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In the telencephalon, Mash1 is expressed at high levels by ventral progenitors, whereas Ngn1 and Ngn2 are expressed exclusively in cells of the dorsal ventricular zone VZ Gradwohl et al. Here, we have addressed the question of whether the restricted patterns of expression of Mash1 and the Ngns in the telencephalon reflect a role for these genes in the specification of distinct neuronal identities in the basal ganglia and cerebral cortex, respectively. We show that Ngn gene function is required to prevent ectopic Mash1 expression in the dorsal telencephalon, ensuring that the pallial and subpallial subdivisions of the telencephalon express different neural determination genes.
The up-regulation of Mash1 in the dorsal telencephalon compensates to some extent for the loss of the determination function of the Ngns , as neurogenesis defects in Ngn mutant cortices are mild, as compared with the more severe defects in the Ngn2;Mash1 mutant cortex. However, the importance of restricting Mash1 expression to the ventral telencephalon for correct neuronal patterning is highlighted by the observation that ectopic Mash1 expression in Ngn mutants results in the mis-specification of a subpopulation of early born cortical neurons, which lose expression of several dorsal markers e.
These data are supported by the results of forced expression of Mash1 in dorsal telencephalic progenitors, which is sufficient to confer ventral characteristics to a subset of cortical neurons. These results indicate that Mash1 and the Ngns are involved in coupling neural determination and the specification of neuronal phenotypes during forebrain development.
Previous expression studies have shown that Mash1 and the Ngns are expressed in a complementary manner in the developing telencephalon and diencephalon, suggesting that these genes may play a role in specifying regional neuronal identity in the forebrain Gradwohl et al. To begin to investigate this hypothesis, we compared the expression patterns of these genes in greater detail.
To better trace Ngn2 -expressing progenitors, a new null allele of Ngn2 Ngn2 lacZKI , in which coding sequences of the bHLH domain were replaced with a lacZ reporter, was introduced in mice by standard embryonic stem ES cell technology Fig. The Ngn2 lacZKI allele behaves as a null allele on the basis of the identical perinatal lethality and cortical phenotype observed in Ngn2 lacZKI and Ngn2 neo null homozygous embryos Fode et al. Mash1 and Ngns are expressed in complementary patterns in the embryonic telencephalon.
Schematic representation of the endogenous Ngn2 locus top , Ngn2 targeting vector middle , and recombined allele bottom. Ngn1 and Ngn2 are expressed in proliferating progenitors of the dorsal telencephalon and dorsal thalamus in an almost completely overlapping fashion. Mash1 is expressed in a complementary manner with transcripts at high levels in the VZ of the ventral telencephalon and ventral thalamus. At embryonic day E Ngn1 is expressed in a graded manner, with higher levels in the lateral VZ, whereas Ngn2 transcripts appear distributed more uniformly throughout the dorsal VZ.
Helene Kern (Translator of Histoire de la philosophie occidentale)
In contrast to Ngns, Mash1 is expressed at high levels in ventral progenitors located in the lateral LGE and medial ganglionic eminences MGE and only at low levels in the dorsal telencephalon Fig. The complementary patterns of expression of these genes are also observed in the dorsal thalamus, in which Ngn1 Fig. Cross-regulatory interactions exist between Ngn1 and Ngn2 in neuronal lineages of the PNS, including the dorsal root and cranial ganglia Fode et al. Thus, we examined whether the overlap in expression of Ngn1 and Ngn2 in the telencephalon reflects cross activation or independent means of activation, by studying Ngn1 expression in Ngn2 mutants and vice versa.
In the dorsal telencephalon of Ngn2 mutants, Ngn1 expression is lost in medial and dorsal domains but is maintained in the lateral VZ Fig. In contrast, Ngn2 expression is detected throughout the dorsal telencephalon of Ngn1 mutants, although the levels appear reduced Fig. Ngns down-regulate Mash1 transcription in the dorsal telencephalon.
A,B Distribution of Ngn1 transcripts on frontal sections of E Ngn1 expression is dependent on Ngn2 function in dorsal and medial cortical domains arrowhead, B and independent of Ngn2 in a lateral cortical domain. C,D Distribution of Ngn2 transcripts on frontal sections of E Ngn2 is expressed at reduced levels in cortical progenitors of Ngn1 mutants, suggesting that Ngn2 transcription is partially dependent on Ngn1 function throughout the dorsal telencephalon.
E,J Distribution of Mash1 transcripts on frontal sections of E Mash1 expression is up-regulated in the VZ of the dorsal telencephalon arrowhead in F and H and dorsal thalamus of Ngn2 single and more strikingly of Ngn1;Ngn2 double mutant embryos. We also investigated potential regulatory interactions between Mash1 and the Ngns in the telencephalon. An up-regulation of Mash1 expression was observed throughout the dorsomedial cortical VZ of Ngn2 mutant embryos at E This up-regulation was less apparent in the lateral VZ, in which Ngn1 expression is maintained in Ngn2 mutants.
No changes in Mash1 expression levels were observed in the forebrain of Ngn1 single mutants Fig. In contrast, Ngn1 and Ngn2 expression patterns were unaltered in the forebrain of Mash1 mutant embryos data not shown. Taken together, these results suggest that the Ngns function in a partially redundant manner to restrict Mash1 expression to limited domains of the forebrain neuroepithelium.
The nature of the regulatory interactions between Ngns and Mash1 remains to be characterized. To address this possibility, expression of the panneuronal marker SCG10 was examined at E In Ngn1 single Fig. However, the presence of neurons in the PP of Ngn mutants suggested that up-regulation of Mash1 could partially compensate for the lack of Ngn activity and permit cortical neurogenesis to proceed. Cortical preplate neurons lose dorsal characteristics and acquire ventral properties in Ngn mutants.
M—P Distribution of Dlx1 transcripts on frontal sections of E This ventral marker is ectopically expressed by PP neurons in the dorsal cortex of Ngn2 mutant embryos arrow, O and in the dorsal and lateral cortex of Ngn1;Ngn2 double mutant embryos arrow, P.
Q,R Expression of Dlx1 in the telencephalon of E High-magnification views of Math2 purple and Dlx1 orange expression in the dorsal cortex of E Math2 and Dlx1 are not coexpressed in the Ngn2 mutant cortex, indicating that mutant PP neurons adopt either a dorsal or a ventral fate. The same mis-specification of cortical neurons was observed in Ngn2 neo and Ngn2 lacZKI homozygous mutants. To examine whether the replacement of Ngns by Mash1 as a determination gene in Ngn mutant cortical progenitors resulted in specification defects, we first analyzed the expression of several dorsal-specific neuronal markers in the telencephalon of Ngn mutants.
Reductions in expression levels of Tbr1 Fig. In contrast to the dorsal postmitotic markers, no changes were observed in the level of expression of dorsal-specific progenitor markers, including Emx1, Emx2 , and Pax6 , in the Ngn2 mutant cortex data not shown , suggesting that with the exception of Mash1 up-regulation, the molecular phenotype of dorsal progenitors is unaltered in Ngn2 mutants.
We hypothesized that the down-regulation of dorsal-specific markers by Ngn mutant cortical neurons might reflect their respecification to a more ventral identity.
To address this possibility, the expression patterns of several ventral telencephalic-specific genes were examined in Ngn mutant embryos. In wild-type E Ectopic Dlx1 and Dlx2 expression was detected in the Ngn2 mutant cortex, beginning at E12 in the rostral and dorsal cortex E Ectopic Dlx1 expression was restricted to the neuronal layer and was not detected in the lateral cortex of Ngn2 single mutants, in which Ngn1 expression is maintained, nor in the cortex of Ngn1 single mutants, which maintain Ngn2 expression Fig.
However, an expansion of ectopic Dlx1 expression into the lateral and caudal cortex was observed in Ngn1;Ngn2 double mutants at E By E Several other ventral telencephalic markers, such as Lhx6 , Lhx7, Islet1 , and Nkx2. Consistent with the idea that the decreased expression of dorsal markers correlates with a change to a ventral-like fate by Ngn mutant cortical neurons, double-labeling experiments revealed that Dlx1 and Math2 are expressed in largely nonoverlapping and complementary populations of neurons in the Ngn2 mutant PP Fig.
Up-regulation of Mash1 is required for the mis-specification of cortical neurons in Ngn2 mutants. NeuroD expression is dramatically reduced in the dorsal and medial cortex of Ngn2;Mash1 double mutants arrow, F but is not affected in Ngn2 single mutants E. G—I Distribution of Hes5 transcripts in E Hes5 expression is strongly down-regulated in the VZ of the dorsal and medial cortex of Ngn2;Mash1 double mutants arrow, I , suggesting defects in Notch signaling in these progenitors.
J—L Distribution of Dlx1 transcripts in E M—O Distribution of Math2 transcripts in E The reduction in Math2 expression levels observed in the Ngn2 mutant cortex N is also found in Ngn2;Mash1 double mutants O , thus confirming the overall reduction in number of medial and dorsal PP neurons in Ngn2;Mash1 double mutants. To determine whether neural bHLH genes are involved in specification of neuronal phenotypes throughout the forebrain, we examined neuronal patterning in the thalamus, a region in which Mash1 and the Ngns are also expressed in a complementary manner.
Ngn1 and Ngn2 are highly expressed in the dorsal thalamus Fig. Ectopic Mash1 expression was observed in the dorsal thalamus of Ngn2 mutants Fig. Therefore, the Ngns function to restrict Mash1 expression to limited domains throughout the forebrain neuroepithelium. In addition, consistent with the idea that altered neuronal identities arise in regions where Mash1 expression levels are up-regulated, ectopic GAD67 transcripts were detected in postmitotic neurons in the dorsal thalamus of Ngn2 single mutants Fig.
Taken together with our observations in the cortex, these results indicate that the proper expression of Ngn and Mash1 is required for the correct specification of neuronal identity in several subdivisions of the forebrain. In addition, the dorsal thalamus of Ngn1;Ngn2 mutant embryos appears highly abnormal, with an enlargement of the VZ and a reduction in size of the mantle zone Fig.
Mis-specification of neurons in the dorsal thalamus of Ngn mutants. A—C Distribution of Mash1 transcripts on frontal sections of E Distribution of GAD67 transcripts on frontal sections of E Morphological defects are also observed in the thalamus of Ngn1;Ngn2 double mutant embryos F , consistent with a role for the Ngn genes in neural determination in this region of the forebrain. The presence of cortical neurons with a ventral phenotype in Ngn mutants may arise from the mis-specification of dorsal progenitors or, alternatively, from premature and excessive tangential migration of ventrally derived neurons.
Cortical neurons labeled with both X-Gal and Dlx1 were detected Fig. A,B Double labeling of frontal sections through the telencephalon of E C—K Explants from E11 wild-type top and Ngn2 mutant bottom telencephalic vesicles dissected into ventral—lateral E,I , dorsal F,J , and medial G,K domains, as defined in brackets in C , cultured for 4 days on floating filters and labeled for Dlx1 transcripts.
These neurons differentiate in dorsal explants isolated from ventral telencephalon and thus originate from dorsal progenitors. Confirmation that explants were dissected prior to the onset of ectopic Dlx1 expression was obtained from the lack of Dlx1 expression in uncultured contralateral dorsomedial explants from wild-type D and Ngn2 mutant H embryos.
Medial, dorsal, and ventrolateral domains were dissected out and cultured separately on filters for 4 days Fig. To determine to what extent the up-regulation of Mash1 compensates for the loss of Ngn2 function, we examined neurogenesis in the dorsal telencephalon of Ngn2;Mash1 double-mutant embryos.
The number of neurons expressing the panneuronal marker SCG10 is not significantly affected Fig.
The anatomical substrate for telencephalic function.
As reported previously, no defect in neurogenesis is apparent in the dorsal telencephalon of Mash1 mutants Casarosa et al. These results indicate that Ngn2 normally has a determination function for early born cortical neurons, and that the up-regulation of Mash1 can partially compensate for the loss of this activity in Ngn2 mutants. Although reduced in number, neurons are present in the PP of Ngn2;Mash1 double mutant embryos, allowing us to assess whether the mis-specification of PP neurons in Ngn2 mutants is due to the loss of Ngn2 function or to the up-regulation of Mash1.
We thus determined whether Mash1 is required for the ventralization of cortical neurons by examining Dlx1 expression in double mutant embryos.
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