In this study we have used a conditional genetic strategy to examine the separate and combinatorial roles of two transcription factors, Brn3a and Islet1, which together regulate the core gene expression program of developing sensory neurons at all levels of the neural axis. This and prior studies of Brn3a and Islet1 in the sensory system (Dykes et al., 2010
; Eng et al., 2004
; Huang et al., 1999a
; Lanier et al., 2009
; Lanier et al., 2007
; Sun et al., 2008
) define four essential functions for these factors in which they: 1) repress early regulators of neurogenesis, including bHLH factors of the Neurog and Neurod classes, 2) repress gene expression programs characteristic of other tissues, including expected cell types, such as the dorsal spinal cord and hindbrain, and surprising ones, such as cardiac/cranial mesoderm, 3) create a permissive condition for the expression of Ntrk, Runx, and Ets factors that specify the principal sensory subtypes mediating pain, touch, and proprioception, and 4) activate an extensive set of genes essential for specific sensory functions but not for generic neural differentiation.
The developmental phenotype observed in the sensory system of Brn3a/Islet1 DKO mice, including defective neuronal migration and axonogenesis, is likely to reflect a composite of these defects in gene regulation, rather than the effect of a single target gene. The DRG in these embryos fail to migrate to their correct position and arrest near the dorsal aspect of the spinal cord, especially in the cervical region. It is possible that this represents an aberrant response to dorsal/ventral patterning signals. In mice lacking Shh, an opposite effect is observed, in which TG and DRG neurons migrate excessively, sometimes reaching the floorplate region (Fedtsova et al., 2003
). Recently Sema3/neuropilin signaling has been shown to be necessary for correct DRG neuron migration (Schwarz et al., 2009
), although the principle effect of disruption of this pathway is the fusion of adjacent DRG, not aberrant dorsoventral migration. We are not aware of a defect in a single signaling pathway that phenocopies the aberrant migration pattern of the DKO ganglia.
Brn3a and Islet1 exhibit epistatic interactions on nearly all of their targets, with a few notable exceptions. Brn3a exclusively regulates the other members of its own class: Brn3b and Brn3c depend completely on Brn3a, and are unaffected in the Islet1 CKO. Islet1 also plays the predominant role in repression of spinal cord/brainstem transcription factors of the Lhx and Olig classes. Finally, these factors have a partially selective role in subtype specification, in that Brn3a and Islet1 are the principal regulators of Runx3 and Runx1 expression, respectively, but loss of both factors is required to completely extinguish expression of these subtype markers.
For nearly all other target gene classes, Brn3a and Islet1 exhibit epistasis, but this does not imply a common mechanism for all downstream effects. The effects of Brn3a and Islet1 knockouts are less than additive on a majority of the target genes, but for negatively regulated targets, this implies that either factor is sufficient to repress transcription (“redundancy”, e.g. Eya1), while for positively regulated targets, it suggests that both factors are required to activate expression (“cooperativity”, e.g. Trpv1). This may indicate different molecular mechanisms of interaction on positively and negatively regulated targets. In addition, because a large number of the Brn3a/Islet1 regulated genes are themselves transcription factors or other classes of developmental regulators, it is likely that some of the downstream effects are indirect.
The highly conserved roles of the Pou4 and LIM transcription factor classes in sensory development in widely separated phyla are illustrated by the C. elegans
Brn3a and Islet1 orthologs, Unc86 and Mec3, which are required for the differentiation of specific touch receptor neurons in nematodes. Unc86 and Mec3 cooperate to regulate the Mec3 gene (Xue et al., 1993
) and downstream sensory targets (Duggan et al., 1998
), suggesting a model for Brn3a/Islet1 epistasis. However, there is a fundamental difference in the regulatory relationship between Brn3a and Islet1 and their nematode orthologs in that Mec3 expression requires Unc86 in the lineages that generate touch receptor neurons (Finney and Ruvkun, 1990
), whereas the generation of Islet1-expressing neurons and Islet1 expression do not depend on Brn3a. Instead, Brn3a and Islet1 are regulated independently and appear to interact only at the target gene level.
Transcription factors regulating neural development are rarely restricted to a single cell type, and whether these factors regulate the same gene expression programs in different cellular contexts remains a central question. Brn3a is expressed in multiple CNS loci, including the spinal cord, inferior olivary nucleus, superior colliculus, red nucleus, and habenula (Fedtsova and Turner, 1995
). Global examination of gene expression downstream of Brn3a in the habenula has revealed very few target genes in common with sensory neurons (Quina et al., 2009
). Islet1 is not expressed in the habenula, providing a potential explanation for these differences, and it is thus important to consider target genes in other cell types which co-express Brn3 and Islet factors.
Brn3/Islet interactions have been examined in the only major CNS-derived cell type expressing both factors, the retinal ganglion cell (RGC). In RGCs the regulatory relationship of Brn3a and Brn3b is reversed compared to sensory neurons such that Brn3b is expressed earlier in development, has more profound effects on RGC gene expression, and is required for the expression of Brn3a (Badea et al., 2009
; Erkman et al., 2000
; Gan et al., 1996
). Both Brn3b and Islet1 knockout embryos exhibit a partial loss of RGCs, and the DKO phenotype is more severe than either single mutant (Pan et al., 2008
Array and candidate gene methods have been used to identify genes downstream of Brn3b (Mu et al., 2004
; Qiu et al., 2008
) and Islet1 (Mu et al., 2008
; Pan et al., 2008
) in developing RGCs. As in the sensory system, genes downstream of Brn3b and Islet1 overlap considerably in RGCs, yet few of the Brn3/Islet1 targets are conserved between the sensory system and the retina. Brn3a/b and Islet1 knockouts in both cell types show dysregulation of Neurod-, Irx- and Lim- class transcription factors, which suggests some conservation of core regulatory programs. However, key identified targets of Brn3b in the retina, including Ablim1, Ebf1, Mstn (Gdf8), Eomes (Tbr2) and Dlx1/2, are not regulated in the DRG, and conversely the large majority of genes downstream of Brn3a and Islet1 in the DRG have not been identified as target genes in RGCs.
How can the Brn3- and Islet-class factors regulate largely distinct gene expression programs in different cell types? It is unlikely the differences arise from distinct properties of Brn3a and Brn3b proteins, as gene swapping experiments have shown these to be largely interchangeable, at least in retinal development (Pan et al., 2005
). The conventional combinatorial model of gene regulation would imply that specific DNA-binding partners interact with Brn3a/b and Islet1 in each cell type, allowing the discrimination of enhancers in genes expressed specifically in sensory and retinal neurons. However, a recent study of genes that are regulated by Brn3a in the TG, but not the DRG, of Brn3a KO mice suggests a parallel epigenetic mechanism for cell-specific gene regulation (Eng et al., 2007
). The TG-specific target genes bear repressive histone marks in the DRG suggesting that they are constitutively silenced there, and thus cannot be regulated by Brn3a. These results imply that chromatin modifications accumulated during the developmental history of each cell type may control the range of genes available for regulation by a given transcription factor. Sensory and retinal precursors, for example, may have distinct “sub-genomes” accessible to Brn3a/b and Islet1, leading to very different loss-of-function effects. In future studies, combined use of global gene expression assays and genome-wide chromatin analysis in diverse cell types may more fully reveal such mechanisms.