Divergent functions of the proneural genes Mash1 and Ngn2 in the specification of neuronal subtype identity

Abstract

The neural bHLH genes Mash1 and Ngn2 are expressed in complementary populations of neural progenitors in the central and peripheral nervous systems. Here, we have systematically compared the activities of the two genes during neural development by generating replacement mutations in mice in which the coding sequences of Mash1 and Ngn2 were swapped. Using this approach, we demonstrate that Mash1 has the capacity to respecify the identity of neuronal populations normally derived from Ngn2-expressing progenitors in the dorsal telencephalon and ventral spinal cord. In contrast, misexpression of Ngn2 in Mash1-expressing progenitors does not result in any overt change in neuronal phenotype. Taken together, these results demonstrate that Mash1 and Ngn2 have divergent functions in specification of neuronal subtype identity, with Mash1 having the characteristics of an instructive determinant whereas Ngn2 functions as a permissive factor that must act in combination with other factors to specify neuronal phenotypes. Moreover, the ectopic expression of Ngn2 can rescue the neurogenesis defects of Mash1 null mutants in the ventral telencephalon and sympathetic ganglia but not in the ventral spinal cord and the locus coeruleus, indicating that Mash1 contribution to the specification of neuronal fates varies greatly in different lineages, presumably depending on the presence of other determinants of neuronal identity.

Figure 1

Targeting strategy to introduce the Ngn2 sequence into the Mash1 locus. (A) Targeting vector designed to substitute the coding sequence of Mash1 with the coding sequence of Ngn2, leaving the 5′ and 3′ untranslated regions (UTR) of Mash1 intact. Following transmission of the mutation to the germline, the neomycin resistance gene (Neo) was excised, giving rise to the Mash1^KINgn2 allele. (B) Hybridization of RNA probes for the Mash1 3′UTR sequence and the Ngn2 coding sequence to frontal sections of the telencephalic region of E12.5 wild-type and Mash1^KINgn2homozygous mutant embryos, showing that Mash1^KINgn2transcripts are detected in the ventral telencephalon.

Figure 2

Ngn2 rescues ventral telencephalic development and does not promote dorsal telencephalic fates when replacing Mash1 in ventral telencephalic progenitors. Frontal sections at telencephalic level of E12.5 wild-type (A–F), homozygous Mash1^Δ (A‘–F‘), and homozygous Mash1^KINgn2(A"–F") embryos were hybridized with RNA probes for the genes shown on the left. Progenitors in the medial ganglionic eminence (MGE) are missing in Mash1 ^Δ embryos (A‘–F‘), and MGE formation is rescued by Ngn2 expression from the Mash1 locus (A"–F"). Differentiation of MGE progenitors is also restored, as shown by expression of Prox1 in subventricular zone (SVZ) progenitors (A") and of SCG10 in mantle zone neurons (B"). Ngn2 expression in ventral telencephalic progenitors also restores Notch signaling, as shown by expression of the Notch effector Hes5 in ventricular zone (VZ) progenitors (C") and by the “salt and pepper” pattern of expression of Mash1, as detected by a Mash1 3′UTR probe (D"), whereas Mash1 is transcribed more uniformly by VZ cells in Mash1 ^Δ embryos (D‘). Oulined areas in D–D" are magnified in the insets. Neurons differentiating from Ngn2-expressing progenitors in the ventral telencephalon acquire a normal phenotype, as shown by their expression of ventral markers such as GAD67 (E") and by the lack of misexpression of dorsal-specific genes such as NeuroD (F").

Figure 3

Ngn2 rescues the formation of tangentially migrating interneurons in the cerebral cortex. Frontal sections through the telencephalon of E15.5 embryos hybridized with RNA probes for markers of tangentially migrating interneurons. Interneurons expressing Dlx1 (A–A") and GAD67 (B–B") are born in the ventral telencephalon and migrate tangentially into the cortex. Many of these neurons are missing in absence of Mash1 (A‘, B‘), and are rescued in Mash1^KINgn2 embryos (A", B"). Outlined areas are magnified in the insets.

Figure 4

Mash1 and Ngn2 LOF and GOF phenotypes in the ventral spinal cord. (A–F) Double immunolabeling for the motor neuron marker Isl1 (red) and the V2 interneuron marker Chx10 (green) in transverse sections at brachial level of the ventral spinal cord of E10.5 embryos. There is a drastic reduction in number of V1 interneurons produced in the Mash1 mutant spinal cord (B), and expression of subtype-specific homeodomain proteins is affected in many neuronal populations of Ngn2 mutant embryos (Scardigli et al. 2001), including Isl1 in motor neurons and Chx10 in V1 interneurons (C). Expression of Isl1 is rescued by the ectopic expression of Mash1 in most motor neurons of homozygous Ngn2^KIMash1 embryos (D), whereas a few cells in the motor neuron population fail to express Isl1 and instead express Chx10 (D). Ectopic expression of Chx10 in cells ventral to the V2 interneuron population is also observed in embryos heterozygous for the Ngn2^KIMash1 allele (E). Expression of Ngn2 from the Mash1 locus in Mash1^KINgn2homozygous mutant embryos does not rescue the defect in V2 interneurons resulting from the loss of Mash1 function (F). (G–L) Sections of the brachial spinal cord of E10.5 embryos hybridized with an RNA probe for Chx10. Loss of Mash1 results in a severe reduction in Chx10 expression (H) which is not rescued by ectopic Ngn2 expression in Mash1-expressing progenitors (L). In contrast, ectopic expression of Mash1 in Ngn2-expressing progenitors leads to a ventral expansion of the Chx10 expression domain (J,K).

Figure 5

Ngn2 rescues the differentiation of noradrenergic neuronal precursors in sympathetic ganglia but does not support their proliferation. Transverse sections at forelimb level of wild-type and mutant embryos at E10.5 (A–F), E13.5 (G–H) and E12.5 (I–J). In Mash1^Δ embryos, Phox2b is expressed independently of Mash1 in sympathetic ganglia (B‘), whereas other genes associated with the noradrenergic phenotype, including Phox2a (C‘) and DBH (D‘), are not expressed, and postmitotic SCG10+ neurons (E‘) are not generated. In Mash1^KINgn2embryos, Ngn2 supports the noradrenergic differentiation of sympathetic precursors, as shown by expression of Phox2a (C") and DBH (D"), and the generation of postmitotic SCG10+ neurons (E"). Hybridization on sagittal sections of E13.5 embryos with probes for Phox2a (G–G") and SCG10 (H–H") shows that the sympathetic ganglia of Mash1^KINgn2embryos (G", H") are reduced compared with wild-type ganglia (G, H), to a level similar to that seen in Mash1^Δ embryos (G‘, H‘). Antibody staining for the mitotic marker Ki67 reveals a reduced number of dividing cells in sympathetic ganglia of Mash1^KINgn2embryos at E12.5 (I‘) as compared with wild-type embryos (I). TUNEL analysis shows that there is no increase in cell death in sympathetic ganglia of Mash1^KINgn2embryos at E11.5 (J‘). Arrowheads indicate sympathetic ganglia

Figure 6

Ngn2 does not rescue the development of noradrenergic neurons of the locus coeruleus. (A–D, A‘–C‘) Hybridization of E10.5 wild-type (A–D) and Mash1^KINgn2embryos (A‘–C‘) embryos with probes for Mash1 (A), Phox2b (B) and DBH (C), showing expression of these genes in precursors of the locus coeruleus, located in the rostral hindbrain near the rhombic lip (arrowheads). Ngn2 is expressed in these precursors in Mash1^KINgn2embryos (A‘) and not in wild-type embryos (D). Nevertheless, noradrenergic traits, including expression of Phox2b (B‘) and DBH (C‘) are not induced by Ngn2 in Mash1^KINgn2embryos. (E–E", F–F") Sagittal sections at the level of the fourth ventricle in wild-type and mutant embryos at E13.5. Phox2a and DBH are normally expressed by the neurons of the locus coeruleus located anterior and dorsal to the fourth ventricle (arrowheads in E and F, respectively), whereas expression of the two genes is missing at the same location in Mash1^Δ (E‘, F‘) and Mash1^KINgn2embryos (E", F"). (G, G‘) Nissl stained coronal sections at the level of the pons at birth. The locus coeruleus is recognizable as a compact group of darkly stained neurons lateral to the fourth ventricle (arrowhead in G), which is missing in Mash1^KINgn2newborns (G‘).

Figure 7

Mash1 does not rescue the development of Ngn2-dependent sensory neurons in dorsal root ganglia. (A, B). Transverse section of E9.5 embryos sectioned following whole mount in situ hybridization with a Mash1 probe. Mash1 is expressed in sympathetic ganglia (arrowheads) of both wild-type and Ngn1^Δ; Ngn2^KIMash1 double mutant embryos. Mash1 expression is also observed in migrating neural crest cells entering DRGs in a Ngn2^KIMash1 embryo. (C–F) Mouse embryos at E10.5 are probed with the pan-neuronal marker SCG10. A wild-type embryo shows DRG staining well beyond the 20^th somite (C, arrow). SCG10 expression in Ngn2^KIMash1 (E) is delayed in comparison to the wild-type embryo, as also observed in Ngn2^Δ mutants (D). Arrowhead points to the eighth somite (C–E). Ngn1^Δ, Ngn2^KIMash1 embryo (F) shows no SCG10 staining in DRGs, indicating that no sensory neurons are generated by Mash1 expression from the Ngn2 locus.

Figure 8

. Summary of the phenotypic analysis of Ngn2^KIMash1 and Mash1^KINgn2mice. The different regions of the CNS and PNS analyzed in the two KI mouse strains are schematized. Neurogenesis defects observed in null mutant mice are rescued in KI mice (indicated by RN +), except in tissues that are missing due to specification defects (indicated by RN nd). The rescue of specification defects (indicated by RSS +) varies from lineage to lineage in both KI strains, reflecting the variable contribution of proneural genes to the specification of neuronal subtype identity in different lineages. Respecification of neuronal subtypes caused by ectopic proneural gene expression (indicated by RNS +) is observed only in the CNS of Ngn2^KIMash1 mice, indicating that Mash1 but not Ngn2 has an instructive role in the specification of neuronal identity in the CNS.