Healthcare; NA931V).

Data analysis
For ISH data, embryos were scored for the extent and pattern of gene expression on the injected side relative to uninjected side and to uninjected embryos. Scores were assigned as 0, no difference; +1, mild increase in expression within the neural tube with or without occasional ectopic expression on the injected side; +2, moderate increase with ectopic expression occurring in patches on the injected side and sometimes bilaterally; +3, substantial increase with extensive ectopic expression in a more homogenous pattern on the injected side and sometimes bilaterally. Experiments were conducted in independent duplicate in different batches of eggs and the N numbers refer to the range of total numbers of embryos in each injection category.
For qPCR data, mRNA expression was normalised to expression of reference gene EF1α and mRNA levels in the injected embryos were calculated relative to stage-matched uninjected controls. Mean values are plotted and error bars show the standard error of the mean from three independent experiments. Statistical significance was calculated by a paired two-tailed student T test; NS = not significant; * = p< 0.05; ** = p< 0.025; *** = p< 0.0125. Western blots show two independent experiments. For protein quantification, Image J software was used as described in 12 .

Results
Mouse Ascl1 is more active than Xenopus Ascl1 in the Xenopus primary neurogenesis assay Xenopus primary neurons endogenously develop in the trigeminal ganglia and three bilateral stripes on the neural plate 18 , but over-expression of proneural proteins by microinjection of mRNA can lead to expansion of the endogenous domains and induction of neural-β-tubulin expression across the lateral and ventral epidermis 12 . In order to directly compare the effects of over-expression of the mouse and Xenopus Ascl1 proteins, equivalent plasmids were constructed with the coding region of each wild-type gene placed directly after the SP6 promoter and with a single C terminal HA tag.
Two cell-stage embryos were unilaterally injected with 100pg mRNA encoding either mAscl1 or xAscl1 and with βgal as a lineage tracer. At stage 18, embryos were assayed by in situ hybridisation (ISH) for expression of N-β-tubulin as a marker of primary neurogenesis 19 , and for xMyt1 that is induced downstream of proneural proteins and mediates resistance to lateral inhibition 17 ( Figure 1A-C). xMyt1 expression mirrors N-β-tubulin expression, consistent with both of these genes being induced by the Ascl1 proteins. In comparison, mouse Ascl1 produces a moderate increase in expression of N-β-tubulin and xMyT1 across the lateral epidermis, while the Xenopus protein results in less pronounced ectopic neurogenesis that is concentrated in the region of the developing neural tube. As these experiments involve protein translation in vivo from over-expressed coding sequence mRNA, the observed differences in Ascl1 activity must be mediated at a post-translational level, rather than differences in Ascl1 gene expression or mRNA processing.

Mouse and Xenopus Ascl1 proteins are both phosphorylated and show similar protein stability
Proneural transcription factors are known to be unstable proteins, which are turned over rapidly by the ubiquitinproteosome system 20 . To explore potential species differences in protein accumulation/stability following mRNA expression, Ascl1 levels in whole embryo extracts were compared by western blot, probing for the HA-tag at the C terminus of each protein ( Figure 1D). Mouse Ascl1 migrates as a single band and at a higher molecular weight than xAscl1, in part expected from the extended N terminus in the mouse protein (see below). In contrast, Xenopus Ascl1 migrates as a broad or multiple bands that may represent various post-translationally modified isoforms. When the amount of mAscl1 and xAscl1 is measured relative to the tubulin loading controls in two independent experiments (shown), there are no significant differences in protein accumulation; 1.60 +/-0.11 units for xAscl1 and 1.54 +/-0.08 units for mAscl1, indicating that accumulation and stability are similar for the two proteins and cannot account for the differences in activity.
Multi-site phosphorylation has been shown to have a marked inhibitory effect on Ascl1 proneural activity 21 . To determine whether there are differences in phosphorylation between mAscl1 and xAscl1, extracts from embryos expressing the two proteins were treated with lambda protein phosphatase prior to separation and western blot ( Figure 1E). The multiple bands of xAscl1 protein collapse to a single faster migrating band in the presence of phosphatase enzyme, suggesting that the multiple bands in the untreated samples represent xAscl1 phospho-forms. Similarly, the single band of the mouse protein also undergoes a prominent increase in migration after phosphatase treatment, indicating that mouse Ascl1 is also phosphorylated, and indeed may be more uniformly phosphorylated than the Xenopus protein. Thus both proteins accumulate to comparable levels following over-expression and both undergo phosphorylation, so these properties cannot account for the observed in vivo differences in activity.

Mouse and Xenopus proteins are highly conserved except for the N terminus
Protein sequence alignment of mAscl1 and xAscl1 reveals a high degree of species conservation in all but the N terminus ( Figure 2A). The bHLH domains of mouse and Xenopus proteins (shown in blue and green) are identical, and the C terminus (purple) differs by just four amino acids that are broadly conserved alternative residues. Furthermore, the 35 residues in N terminal fragment adjacent to the bHLH domain are identical in all but two residues between species. However, outside of this, the most N terminal regions of the two proteins diverge in both sequence and length.
To investigate the potential role of the N terminus in conferring Ascl1 protein activity, a chimeric construct was made  The non-conserved region of the N terminus mediates species differences in Ascl1 activity Using the Xenopus neurogenesis assay as before, mRNA encoding chimeric and truncated forms of Ascl1 ( Figure 2B) was injected unilaterally in two-cell stage embryos, and expression of N-β-Tubulin was detected in stage 18 embryos by qPCR and ISH (Figure 3). Exchange of the non-conserved N terminal region of mouse Ascl1 in place of the shorter N-terminal region of xAscl1 results in enhanced activity of the chimera to the level of native mAscl1; by qPCR, both mAscl1 and N-m/bHC-xAscl1 induce around two-fold higher level of N-β-Tubulin expression than wild-type xAscl1. This indicates that the non-conserved region of the N terminus of the mouse protein mediates its relatively greater potency. Interestingly, deletion of the corresponding nonconserved region of the Xenopus protein without its replacement by the respective region from mAscl1 also increases N-β-Tubulin expression relative to wild-type xAscl1, although this is not significant on qPCR analysis. This may suggest that while the N terminus of the mouse protein enhances Ascl1 activity, the corresponding region of the Xenopus protein may have an inhibitory influence.

Discussion/conclusions
In this study we have determined that mouse Ascl1 protein is approximately twice as active as the Xenopus protein at inducing ectopic primary neurons in Xenopus embryos. This requires the presence of the non-conserved N terminal residues in the mouse protein, but is not mediated through changes in protein accumulation or Ascl1 phosphorylation.
Different activity between mouse and Xenopus homologues in Xenopus embryos has previously been reported for another master regulatory bHLH protein in muscle, MyoD. The C terminus of xMyoD causes cytoplasmic retention of the protein until mesoderm induction, while mMyoD is constitutively nuclear 22 . The differing activity of mouse and Xenopus Ascl1 in vivo in Xenopus embryos may similarly be due to differences in nuclear localisation, although we have yet to explore this possibility. Species differences in activity could also be due to cofactor binding involving the N terminus of the protein, for instance, association of mouse Ascl1 with an activating partner or reduced association with an inhibitory factor. As such, if using Xenopus to dissect the molecular mechanisms of proneural activity, consideration should be given to potential differences in activity between homologous proteins from different species.

Data availability
Raw data files are available in Open In this research note, the authors have compared the proneural activity of and mouse Xenopus transcription factor Ascl1, by mRNA injection in embryos. They demonstrated that the mouse Xenopus Ascl1 protein is surprisingly more active in inducing ectopic neurons than the one. Using Xenopus chimeric and truncated forms of Ascl1, the authors determined that this species difference in Ascl1 activity is mediated by the N terminus of Ascl1. This is interesting considering that this region was identified as poorly conserved between the two species. The molecular mechanisms underlying its distinct influence in and mouse remain to be investigated. Xenopus Overall, the research note is clearly written, the methodology is sound and the data are convincing. The illustration in Fig. 1C could however be improved. It is explained that Ascl1 mRNA injection Xenopus leads to ectopic neurogenesis that is concentrated in the region of the developing neural tube (compared to broader ectopic neurogenesis observed following injection of the mouse mRNA). The figure indeed shows an embryo injected with Ascl1 mRNA, displaying restricted ectopic N-tubulin expression. Xenopus However, it also has a restricted ß-gal staining to the same region. It would have been more convincing to show an embryo with ß-gal staining on the whole side of the embryo and ectopic N-tubulin expression restricted to the neural tube region.
Although the data appear convincing in graphs of Fig. 1A On western blots, it should be written alpha-tubulin since it could be confusing with the N-tubulin expression that is analyzed elsewhere in the manuscript.
The authors determined that mouse Ascl1 protein is approximately twice as active as the protein Xenopus at inducing ectopic primary neurons in embryos. It is not clear how this two-fold difference has Xenopus been calculated.
Is the work clearly and accurately presented and does it cite the current literature?

Is the study design appropriate and is the work technically sound? Yes
Are sufficient details of methods and analysis provided to allow replication by others? Yes

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
No competing interests were disclosed.

Competing Interests:
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. In this Research Note, Hardwick and Philpott use a simple assay to assess the ability of mouse and Xenopus Ascl1 to generate primary neurones in Xenopus. It is already known that Xenopus Ascl1 mRNA injected into an early stage Xenopus embryo will cause an increase in neurogenesis, the interesting result here is that the mouse version, injected into Xenopus, is even more active in this process. By using a chimeric mouse-Xenopus Ascl1 and a deleted version of the Xenopus protein they provide evidence that the poorly conserved N-terminal part of the protein is responsible for differences in its activity.
The report is concise and informative and the approach technically sound. The results clearly implicate the N-terminal region of the proteins. Other factors are excluded, since experiments on the broad phosphorylation status of the proteins indicate both are to some degree phosphorylated within the embryo and there are no significant differences in the accumulation of the Ascl1 proteins, suggesting that they have similar stabilities.
The inclusion of two additional constructs, a truncated version of the mouse protein and a chimeric form with the Xenopus N-terminus in front of the rest of the mouse protein, would have provided a more complete comparison. The results as they stand, however, give a clear indication of the significance of the N-terminal region, which is the main aim of this short report, even if the underlying molecular mechanism remains uncertain.