Oligodendrocyte development in the embryonic tuberal hypothalamus and the influence of Ascl1
- Candace M. Marsters†1, 3, 4, 5,
- Jessica M. Rosin†1, 5,
- Hayley F. Thornton1, 5,
- Shaghayegh Aslanpour1, 5,
- Natasha Klenin1, 5,
- Grey Wilkinson2, 4, 5,
- Carol Schuurmans2, 4, 5, 6,
- Quentin J. Pittman3, 4, 5 and
- Deborah M. Kurrasch1, 5Email author
© The Author(s). 2016
Received: 3 May 2016
Accepted: 8 November 2016
Published: 18 November 2016
Although the vast majority of cells in our brains are glia, we are only beginning to understand programs governing their development, especially within the embryonic hypothalamus. In mice, gliogenesis is a protracted process that begins during embryonic stages and continues into the early postnatal period, with glial progenitors first producing oligodendrocyte precursor cells, which then differentiate into pro-oligodendrocytes, pro-myelinating oligodendrocytes, and finally, mature myelinating oligodendrocytes. The exact timing of the transition from neurogenesis to gliogenesis and the subsequent differentiation of glial lineages remains unknown for most of the Central Nervous System (CNS), and is especially true for the hypothalamus.
Here we used mouse embryonic brain samples to determine the onset of gliogenesis and expansion of glial populations in the tuberal hypothalamus using glial markers Sox9, Sox10, Olig2, PdgfRα, Aldh1L1, and MBP. We further employed Ascl1 and Neurog2 mutant mice to probe the influence of these proneural genes on developing embryonic gliogenic populations.
Using marker analyses for glial precursors, we found that gliogenesis commences just prior to E13.5 in the tuberal hypothalamus, beginning with the detection of glioblast and oligodendrocyte precursor cell markers in a restricted domain adjacent to the third ventricle. Sox9+ and Olig2+ glioblasts are also observed in the mantle region from E13.5 onwards, many of which are Ki67+ proliferating cells, and peaks at E17.5. Using Ascl1 and Neurog2 mutant mice to investigate the influence of these bHLH transcription factors on the progression of gliogenesis in the tuberal hypothalamus, we found that the elimination of Ascl1 resulted in an increase in oligodendrocyte cells throughout the expansive period of oligodendrogenesis.
Our results are the first to define the timing of gliogenesis in the tuberal hypothalamus and indicate that Ascl1 is required to repress oligodendrocyte differentiation within this brain region.
KeywordsGliogenesis Oligodendrogenesis Astrocyte Ascl1 Neurog2 Sox9 Olig2 PdgfRα
The tuberal hypothalamus, consisting of the ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH), and arcuate nucleus (ARC), is a key regulator of many important biological functions, such as energy balance, sexual behavior, thermoregulation, and affective functioning [1–4]. Although most of the research within this brain region is focused on understanding neuronal differentiation and function, the glial cells that interact with hypothalamic neurons also play a critical role in controlling homeostatic mechanisms, particularly aspects of feeding regulation [5, 6].
Gliogenesis is the developmental process of generating the supportive and active signalling central nervous system (CNS) glial cells, namely oligodendrocytes and astrocytes. Temporally, gliogenesis has been shown to follow embryonic neurogenesis in the CNS. Indeed, the differentiation of oligodendrocytes, which are the last cell type to differentiate in the CNS, has been well defined in the cortex and spinal cord and begins around embryonic day (E) 12.5, occurring in three consecutive waves each from its own distinct domain, as has been extensively reviewed elsewhere [7, 8]. Briefly, oligodendrocytes arise from Sox9+ progenitor cells that express the transcription factor Olig2 within restricted regions of the ventral neuroepithelium throughout the rostrocaudal axis. These progenitors further mature and start to migrate outwards after starting to express Sox10 and PdgfRα. Once in the mantle region, these glioblasts begin to actively proliferate and give rise to the many oligodendrocyte precursor cells (OPCs) that are necessary to adequately populate the mantle region. Temporally, these OPCs continue to mature and upon reaching their final maturation, these oligodendrocytes begin to myelinate axons [9–12]. Within the tuberal hypothalamus, neural progenitors that line the third ventricle are known to give rise to both neurons and glia . Yet, despite considerable understanding of glial development in other brain regions, primarily the cortex and spinal cord, the timing for gliogenesis in the hypothalamus remains undefined.
Interestingly, genes of the basic helix-loop-helix family (bHLH), particularly Olig2 and proneural genes Neurogenin 2 (Neurog2) and Achaete-scute homolog1 (Ascl1) also influence gliogenesis . For example, during neurogenesis Olig2 has well-defined roles in the development of motor neurons in the spinal cord  and GABAergic neurons in the cortex [16, 17], while later it is required for the development of oligodendrocytes in both brain regions and, to a much lesser extent, ventrally-located astrocytes as observed in the forebrain and spinal cord [15, 18, 19].
In the case of proneural genes, their function during gliogenesis is more varied. For instance, ectopic expression of Ascl1 in the cerebellum has been shown to increase the numbers of interneurons while concomitantly supressing an astrocytic fate; the loss of Ascl1 exhibits the opposite phenotype , suggesting that Ascl1 restricts the differentiation of a shared progenitor pool into astrocytic lineages. Similarly in the cortex, Neurog2 and Ascl1 double knockout animals show increases in an astrocytic fate at the expense of neurons , while a single Ascl1 knockout shows defects in populations of early-born Pdgfrα+ OPCs but not of late born OPCs . Comparatively, in the developing spinal cord loss of Ascl1 in progenitor cells that would normally produce neurons leads to a reduction in neurons and an increased expression of immature glial markers of both astrocyte and oligodendrocyte origin, but with no change in the OPC marker, Sox10 . Consistently, Ascl1 overexpression in the spinal cord has been shown to promote the maturation of OPCs into myelin forming oligodendrocytes . Compounding the heterogeneity of the influence of Ascl1 on glial progenitors, it was recently shown in the spinal cord that Ascl1 affects both astrocytes and oligodendrocytes differentially in grey matter and white matter. In Ascl1 knockouts, an increase in NFIA+, Olig2+, and Sox10+ glioblasts was observed in the grey matter, which is opposite to that observed in the white matter glial progenitor populations during later embryonic stages . Interestingly, both Neurog2 and Ascl1 are expressed within progenitors within the tuberal hypothalamus but their role during hypothalamic gliogenesis has not yet been defined .
In this study we determined the spatiotemporal timing of gliogenesis in the tuberal hypothalamus by quantifying the timing and location of maturing oligodendrocyte, and to a lesser extent, astrocytes. We also employed Ascl1- and Neurog2-null mice to investigate the influence of these bHLH transcription factors in the progression of gliogenesis in this brain region. By characterizing the development of oligodendrocytes in this important brain region, we will be poised to better understand how disruption of gliogenesis might contribute to hypothalamic disease states, such as obesity.
Mouse strains and tissue preparation
Timed-pregnant wildtype CD1, Neurog2 GFPKI [26, 27] and Ascl1 GFPKI [26–28] were bred to obtain embryonic tissue samples. For embryonic staging, female mice were plug checked in the morning and those with a positive vaginal plug were assigned embryonic day (E) 0.5. For postnatal staging, the day of birth was assigned as postnatal day (P) 0. Genotyping was confirmed by embryonic tissue sampling using PCR with Neurog2 GFPKI primers; mutant forward 5′-GGACATTCCCGGACACACAC-3′, mutant reverse 5′-GCATCACCTTCACCCTCTCC-3′, wildtype forward 5′-TAGACGCAGTGACTTCTGTGACCG-3′, wildtype reverse 5′-ACCTCCTCTTCCTCCTTCAACTCC-3′; and Ascl1 GFPKI primers; mutant forward 5′-AACTTTCCTCCGGGGCTCGTTTC-3′, mutant reverse 5′-TGGCTGTTGTAGTTGTACTCCAGC-3′, wildtype forward 5′-TCCAACGACTTGAACTCTATGG-3′, wildtype reverse 5′-CCAGGACTCAATACGCAGGG-3′. Animal protocols were approved by the University of Calgary Animal Care Committee and follow the Guidelines of the Canadian Council of Animal Care.
For sample preparation, gravid females were anaesthetized with isoflurane and immediately decapitated. Embryos were removed and embryonic brains were extracted for E15.5 and E17.5 time points while whole embryonic heads were taken for E11.5 and E13.5 time points. For mutant samples, which have been outcrossed onto a CD1 background, CD1 wildtype samples were used as controls, with the exception of Fig. 7e, h where heterozygous Ascl1 +/GFPKI animals were used as controls. For 5-Bromo-2′-deoxyuridine (BrdU) samples, 200 μl of 10 μg/μl BrdU was injected intraperitoneally into the pregnant dam. For neuronal birthdating studies, BrdU was injected at E11.5, E13.5 and E15.5 into the pregnant dam and resulting pups were sacrificed at P0. For proliferation studies, BrdU was injected at E13.5 and 30 min before decapitation of dam. Samples were fixed overnight with 4% paraformaldehyde in phosphate buffered saline (PBS), washed in PBS, then treated with 20% sucrose before being embedded in O.C.T. for cryosectioning.
Brain samples were cryosectioned at 10 μm with a selection sampling fraction of 1 for every 8 serial sections within our region of interest. Sections were treated with primary antibody overnight at 4 °C in 5% normal donkey or goat serum/PBS with 0.1% Tween-20 or Triton-X 100 followed by the appropriate fluorescently conjugated secondary antibody. Primary antibodies were as follows: Mouse anti-NeuN (Millipore; 1:400), Rat anti-BrdU (Cedar Lane; 1:300), Goat anti-Ki67 (Santa Cruz; 1:300), Rabbit anti-Ki67 (Abcam; 1:100), Goat anti-Sox9 (R&D systems; 1:40); Rabbit anti-Olig2 (Millipore; 1:500); Mouse anti-Olig2 (Millipore; 1:300), Goat anti-PdgfRα (R&D Systems; 1:150); Rat anti-SF-1 (graciously provided by Dr Taro Tachibana, Osaka City University JAPAN, 1:800); Rabbit anti-TTF-1 (alternatively Nkx2.1; Santa Cruz; 1:500), Goat anti-Sox10 (Santa Cruz, 1:500), Rabbit anti-pHH3 (Millipore; 1:500), Rabbit anti-Cyclin E (Santa Cruz; 1:200), Rabbit anti-Cyclin B1 (Santa Cruz; 1:200), Mouse anti-Cyclin D2 (ThermoFisher Scientific; 1:200), Rabbit anti-p57kip (Sigma; 1:200), Rabbit anti-Aldh1L1 (Abcam; 1:500), Rat anti-MBP (Millipore; 1:50), and Rabbit anti-Cleaved Caspase 3 (Abcam; 1:800). All appropriate secondary antibodies were Donkey or Goat anti-IgG and Alexa Fluor conjugated (ThermoFisher Scientific; 1:200–1:400). All samples were counterstained with Hoechst nuclear stain (ThermoFisher Scientific; 1:1000).
Quantification and statistical analysis
For cell number quantification, images were taken using a Ziess Axioplan 2 manual compound microscope with a Zeiss Axiocam HRc camera. Adobe Photoshop CS6 counting software was used to manually count individual and co-labeled cells. SF-1 staining-which marks the ventromedial hypothalamic nuclei-was used to denote the beginning and end of the tuberal hypothalamus across adjacent brain sections  and Nkx2.1 was used to verify the hypothalamic sulcus border at the dorsal edge of the tuberal hypothalamus. Cells were counted from 3 brain sections (descriptive counts) or 2 brain sections (mutant counts) in the rostral to mid tuberal hypothalamus for WT and mutant brains, respectively. Aldh1L1 cell counts were taken from the mid to caudal tuberal hypothalamus for control and mutant brains. Embryonic samples from more than one pregnant dam were used for each experimental group. Statistical differences between controls and mutants and between age time points were assessed using an ANOVA statistical test with Tukey post-hoc analysis or a Student’s t-test when applicable. Results are displayed as mean±standard deviation (SD).
Glial progenitors first appear after E13.5 in the tuberal hypothalamus
Next we asked when glioblasts first appeared in the tuberal hypothalamus by assaying Sox9 expression, a transcription factor required to specify a glial identity but that also labels neurogenic progenitors at the end of neurogenesis [35, 36]. At E11.5 and E13.5, Sox9 expression was mainly restricted to the ventricular zone (VZ) where multipotent neural progenitors are located, labeling the entire dorsal-ventral and rostral-caudal extent of the tuberal hypothalamic VZ (Additional file 1: Figure S2A and Fig. 1b). In contrast, by E15.5 and at E17.5, Sox9 expression was detected both in the VZ, representing a mix of neural and glial progenitors, and in the mantle zone (MZ; Fig. 1b), representing likely glial precursors, as the loss of ventricular contacts by dividing progenitors is a hallmark feature of glial precursors . We thus conclude that the first glial precursors appear in the tuberal hypothalamus between E13.5 and E15.5.
Olig2+ cells are restricted to a tight domain along the third ventricle in the tuberal hypothalamus
Olig2+ progenitors along the VZ of the tuberal hypothalamus are not actively dividing
As progenitor cells lining ventricles are well known to be mitotically active , we next determined whether Olig2+ cells lining the third ventricle were mitotically active. To label rapidly proliferating S-phase cells, we injected BrdU at E13.5 and sacrificed the animals 30 min later. Interestingly, very few Olig2+/BrdU+ double labeled cells were observed within the Olig2+ domain along the tuberal hypothalamic VZ, whereas many BrdU+ cells were detected in the VZ directly dorsal and ventral to the Olig2+ enriched domain (Fig. 2d). In addition, Olig2+ cells that had migrated into the MZ were BrdU+, consistent with a proliferative glioblast fate (Fig. 2d).
Changes in glial progenitor and precursor cell populations in the tuberal hypothalamus across development
Given that glial precursors migrate away from the VZ and into the MZ where they proliferate and mature, we next examined the number of progenitors expressing Sox9 and/or Olig2 in the MZ, observing all three possible populations of glioblasts, namely Sox9+/Olig2-, Sox9+/Olig2+, Sox9-/Olig2+ (Fig. 4c, e). We first examined Sox9+/Olig2+ cells, considered to be early-stage glioblasts and OPCs, and found a significant and dramatic increase in number of these cells from E13.5 to E15.5 and a further increase at E17.5: 76±43 cells at E13.5, 826±134 cells at E15.5, and 1159±188 cells at E17.5 (Fig. 4c). Moreover, we also quantified the relative proportion of the Sox9+/Olig2+ population within the total Sox9+ and Olig2+ glioblast populations in the MZ and found Sox9+/Olig2+ population likewise increased from E13.5 to E15.5 and then remained constant to E17.5: 24%±14% at E13.5, 72%±6% at E15.5, and 58%±6% at E17.5 (Fig. 4e). We next examined Sox9+/Olig2- cells in the MZ and found this population to be relative low and maintained from E13.5 to E15.5 with a significant increase at E17.5: 342±123 cells at E13.5, 206±cells at E15.5, and 711±138 cells at E17.5 (Fig. 4c). This was in contrast to the relative proportion of Sox9+/Olig2- glioblasts within the total Sox9+ and Olig2+ populations, which decreased significantly from E13.5 to E15.5 but remained constant to E17.5: 72%±18% at E13.5, 18%±4% at E15.5, and 36%±8% at E17.5 (Fig. 4e). Finally, we quantified the population of Sox9-/Olig2+ cells, which are likely maturing OPCs (see next section). Unlike in the VZ where no Sox9-/Olig2+ cells were detected from E13.5 to E17.5, within the MZ we identified a distinct population that significantly increased from E13.5 to E15.5 and remained constant thereafter to E17.5: 17±14 cells at E13.5, 111±32 cells at E15.5, and 138±44 cells at E17.5 (Fig. 4c). In contrast, the relative proportion of Sox9-/Olig2+ glioblasts remained constant across E13.5, E15.5 and E17.5: 4%±5% at E13.5, 10%±3% at E15.5, and 7%±2% at E17.5 (Fig. 4e). Taken together, these data demonstrate a major expansion in the MZ from E13.5 to E15.5 of the Sox9+/Olig2+ population, thought to be OPCs, as well as a pool of Sox9-/Olig2+ cells that may correspond to differentiating OPCs. We also observe a second wave of later expansion of the Sox9+/Olig2- population, thought to be early-stage glioblasts and/or astrocyte precursors between E15.5 and E17.5.
Oligodendrocyte progenitor and precursor cell populations in the tuberal hypothalamus
Maturing oligodendrocyte and astrocyte populations in the developing tuberal hypothalamus
Altered glial progenitor and precursor cell populations in Ascl1 and Neurog2 mutant embryos
To resolve whether the decrease in Sox9+ cells in the developing tuberal hypothalamus in Neurog2 GFPKI/GFPKI and Ascl1 GFPKI/GFPKI mutants affected oligodendrocyte development, we quantified changes in Olig2, a general marker for glioblasts and OPCs, in the MZ (Fig. 7c, d). In the MZ there was an almost 2-fold increase in Olig2+ cells in Ascl1 GFPKI/GFPKI mutants at E15.5 (197±24 cells) that was also observed at E17.5 (349±60 cells; Fig. 7d), relative to controls at E15.5 (102±19 cells) and E17.5 (212±47 cells). In contrast, no difference was observed in Olig2+ cells in Neurog2 GFPKI/GFPKI mutants relative to controls at E15.5 (93±4 cells) and E17.5 (178±14; Fig. 7d).
Given that a percentage of Olig2+ cells can become astrocytes, we asked if the increase in the Olig2+ population in Ascl1 GFPKI/GFPKI mutants was giving rise to an oligodendrocyte or astrocyte lineage. To do so, we performed immunohistochemistry with Sox10 (Fig. 7e, f) and showed a statistical increase in Sox10+ cells at both E15.5 (120±6 cells) and E17.5 (212±34 cells) in the Ascl1 GFPKI/GFPKI mutant relative to controls at E15.5 (98±9 cells) and at E17.5 (119±6 cells; Fig. 7f). Although an increase in Sox10+ cells in the Ascl1 GFPKI/GFPKI mutant (Fig. 7f) suggested an increase in the oligodendrocyte lineage, we also examined astrocytes in Ascl1 GFPKI/GFPKI mutants using Aldh1L1 to mark astrocytes. Co-labeling with Olig2 and Aldh1L1 demonstrated a decrease in the Aldh1L1+ astrocyte population in Ascl1 GFPKI/GFPKI mutants (145±12 cells) as compared to controls (203±10 cells; Fig. 7g). The decrease in astrocytes was found in the MZ just adjacent to the VZ where Olig2+ glioblasts are migrating outward (Fig. 7e, data not shown), thereby supporting a role for Ascl1 in directing oligodendrocyte versus astrocyte fates. It is interesting to note that these changes also coincided with the appearance of Sox10+ cells in the VZ in Ascl1 GFPKI/GFPKI mutants (Fig. 7h, arrows), which were also found to be Olig2+ (Fig. 7h). Together, these data suggest that Ascl1 but not Neurog2 plays a key role in late embryonic oligodendrogenesis in the developing tuberal hypothalamus.
Spatiotemporal progression of gliogenesis in the embryonic tuberal hypothalamus
We report that Olig2+ cells are restricted to a dorsal domain adjacent to the third ventricle near the hypothalamic sulcus of the tuberal hypothalamus, and propose that OPCs are formed and migrate out from this domain to establish the MZ glioblasts, which then proliferate to generate the expansion of oligodendroctyes. Previous studies have described similar OPC-producing domains in other CNS regions, as nicely reviewed by Nicolay et al. . Moreover, this Olig2+ domain demonstrates a reduced proliferative capacity at E13.5 in comparison to the surrounding ventricle progenitors that appear as a pseudo-stratified layer of Ki67+ and BrdU+ cells likely in various stages of an active cell cycle due to the appearance of interkinetic nuclear migration . Interestingly, we did observe a small population of Ki67+ cells within the Olig2+ domain but they were largely Olig2- in expression and were tightly localized to the ventricular wall and did not assume a pseudo-stratified organization. Here we propose two possible populations of progenitor cells might co-exist within this Olig2+ domain region, which may have distinctive purposes. Firstly, the Ki67+/Olig2- progenitor cells directly adjacent to the ventricle (Fig. 2e) may be in the process of slowing their cell cycle, inferred by their lack of BrdU accumulation but positive expression of Ki67, and ultimately becoming an alternative neural progenitor type. In fact, it was recently discovered that adult stem cells are derived from a subset of the rapidly dividing neural progenitor pool of cells that have slowed their cell cycle starting between E13.5 and E15.5 in order to elongate their cycling potential into adulthood . Coincidentally, overlapping this region of the Olig2+ domain in the tuberal hypothalamic VZ are also progenitors that later generate α-tanycytes, which are known to have adult stem cell properties [44, 45], raising the exciting notion that these Ki67+/Olig2- progenitor cells are being reserved for tanycytic adult neurogenesis.
Secondly, we also propose that the Ki67-/Olig2+ progenitor population at the VZ are slowing their cell cycle to become specified as glioblasts, and have paused their mitotic activity in anticipation of movement as they prepare to exit the VZ. Consistent with this idea, we observe accumulation of the cell cycle arrest marker, p57kip, in cells at the outer edge of the VZ that co-label with Olig2 (Fig. 3e). This notion would be consistent with our findings here that Olig2+ cells expand significantly out in the MZ after E13.5 and acquire a highly proliferative state in the mantle. Future studies are needed to fully understand the proliferative capacity of cells within this Olig2+ domain, although here we demonstrate that at least two distinct populations reside within this dorsal VZ domain.
Ascl1 influences gliogenesis in the tuberal hypothalamus
Here we uncovered a unique role for Ascl1 in gliogenesis in the tuberal hypothalamus. Both Neurog2- and Ascl1-null mutants showed a marked reduction in E17.5 Sox9+ cells, although only the Ascl1 single mutant showed an increase in the Olig2+ and Sox10+ cells at both at E15.5 and at E17.5 and a decrease in astrocytes at E17.5, suggesting that oligodendrocyte populations are increased in the Ascl1 mutant. Since Sox9 is co-expressed with Olig2 in early OPC populations, this increase in Olig2+ and Sox10+ cells and concomitant decrease in the Sox9+ is likely due to a precipitous increase in OPC maturation in the Ascl1 mutant background. These changes also coincide with the appearance of Sox10+ cells in the VZ in Ascl1 GFPKI/GFPKI mutants, which were also found to be Olig2+. This suggests that Ascl1 is required to repress precocious OPC development and oligodendrocyte lineage commitment of Olig2+ cells lining the ventricle [46–48]. This is further supported by the overlap of Ascl1 and Olig2 expression embryonically in progenitors lining the ventricle. However, it is important to note that given immature glia cells divide during their migratory routes, and the number of divisions determine the number of mature glia cells, it is also possible that in the absence of Ascl1 astrocytes do not divide to the same extent. Interestingly, these findings in the tuberal hypothalamus are in direct contrast to those in the cerebellum whereby the Ascl1 loss-of-function mutants were shown to have a reduction in Olig2+ cells and an increase in Sox9+ cells at E18.5 . Furthermore, a study by Parras et al. using Ascl1-null mice in the telencephalon demonstrate a decrease in PdgfRα+ OPCs during early gliogenesis at E13.5 that later resolved by E17.5. The OPC population that was restored late embryonically in their study was suggested to be due to either an increase in the rate of proliferation of OPCs or, more likely, an increase in specification of OPCs from uncommitted progenitors that reflect a compensatory mechanism for OPC specification in the absence of Ascl1 . Here we only examined the mid- (E15.5) to late- (E17.5) stages of embryonic gliogenesis, and thus, it is possible we missed an initial decrease in OPCs earlier (e.g., E13.5), making it possible that we misinterpreted what was ultimately simply overcompensation at later stages. However, given that we show very few PdgfRα OPCs in the developing tuberal hypothalamus at E13.5, we believe it unlikely that we missed an OPC phenotype earlier than E15.5. Moreover, in agreement with our findings here, reports in the spinal cord also using an Ascl1 knockout show an increase in Olig2+ cells at E17.5, which the authors attribute to aberrant proliferation as Ki67 and BrdU were also increased , and are further supported by a more recent report in the spinal cord showing an increase in Olig2+ and Sox10+ cells at E18.5 in grey matter oligodendrocytes . Taken together, our data are consistent with a role for Ascl1 in oligodendrogenesis, and suggests that whether Ascl1 is required or represses an OPC fate might be CNS region-specific.
Mechanistically, we propose that with Ascl1 eliminated, the competency of progenitor cells to maintain a proliferative state may be reduced since oscillatory Ascl1 cannot cross-repress the other bHLH factors that are required to maintain a true progenitor state. Indeed, Ascl1 and Neurog2 are both key components of a progenitor transcription factor oscillatory process whereby progenitors continue to divide instead of exiting the cell cycle  because key transcription factors are expressed for short periods to cross-repress other transcription factors in an oscillatory manner. A lineage becomes committed once a transcription factor stops oscillating and experiences sustained expression . Specifically, when considering the oscillatory activity of Hes1, Ascl1 and Olig2 in neural progenitor cells, Ascl1 oscillations are required to maintain a proliferating neural progenitor state, while sustained Ascl1 expression promotes a neuronal fate. Alternatively, a decrease in expression of Ascl1 with a sustained expression of Olig2 or Hes1 promotes an oligodendrocyte and astrocyte fate, respectively [47, 48]. Thus, in the tuberal hypothalamus, we postulate that the elimination of Ascl1 in Olig2+ progenitor cells restricts the neuronal fate potential of these cycling cells, thereby inducing these progenitors to initiate precocious OPC development.
In summary, this study provides insight into the spatiotemporal timing of gliogenesis in the tuberal hypothalamus revealing that peak production of developing oligodendrocytes occurs between E15.5 and E17.5. We also show a role for Ascl1 in this region that is more consistent with Ascl1 requirements in the developing spinal cord than in the cortex and cerebellum.
Basic helix-loop-helix family
Central nervous system
Green fluorescent protein
Oligodendrocyte precursor cell
Phosphate buffered saline
Steroidogenic factor 1
We would like to thank Dr Taro Tachibana from Osaka City University for generously providing the anti-SF-1 antibodies. This work was supported by funding from the Canadian Institute for Health Research (DMK, QP, JR), The Hotchkiss Brain Institute (CM), and Alberta-Innovates Health Solutions (CM).
DK (CIHR; MOP-275053), QP (NSERC; PGPIN-2014-05791), CMM (AI-HS MD/PhD Fellowship), JR (CIHR; MEF-140891).
Availability of data and materials
Any datasets used and/or analyzed during the current study that are not included in this published article [and its supplementary information files], are available from the corresponding author on reasonable request.
CM, DK designed experiments and performed data analysis. CM, JR, DK wrote manuscript. QP, CS edited manuscript. CM, JR, HT, SA obtained samples, performed imaging and cell counting. GW, CS provided mutant mouse lines. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Animal protocols were approved by the University of Calgary Animal Care Committee and follow the Guidelines of the Canadian Council of Animal Care.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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