Mash1 and Ngn2 partially overlap in the ventricular zone adjacent to dI3, dI4, and dI5 neuronal populations
Mash1 and Ngn2 are present in the ventricular zone of the neural tube at E10.5. Mash1 is in cells adjacent to where dI3, dI4 and dI5 neurons form and in a smaller ventral domain (Gowan et al., 2001
; Gross et al., 2002
; Müller et al., 2002
). In contrast, Ngn2 is present in the ventral and dorsal neural tube, and in the dorsal domain it spans the region where dI2-dI5 are formed (). As a first step in examining the roles of Mash1 and Ngn2 in dorsal neuron specification, we examined their co-localization in detail at E10.5 using previously characterized rabbit polyclonal antibodies against Mash1 (Horton et al., 1999
) and mouse monoclonal antibodies against Ngn2 (Lo et al., 2002
). There are many cells where Mash1 and Ngn2 are co-localized, and these are enriched but not restricted to the more lateral ventricular zone (, arrows). Thus, contrary to the distinct cross-inhibited domains that were observed between Math1, Ngn1, and Mash1, the other bHLH factors present at this time (Gowan et al., 2001
), Ngn2 overlaps with Mash1 in a sub-population of cells in the dorsal neural tube.
Mash1 is present throughout the mediolateral extent of the ventricular zone whereas Ngn2 is enriched more laterally (). This spatial pattern suggests these two factors are acting at different times during neuronal development since cells move laterally out of the ventricular zone when they exit the cell cycle and initiate a program of neuronal differentiation. To characterize Mash1 and Ngn2 relative to cell proliferation, we used BrdU incorporation to detect cells in S-phase. In E10.5 embryos exposed to BrdU for 1h before analysis, a subset of Mash1+ cells were detected that incorporate BrdU (, arrows). In contrast, Ngn2+ cells did not score positive for BrdU incorporation (). These results suggest that Mash1 is present at an earlier stage than Ngn2 during neuronal differentiation, and that temporal regulation of these factors may be important for their activities. Alternatively, Mash1 and Ngn2 could be revealing a code for dorsal interneuron specification such that ventricular zone cells containing each bHLH singly, or in combination, give rise to a distinct neuronal population. In this study, we used both loss-of-function and gain-of-function experiments to address the role of Mash1 and Ngn2 in specification of dorsal neuronal sub-types dI2-dI5.
Mash1 is required for the formation of dI3 and dI5 neuronal populations
Mash1 null embryos were analyzed for alterations in the number of dI2-dI6 neurons using multi-label immunofluorescence at E10.5. The neurons are defined by the presence of HD transcription factors, their position in the dorsoventral axis, and a birthdate prior to E11.5 (). Although Mash1 is in ventricular zone cells adjacent to dI3, dI4, and dI5 neurons, in the Mash1 null there is a striking loss of only two of these populations. There is a 70% loss of dI3 neurons (Isl1) and a complete loss of dI5 neurons (Lmx1b) (, compare E,F,I,J). In contrast, there is a dramatic increase in dI2 (Lhx1/5;Brn3a) and dI4/6 (Pax2) neurons, which appear to fill in at the location of the missing dI3 and dI5 neurons (, compare A,B,I,J). Although in the Mash1 null dI4 and dI6 are not distinguishable, overexpression and lineage analysis (see below) plus the lack of expansion of the ventral ngn1 domain, which marks dI6 progenitors (), suggest the increase in Pax2 is due to an increase in dI4 neurons. These results demonstrate the requirement for Mash1 in the formation of both dI3 and dI5 neurons, and the repression of dI2 and dI4/6 neurons.
Homeodomain (HD) factors used to define dI1-dI6 dorsal interneuron populations at E10.5*
Mash1 is required for dI3 and dI5 neuron formation while Ngn2 represses formation of these populations
Mash1 inhibits Ngn1 but not Ngn2 expression in the dorsal neural tube
To corroborate the conclusions from the Mash1 loss-of-function analysis, over-expression of Mash1 in the chick neural tube was used to test whether Mash1 is sufficient to promote dI3 and dI5 neuronal fates. A Mash1 expression plasmid (pMiWIII-Mash1) was electroporated into chicken embryos at HH13-14 and its effect on dorsal neuronal markers was analyzed 24 hours later. Excess levels of Mash1 increased the number of dI3 (Isl1) and dI5 (Lmx1b) cells as compared to the control side (, right panel of each pair is the injected side). Furthermore, excess Mash1 decreased the number of dI2 (Lhx1/5;Brn3a) and dI4 (Pax2) neurons (). These results are opposite of that seen in the Mash1 null, and demonstrate that Mash1 is sufficient for the generation of dI3 and dI5 neurons. Interestingly, the increase observed for each marker does not generally extend over the whole dorsoventral length of the neural tube, but rather it is localized around the area of its normal expression domain. This suggests that regional differences along the dorsoventral axis modulate the activity of Mash1, and demonstrate the context dependent nature of its specification function.
Excess Mash1 promotes dI3 and dI5 populations while excess Ngn2 represses them in the chick neural tube
Ngn2 acts to limit Mash1 activity in dI3 and dI5 neuron formation
The role of Ngn2 in specification of dorsal neuronal populations was also examined to test the hypothesis that Mash1 and Ngn2 provide a combinatorial code for dorsal neuron identity. In E10.5 embryos null for Ngn2, all neuronal populations, dI1-dI6, were generated () demonstrating that Ngn2 is not required for any specific dorsal cell-type. However, the composition of neurons that formed was altered with a subtle increase in the number of dI3 (Isl1) and dI5 (Lmx1b) neurons relative to wild type embryos (), and no significant changes in dI2 (Lhx1/5;Brn3a) and dI4/6 (Pax2) populations were detected (). Excess levels of Ngn2 in the chick neural tube resulted in a complementary phenotype to the Mash1 experiments in that dI3 (Isl1) and dI5 (Lmx1b) were dramatically decreased but dI4 (Pax2) was slightly increased relative to the non-injected side (, see graph for cell counts). Together these results demonstrate that Ngn2, although not required for any individual neuronal sub-type, limits the generation of dI3 and dI5 neurons, two populations that require Mash1 activity.
Mash1 is epistatic to Ngn2 in the formation of dI3 and dI5 neurons
The single mutants described above demonstrate that Mash1 and Ngn2 have opposite effects on the number of dI3 and dI5 neurons that form. Furthermore, the expression pattern suggests that Ngn2 may function downstream of Mash1 rather than setting up a combinatorial code for neuronal sub-type specification. If this interpretation of temporal expression is correct, the prediction is that the loss of both Mash1 and Ngn2 should phenocopy the Mash1 null. In support of this hypothesis, embryos null for both Mash1 and Ngn2 have a 70% loss of dI3 and complete loss of dI5 neurons, just as seen in the single Mash1 null (). Furthermore, the increase in dI2 neurons detected in the Mash1 null is also seen in the double mutant (). These results are consistent with Mash1 functioning upstream of Ngn2 in these populations.
Surprisingly, embryos null for both Mash1 and Ngn2 have an apparent loss of dI4 neurons (), a phenotype not predicted from the single mutants. Although dI4 and dI6 cannot be distinguished in the absence of dI5, the position of the Pax2-positive cells in the Mash1/Ngn2 double knockout strongly suggests a complete loss of dI4 neurons, and possibly dI6 neurons as well (). This loss of dI4 in the double knockout is in contrast to the single knockouts where there was no indication that either Mash1 or Ngn2 are required for dI4 generation. In fact, dI4/6 cells are significantly increased in the Mash1 null (). Thus, there is an apparent redundant function for Mash1 and Ngn2 in the generation of dI4 neurons.
Mash1-positive cells give rise primarily to dI3 and dI5, but not dI4 neurons
Given the presence of Mash1 in the ventricular zone throughout the dorsoventral domain adjacent to dI3, dI4, and dI5 neurons, it was surprising that dI4 neurons increased in the Mash1
knockout while dI3 and dI5 were lost. This finding suggests either Mash1+ cells become dI4 neurons but do not require Mash1, or that there are distinct low- or non-Mash1+ cells in the ventricular zone that give rise to the dI4 neurons. We used recombination based lineage tracing in vivo to distinguish between these two possibilities. A transgenic mouse was utilized (M1-GIC
) that expresses both GFP and Cre in the Mash1
expression pattern from a bacterial artificial chromosome containing 300 kb of genomic sequence surrounding the Mash1
protein coding region (). By crossing the M1-GIC
mouse line with a Cre reporter line R26R-YFP
, any cell that has expressed the transgene will be permanently labeled with YFP (Srinivas et al., 2001
Embryos at E11.5 were examined by triple-label immunofluorescence to determine the fate of Mash1+ cells in the dorsal neural tube. An antibody to GFP detects GFP and YFP simultaneously, and thus, these cells are referred to as GFP/YFP-positive cells. GFP/YFP-positive cells in the marginal zone were counted and scored for co-labeling with markers of dI2-dI6 neurons (, ). The majority of GFP/YFP+ cells become dI5 (Lmx1b, 73%) and dI3 (Isl1, 17%), rarely, dI2 (Lhx1/5;Brn3a, 2%) and no dI6 (Lhx1/5, 0%), consistent with the requirement for Mash1 specifically in dI3 and dI5. A notable percentage of GFP/YFP-expressing cells co-label with dI4 (Lhx1/5, 8%) suggesting a Mash1+ cell can become a dI4 neuron. However, this only represents a small percentage of the dI4 cells generated (~3%) and when identified, these co-labeled cells border the dI3 and dI5 domains (, arrow). We estimate that Cre induced recombination of the reporter gene in the M1-GIC embryos is approximately 50% since essentially all dI5 neurons require Mash1, but we detect only 52% of the dI5 neurons co-labeled with GFP/YFP (44 of 85 total Lmx1b cells). Furthermore, since we detect 25% of the dI3 neurons co-labeled with GFP/YFP (9 of 35 total Isl1 cells), using the efficiency factor we predict ~50% of dI3 neurons are derived from Mash1+ cells, consistent with the partial loss of dI3 detected in the Mash1 null (). Taken together, these results suggest that the function of Mash1 in the generation of dI3 and dI5 neurons is cell-autonomous, and demonstrate that dI2, the majority of dI4, and dI6 neurons develop from cells that contain Mash1 at levels not detected using this transgenic reporter mouse line. Indeed, heterogeneity in endogenous Mash1 levels is detected by immunofluorescence where ventricular zone cells adjacent to dI3 and dI5 have Mash1 at higher levels than cells adjacent to dI4 ().
Mash1 cells primarily become dI3 and dI5 neurons
Distribution of Total GFP/YFP Expressing Marginal Zone Cells in M1-GIC E11.5 Dorsal Neural Tube
Mash1 null cells are stalled in the ventricular zone at E11.5 and do not appear to trans-fate to dI4 neurons
Analysis of the Mash1 knockout revealed the loss of dI3 and dI5 with an increase in dI2 and dI4/6 that appeared to complement the loss (). To determine if the cells that would have become dI3 and dI5 switch their fate to dI2/dI4 in the absence of Mash1, we examined these neuronal populations in M1-GIC;R26R-YFP;Mash1−/−
embryos at E11.5. The first phenotype noted was that the GFP/YFP level is much higher in the mutant as compared to wild-type (see ). This difference in level reflects the negative autoregulation at the Mash1
locus that has previously been reported (Casarosa et al., 1999
; Horton et al., 1999
; Meredith and Johnson, 2000
). The Mash1 null sections showing GFP/YFP () were imaged at much lower GAIN than similar sections from embryos wild-type for Mash1 () (see for images with matched GAIN).
Other than negative autoregulation, the most dramatic phenotype detected using these mouse strains is that the cells that should have expressed Mash1 (GFP/YFP) appear stalled in the ventricular zone with only a few cells detected in the marginal zone (). These stalled cells aberrantly located in the ventricular zone have at least partially initiated a differentiation program; they express Lbx1 and Tuj1, markers normally restricted to the marginal zone, and they rarely incorporate BrdU, demonstrating that many have exited the cell-cycle (). TUNEL labeling shows no detectable increase in cell death in the neural tube at E10.5 and E11.5 (data not shown). Taken together, the precursors to dI3 and dI5 are not leaving the ventricular zone in the Mash1 null at E10.5/E11.5, and do not appear to significantly contribute to the increase in dI2 and dI4 since there was no increase in the proportion of GFP/YFP cells co-labeled with dI2 and dI4 markers (). Thus, the ectopic dI2 and dI4/6 neurons in the Mash1 null at E10.5 cannot be accounted for by a switch in fate of dI3 and dI5 precursor cells. Furthermore, cells with undetectable levels of Mash1 must give rise to dI2 and dI4/6 neurons, and the number of these cells increase in the Mash1 knockout.
Supplemental Figure 2
Expanded ventricular zone and premature differentiation in the Mash1 null neural tube
Mash1 and Ngn2 levels are independent of each other
Previously it was demonstrated that cross-inhibition between the three bHLH family members, Mash1, Math1, and Ngn1, was used to control cell number and cell-type formed (Gowan et al., 2001
; Scardigli et al., 2001
). To determine whether this type of regulation is occurring between Mash1 and Ngn2, we examined the levels of Ngn2 in the Mash1 null at E10.5 and vice versa. We observed no significant change in the number of Ngn2+ cells in the dorsal neural tube of Mash1 nulls () and no significant change in the number of Mash1+ cells in Ngn2 nulls (). These results suggest that Mash1 and Ngn2 do not use cross-inhibition as a mechanism to control the number of dorsal neurons formed, consistent with the co-expression seen with these two bHLH factors. Although difficult to quantify, there may be increased levels of Mash1 in individual cells in the most dorsal region in the Ngn2 null and vice versa in the Mash1 null (, compare A, B, and D,E) and this may account for the increase in dI3 neurons in the Ngn2 null embryos..
We have previously shown that an increase in Ngn1 in the dorsal neural tube leads to an increase in dI2 neurons (Gowan et al., 2001
). To test whether an increase in Ngn1 could explain the increase in dI2 neurons in the Mash1
and the Mash1/Ngn2
double knockouts, we examined Ngn1
expression. Indeed, in the Mash1
and the Mash1/Ngn2
double knockouts, we detected an increase in Ngn1
expression in the dorsal neural tube at E10.5 relative to wild type (, compare G with H,J). Consistent with the lack of change of the dI2 population in the Ngn2 null, there was no change in Ngn1 detected (). Thus, the excess dI2 cells in the Mash1
knockouts is likely due to loss of cross-inhibition of Ngn1
by Mash1 in its dorsal domain of expression (Gowan et al., 2001
Ngn2 does not directly block Mash1 function in specifying dI3 and dI5 neurons
The preceding data strongly suggest that Ngn2 opposes Mash1 function in generation of dI3 and dI5 neurons, and thus, the levels and timing of Mash1 and Ngn2 determine the number of dI3 and dI5 neurons that form. Possible mechanistic models to explain the phenotypes involve Ngn2 directly opposing Mash1 function by forming a non-functional heterodimer, or by competing with Mash1 on target genes, analogous to interactions between Olig2 and Ngn2 recently reported (Lee et al., 2005
). The ability of Ngn2 and Mash1 to form non-functional heterodimers, or to bind similar DNA recognition sites, has been shown in vitro (Gradwohl et al., 1996
). To test these models in vivo, we utilized mouse mutant lines that contain replacement mutations where either the Ngn2
protein coding region was swapped into the Mash1
locus (Mash1 KI Ngn2
) or the Mash1
protein coding region was swapped into the Ngn2
locus (Ngn2 KI Mash1
) (Parras et al., 2002
). The heterozygous embryos in each strain shift the balance and temporal relationship of Mash1 and Ngn2. If Ngn2 directly opposes Mash1 function as predicted above, then the Mash1KI Ngn2/+
would approximate the Mash1 knockout, and the Ngn2KI Mash1/+
would approximate a Mash1 gain-of-function phenotype. In Ngn2KI Mash1/+
embryos, we see an increase in dI3 and dI5 reflecting the Mash1 gain-of-function phenotype as predicted from the specification function of Mash1 (). However, rather than losing dI3 and dI5 in Mash1KINgn2/+
embryos, dI3 and dI5 are increased (). Due to variability between these mutant embryos, only the dI3 increase was statistically significant. There is also a significant increase in dI2 neurons in Mash1KINgn2/+
embryos, possibly reflecting the role of Ngn2 in generation of these neurons (Gowan et al., 2001
) (). Although the results confirm the importance of Mash1 in specifying dI3 and dI5 neurons, they contradict the model that Ngn2 directly opposes this activity of Mash1. Rather, these results fit a model that highlights distinct functions for Mash1 and Ngn2 where Mash1 has a major role in neuronal specification and Ngn2 has a role in temporal control of neuronal differentiation.
Distinct functions for Mash1 and Ngn2 in specification of dorsal neurons
The Mash1 and Ngn2 knock-in strains have also been used to address whether these related bHLH factors are able to compensate for each other’s function. It has been reported that Ngn2 and Mash1 share the ability to induce neuronal differentiation, but are distinct in specifying neuronal identity (Parras et al., 2002
). The functions of Mash1 and Ngn2 in the dorsal neural tube are similar to those attributed to them in other regions of the nervous system. Ngn2 is unable to compensate for Mash1 function in specification as shown by its inability to rescue the generation of dI3 and dI5 neurons (), and its inability to suppress the ectopic formation of dI2 and dI4/6 neurons (). Similarly, Mash1 does not compensate for Ngn2 since in both Ngn2−/−
and Ngn2KI Mash1/+
embryos the number of dI3 and dI5 neurons is elevated (), reflecting the specification function of Mash1 distinct from Ngn2. The number of dI2 neurons was decreased in this mutant line (), suggesting that ectopic Mash1 in dI2 precursors is inhibitory for dI2 generation as is predicted from Mash1 repression of Ngn1
expression (Gowan et al., 2001
). Together, these results confirm that Mash1 function is critical for the specification of dorsal neuronal sub-types dI3 and dI5, and Ngn2 cannot replace this function. In addition, although no specification function can be ascribed to Ngn2, it functions in controlling the number of different neuronal sub-types that form.