Gata2 is down-regulated by NGF in PC12 cells and influences their neuronal differentiation
Our interest in Gata2 arose from findings that NGF-promoted neuronal differentiation of PC12 pheochromocytoma cells is accompanied by loss of detectable Gata2 transcripts (Greene and Angelastro, 2008). Western immunoblotting confirmed that Gata2 protein levels are undetectable by 4 days of NGF exposure (). To assess Gata2’s role in this system, we over-expressed it either transiently or constitutively and monitored NGF-dependent neuronal differentiation. This reduced the rate of neurite production by 2-fold () and markedly decreased neurite length (). In contrast, constitutive Gata2 expression did not affect induction by NGF of peripherin or neurofilament light protein (). These findings are consistent with the general themes that Gata2 is regulated during neuronal differentiation and may play functionally important roles in this process.
Gata2 is a NGF-responsive gene and suppresses neurite outgrowth in PC12 cells
GATA2 protein is transiently expressed and confined to a periventricular zone during mesencephalic embryonic development
To extend our studies to brain development, we used immunohistofluorescence staining of Gata2 protein. Consistent with prior work (Bell et al., 1999
; Zhou et al., 2000
; Craven et al., 2004
; Herberth et al., 2005
; Kala et al., 2009
), we observed expression in embryonic rat hindbrain and midbrain. We subsequently focused on the dorsal mesencephalon and developing superior colliculus (SC) where expression was robust as well as transient, and in which the developmental role of Gata2 is unclear.
The earliest detectable mesencephalic Gata2-expressing cells appear at e12 in the ventrolateral part of this structure (). At this and all other times between e12 and e18, Gata2 expression is limited to a domain lying between the ventricular zone of neuroprogenitor cells and the β-III-tubulin-expressing neuronal layers neurons (). We will refer to this domain as the “intermediate zone” or IZ. By e14, Gata2 expression spreads to the entirety of the IZ in the ventral mesencephalon (). Gata2 is not detected in the dorsal mesencephalon at these times, indicating that it does not play a role in development of early born SC neurons that are present at e12–e14. This is consistent with a report that expression of Gata2 transcripts is absent in the mouse dorsal mesencephalon as early as e8.5 (equivalent to about e10.5 in rat) (Nardelli et al., 1999
Dynamics of Gata2 expression during embryonic rat midbrain development
After e14, Gata2 expression spreads dorsally and at e15–17 lies in the IZ in a diffuse pattern around the full mesencephalic circumference (), except for a medial gap in the ventral midbrain (). The latter contains cells that are positive for tyrosine hydroxylase and negative for Gata2 and that appear to be immature dopaminergic neuronal cells in process of migration (). By e18, Gata2 is absent from the ventral midbrain but persists in a thin layer immediately beneath the differentiating SC in the dorsal mesencephalon (). The IZ lying between the ventricle and SC is nearly gone by e21 and there is no detectable Gata2 expression there by this time (). By e17 and beyond, a second wave of strong Gata2 expression appears in subsets of mature neurons within the ventral midbrain including the medial geniculate nucleus and the olivary nuclei ().
Gata2 is expressed in the e15 rat midbrain but excluded from catecholaminergic progenitors as well as SC neuronal and VZ layers
Gata2 positive cells in the embryonic dorsal mesencephalon appear to be migrating immature, post-mitotic neuronal precursors
We further characterized Gata2+ cells in the developing dorsal mesencephalon to better understand their identity. At all times, Gata2 was expressed by only a subpopulation in the IZ. For example, at e15 they comprised 29±4% of the cells there, and this proportion appeared to hold through at least e17. Immunostaining for phospho-histone H3 at e15 revealed a layer of mitotic cells along the ventricle from which Gata2 was completely excluded, indicating that the Gata2+ population is post-mitotic (). Co-staining at this age confirmed that the highest-expressing Gata2+ cells lack β-III-tubulin (TuJ1)+ expression in the IZ (; ). However, a small number of neurons that express low levels of Gata2 and TuJ1 were detected near the IZ/SC boundary, suggesting that these may be in transition with Gata2 expression waning while β-III-tubulin expression is induced. These findings and distribution of Gata2+ cells suggest that they are immature, postmitotic neuronal precursors migrating to the differentiating SC.
Gata2-expressing cells in the e15 developing SC are postmitotic and immature
The immature nature of Gata2+ cells was supported by comparison with the expression patterns of Msi1 and Sox2, markers for neuroprogenitor cells and immature neuronal precursors (Kaneko et al., 2000
; Bylund et al., 2003
; Graham et al., 2003
; Sakakibira et al., 2002
). Because available antisera precluded co-staining, we examined contiguous serial sections. Both Msi1 and Sox2 were present with Gata2 in the VZ at e15, as well as in the IZ (). While both Msi1 and Gata2 extended all the way to the TUJ1-positive SC, Sox2 expression ended several cell diameters short of it. Within the central area of the IZ, essentially all cells were Msi1+ and the vast majority were Sox2+. This indicates that most/all Gata2+ cells there also express Sox2 and Msi1.
Pax homeodomain transcription factors confer region-specific patterning in the central nervous system and are crucial for establishing area boundaries (reviewed by Nakamura, 2001
). Pax3 and Pax7 are expressed in dorsal midbrain, specifically in the developing SC, initially together within neural progenitors and early post-mitotic neuronal precursors (Thompson et al., 2008
). In late embryogenesis, their patterns diverge and by birth Pax3 persists in a periventricular arrangement while Pax7 is low around the ventricle and highest in mature SC neurons (Thompson et al., 2004
; Thompson et al., 2008
; Fedtsova et al., 2008
). We observed that in e15 dorsal midbrain, Pax3 and Pax7 expression extends from the VZ into the IZ and ends several cell diameters beneath the neuronal layers (). Thus, Pax3 and Pax7 are expressed in the IZ along with Gata2+ except in the most superficial part of this zone. Of all Gata2+ cells in the IZ, counts revealed that 50±6% (n=3) express Pax3, and that within the part of the IZ where these markers overlap, 76±6% are also Pax3 positive. Conversely, 13±2% (n=3) of Pax3+ cells in the IZ co-express Gata2. With the patterns of Pax3 and Pax7 expression being so similar at e15, the coincidence of Gata2 and Pax7 appears to be very similar. Another homeodomain transcription factor Brn3a (Pou4f1) is expressed throughout the SC neuronal layers at e15 and e17, but not in the IZ and does not co-express with Gata2 (data not shown).
Gata2-expressing cells are early neuron precursors
The bHLH proneural gene Ascl1 (Mash1) is expressed in developing mesencephalon (Gradwohl et al., 1996
; Kala et al., 2009
; Osório et al., 2010
) and is partially co-expressed with Gata2 in the IC (Kala et al., 2009
). We also observed co-expression of Ascl1 and Gata2 in the dorsal mesencephalon. At e15, dorsal mesencephalic Ascl1 is expressed both in the VZ and IZ and overlaps with Gata2 in the upper regions of the IZ (). Counts revealed that where both are present, 46±9% (n=3) of Gata2+ cells co-express Ascl1+ and that 68±1% (n=3) of Mash+ cells are also Gata2+. Taken together, co-expression of Gata2 in the IZ with Sox2, Msi1, Pax3/7 and Ascl1, but not phospho-histone H3 or β-III tubulin supports the idea that Gata2+ cells are post-mitotic neuron precursors that are migrating to the SC. Once in the SC, they lose Gata2 expression and complete their differentiation into mature neurons.
The final marker evaluated was the early neuronal migrating cell marker doublecortin (Dcx). In the e15 dorsal mesencephalon, Dcx expression was essentially indistinguishable from that of β-III-tubulin, and as such, was excluded from the Gata2+ population ().
Gata2 knockdown in dorsal mesencephalon interferes with cell migration
Because our findings indicate that Gata2 is selectively expressed in immature post-mitotic neuronal precursors in the dorsal mesencephalon, we next addressed its functional role there. To achieve this, we designed and characterized independent siRNA constructs targeted against rodent Gata2, but not human Gata2, and expressed them as shRNAs in a vector that also expresses eGFP. Two of these (siGata2-1 and siGata2-2) show effective knockdown of over-expressed rat Gata2 in HEK293 cells, with siGata2-1 being the most robust (). In contrast, they did not knock down human Gata2 ( and data not shown).
Transient Gata2 expression is required for normal migration of early neuron precursors in the developing SC
Because Gata2 is induced in neuronal precursors as they exit the cell cycle and migrate towards the SC, we used in utero electroporation to test its role in this critical period of dorsal mesencephalic development. Plasmids were injected into the 3rd ventricle at e16 and electrodes were oriented so that DNA was selectively delivered to the dorsal mesencephalon. In this approach, plasmids are electroporated mainly into the neuroprogenitor cells of the VZ which do not express Gata2. These continue to divide or radially migrate out from the VZ where the expressed Gata2 siRNA should diminish synthesis of endogenous Gata2. For most experiments, animals were sacrificed 5 days after electroporation and their brains processed by immunofluorescence for expression of eGFP and other markers.
As evidence that our shRNAs were effective, counts revealed an approximate 75% decrease in proportion of electroporated cells in the IZ that were positive for endogenous Gata2 after receiving Gata2 shRNA (shControl, 33% Gata2+ vs siGata2-1, 8.3% Gata2+). Even in cells with detectable Gata2 after electroporation with Gata2 shRNA, the signal was markedly reduced (data not shown).
Visualization of eGFP at e21 (5 days post-electroporation) revealed labeled cells at all levels of the dorsal midbrain for both knockdown and control constructs (). In the chick tectum, deep layer neurons are generated first, followed by a second wave that produces the outermost retinorecipient layers and a third and final wave that generates the interstitial lamina (LaVail and Cowan, 1971
; Gray and Sanes, 1991
; Sugiyama and Nakamura, 2003
). It is unknown whether such neuron generation patterns are similar in rat SC. However, in brains electroporated with control constructs and stained for the neuronal marker NeuN, essentially all eGFP+/NeuN+ cells were confined to the middle and outermost laminae of the SC. Conversely, 75±3% (n=3 brains, 15–35 cells each) of eGFP+ cells in the brains were NeuN+ in these laminae whereas <5% of eGFP+ cells in the lower layers of the SC were NeuN+. These findings thus indicate that cells electroporated at e16 correspond to waves 2 and 3 of chick tectal neurogenesis.
Initial observations of knockdown cells indicated impaired migration (). To facilitate comparison between migration of control and knockdown cells, we partitioned coronal brain sections along the radial axis into three sectors. Sector A included the portion of dorsal mesencephalon 30% of the distance from the aqueduct to the pial surface and contained the VZ, a narrow periventricular zone and the deepest layers of the SC (). Most electroporated cells in the VZ had a morphology and possessed markers (nestin and BLBP) identifying them as radial glia (; and data not shown). These are Gata2- and appear unaffected by Gata2 shRNAs and were therefore excluded from our subsequent quantifications. Most cells in sector A outside the VZ had the appearance of migrating neuronal precursors with a bi- or unipolar morphology (data not shown). Gata2 knockdown caused a robust effect on migration so that a large number of electroporated cells appeared to be arrested in the periventricular area of sector A where endogenous Gata2 is expressed during development (). Quantification indicated that while about 35–45% of control electroporated cells were in sector A, 60–75% of knockdown cells were located there (). The difference was greater with siGata2-1 than for siGata2-2 shRNA which is consistent with their relative efficacy for knocking down Gata2 ().
Sector B included the portion of the dorsal mesencephalon between 31–60% of the distance to the pial surface and contains the central layers of the SC. Sector C comprised the remaining 61–100% of the distance to the pial surface and contains the retinorecipient layers of the SC. While Gata2 knockdown did not significantly affect the proportion of labeled cells in sector B, it markedly reduced (by about 2/3) the proportion of electroporated cells that reached Sector C ().
The loss of cells migrating into the outermost layers of the SC promoted by knockdown of Gata2 could be due to several potential causes, including cell death. Gata2 expression is required for normal survival and expansion of hematopoietic cells (Tsai et al., 1994
; Rodrigues et al., 2005). Additionally, the GATA factor Gata3 is downstream of Gata2 in sympathetic neuron differentiation and loss of Gata3 in developing (Tsarovina et al., 2004
) and in mature sympathetic neurons (Tsarovina et al., 2010) negatively impacts survival. To assess whether the observed effect of Gata2 knockdown in the superior colliculus might be due to excessive cell death, we electroporated at e16 and sacrificed the animals at e18 for a 2 day time-point. Immunostaining of siCon control, siGata2-1, or siGata2-2 electroporated brains were performed using the markers PH2A.X and cleaved caspase 3, which have been shown to correlate well with apoptotic cell death (Holubec et al., 2005
). A small number of PH2A.X+ and cleaved caspase 3+ cells were observed in each 50 μm SC section, which indicates that the staining was successful. However less than 1% of the electroporated cells stained for these markers and no difference was observed for such staining of cells electroporated with either the siGata2-1/2 or control constructs (data not shown). In addition, if the absence of GFP+ cells in the outer SC layers in Gata2 knockdown brains were due to cell death, substantially fewer total labeled cells would be observed in these brains compared to siCon controls. However, the distribution patterns of cells in siRNA electroporated brains () are consistent with redistribution of Gata2 knockdown cells, rather than cell loss. Taken together, these findings support the conclusion that cell death does not account for the absence of Gata2 knockdown cells in the SC outer laminae and suggest the alternative that Gata2 is required for proper migration of SC neuronal precursor cells to these layers.
As a control for possible off-target shRNA effects, we performed rescue experiments in which a plasmid expressing human Gata2 was co-electroporated with siGata2-1 or siGata2-2 shRNA. Pairwise comparison of human (hGata2) and rat Gata2 (rGata2) DNA sequences predicts that siGata2-1 and siGata2-2 should knockdown the rat, but not human form, although the two are 98% homologous at the protein level. As discussed below, expression of hGata2 itself did not perturb migration of cells into the SC.
In contrast to the effect of electroporating cells with either siGata2-1 or siGata2-2 alone, there was no evident accumulation of cells in sector A after electroporation with siGata2-1/hGata2 or siGata2-2/hGata2 ( and data not shown). Moreover, for brains matched for electroporation efficiency, cells receiving siGata2-1/hGata2 or siGata2-2/hGata2 reached sector B/C in numbers comparable to that for control vector and that were approximately 3-fold higher compared with brains receiving only siGata2-1 or siGata2-2 (). Co-immunostaining indicated that 65% of the electroporated cells that reached sectors B/C in the rescue study were NeuN+ (), a level similar to that for brains electroporated with control constructs (75%).
Human Gata2 (hGata2) rescues migration and differentiation of siGata2 knockdown cells
Effects of exogenous Gata2 expression in the dorsal mesencephalon
Because Gata2 knockdown arrests migration of SC precursor cells, we next assessed the potential effects of electroporation with Gata2 expression constructs. Migration of cells electroporated with rat or human Gata2-FLAG pCMS-EGFP at e16 and harvested at e21 showed no significant change in migration relative to control electroporated cells (data not shown). Expression of exogenous Gata2 was verified by immunofluorescence against Gata2 protein and the FLAG epitope, and robust expression was found in many cells at e21 ( and data not shown). However, the expression pattern of exogenous Gata2-FLAG protein in the SC was somewhat unexpected in that not all GFP+ cells were positive for Gata2/FLAG (). Expression was detected only in a subset of electroporated cells in the deep layers (Sector A) of the SC and no exogenously expressed protein was found in electroporated cells within Sectors B or C. Comparable results were found with both rat Gata2 and human Gata2. These observations suggest that expression of Gata2 protein may be subject to differential post-translational mechanisms in the SC and that translation or stability of Gata2 protein may be diminished in specific neuronal populations. This mechanism could explain in part why endogenous Gata2 protein levels dramatically fall when neuronal cells enter the SC and the apparent lack of effect of exogenous Gata2 on neuronal migration. The capacity of hGata2 to fully rescue from knockdown of endogenous Gata2 further indicates that sufficient levels of the human protein are expressed in the IZ during the critical period prior to e21 to promote proper migration and differentiation.
Gata2 knockdown in the dorsal mesencephalon interferes with differentiation of neuronal precursors
The above findings indicate that Gata2 plays an important role in permitting migration of immature neuronal precursors from the IZ into the SC. To discern whether loss of Gata2 limits migration alone or whether this is in addition to or secondary to impingement on postmitotic precursor differentiation, we performed additional marker analyses of the electroporated cells. One possibility was that Gata2 loss causes precocious differentiation of precursors into mature neurons which in turn impairs migration. However, both knockdown and control electroporated cells in Sector A lacked expression of neuronal markers β-III-tubulin or NeuN ().
Gata2 is required for differentiation of SC neural precursors
The appearance of endogenous Gata2 protein in postmitotic neuronal precursors raises the possibility that it is necessary for cell cycle exit, as reported in the chick spinal cord (El-Wakil et al., 2006
), and that its knockdown may induce cells outside the VZ to remain in or re-enter the cell cycle. However, Ki67 staining indicated that Gata2 loss did not block proliferation within the VZ or stimulate mitosis outside the VZ (data not shown and ). Moreover, cells in Sector A outside the VZ that were electroporated with Gata2 shRNAs were negative for the radial glial marker BLBP ().
Because Gata2 is highly expressed in immature neuronal precursors, we reasoned that it might play a role in their differentiation into mature neurons and that Gata2 loss would result in retention of co-expressed markers such as Ascl1 and Pax3/7 that are present before its induction and that are extinguished in SC neurons. At e21, endogenous Ascl1 is sparsely expressed within, and is limited to, a 4–5 cell deep zone in Sector A at the junction between the VZ and the deepest neuronal layers of the SC. This largely corresponds with the area of greatest migration arrest caused by Gata2 knockdown. For cells electroporated with control or Gata2 shRNAs, all Ascl1 expression was within this zone. Strikingly, Gata2 knockdown increased the proportion of electroporated cells in this region that were Ascl1+ by 2–3-fold ().
The endogenous expression pattern of Pax3 at e21 is quite different from Ascl1 and includes nearly all cells within the VZ and within Sector A below the neuronal layers of the SC. In addition, all neuronal layers in the SC (i.e., within Sectors A–C) contain a sparse population of Pax3 cells. Examination of electroporated cells for Pax3 revealed no expression within Sectors B and C, irrespective of whether they received control or siGata2 plasmids (data not shown). However, Pax3 was present in a subpopulation of electroporated cells within the neuronal layers of Sector A and the proportion of such cells was 2.5-fold higher for those receiving siGata2 compared with those receiving control plasmid (). We also examined Pax7 which showed strong expression in a subpopulation of neurons within Sectors B and C, but was absent from Sector A. In contrast to Pax3, Pax7 was absent from cells in Sector A electroporated with either control or Gata2 shRNA plasmids.
We additionally assessed Sox2 and Msi1 which are co-expressed at e15 with Gata2. At e21 Sox2 is present in all VZ cells and in isolated cells distributed throughout the SC. The majority of both knockdown and control cells present in Sector A were Sox2+. In contrast, by e21 Msi1 expression was restricted to the VZ and absent from Gata2 knockdown or control cells (data not shown).
To determine whether Gata2 knockdown cells persist in an immature state, animals electroporated at e16 with siGata2-1 were allowed to develop until p5 when SC neurogenesis is nearly complete. Similarly to e21, the Gata2 knockdown cells in the deep layers of the SC at p5 were still positive for Pax3 and Sox2 (), and exhibited an immature radially-oriented unipolar and bipolar morphology. In contrast, Ascl1 and Msi1 were undetectable. Taken together, these findings support the idea that Gata2 plays a critical role in differentiation of post-mitotic neuronal precursor cells in the dorsal mesencephalon and that its loss arrests them at an early stage of maturation characterized by retention of Pax3 and Sox2.
Pax3 and Sox2 expression are retained in Gata2 knockdown cells postnatally