A key unresolved issue in neurobiology is the nature of the molecular programs that regulate the differentiation of neural precursors into specialized neurons with appropriate connectivity. One family that has emerged as important in this regard is the basic helix-loop-helix (bHLH) containing transcription factors (Bertrand et al., 2002
; Ross et al., 2003
). For instance, studies investigating neocortical development have revealed that members of the bHLH family, including the Neurogenins and NeuroD family members, orchestrate the formation of glutamatergic neurons (Schuurmans and Guillemot, 2002
) and many of these are sufficient to activate a pan-neuronal program of gene expression that drives the differentiation of neural precursors into neurons (Farah et al., 2000
; Lee et al., 1995
; Ma et al., 1996
Like many other bHLH transcription factors, Bhlhb5 is broadly expressed in excitatory neurons in the dorsal telencephalon. However, in contrast to the Neurogenins and NeuroD family members, Bhlhb5 appears not to be a transcriptional activator and does not mediate early steps in neuronal differentiation. Rather, Bhlhb5 belongs to a subfamily of bHLH factors including Bhlhb4 (also known as Bhlhe23) and the Olig proteins (Olig1–3) that function predominantly as transcriptional repressors. As an example, when Olig2 is fused to the repressor domain of Engrailed, this fusion protein, but not an activating Olig2-VP16 fusion protein, recapitulates the function of native Olig2 by specifying neural fate in the chick spinal cord (Zhou and Anderson, 2002
). Bhlhb5 and Bhlhb4 are likewise thought to mediate repression based on their ability to inhibit the transactivation of NeuroD-responsive target genes in luciferase assays (Bramblett et al., 2002
; Peyton et al., 1996
; Xu et al., 2002
). However, while these findings suggest that the Oligs, ff, and Bhlhb5 form a sub-family of bHLH factors that mediate transcriptional repression, the manner in which these repressors function endogenously to repress transcription and orchestrate neural development remains obscure.
Studies in the spinal cord have provided a framework for understanding the cellular function of Olig proteins, and these studies suggest that a common function of the Oligs is to confer the neuronal identity of neural progenitors. For instance, Olig1 and Olig2 are expressed in select progenitors of the ventral spinal cord and, in the absence of these factors, neural precursors are re-specified to an alternate fate: instead of forming motor neurons and oligodendrocytes, these pMN progenitors inappropriately generate V2 interneurons and astrocytes (Lu et al., 2002
; Takebayashi et al., 2002
; Zhou and Anderson, 2002
). It is thought that this type of mis-specification occurs because the Oligs function, at least in part, to promote the generation of one subtype of neuron over another by inhibiting the expression of transcription factors that mediate the alternative cell fate choices (Marquardt and Pfaff, 2001
Though Bhlhb4 and Bhlhb5 are closely related to the Oligs, their expression is almost exclusively limited to post-mitotic neurons rather than proliferating neural progenitors, hinting at the possibility that Bhlhb4 and Bhlhb5 regulate later aspects of neuronal differentiation (Bramblett et al., 2002
; Joshi et al., 2008
; Ross et al. 2010
). Further evidence in support of this idea comes from studies in the retina where loss of either Bhlhb4
results not in the mis-specification of retinal progenitors to alternate fates but rather the loss of subsets of neurons, presumably due to apoptosis. Thus, mice lacking Bhlhb4
have an absence of rod bipolar cells, whereas Bhlhb5
mutants are lacking cone bipolar and selective amacrine cells (Bramblett et al., 2004
; Feng et al., 2006
). Similarly, in the spinal cord, disruption of Bhlhb5
function results in the selective apoptosis of a specific population of inhibitory interneurons in the superficial dorsal horn that are required for the normal sensation of itch (Ross et al., 2010
). Together, these studies raise the possibility that Bhlhb4 and Bhlhb5 are involved in late aspects of neuronal differentiation, such as neural circuit assembly, that may be essential for neuronal survival. However, why certain neurons die in the absence of these transcriptional repressors is unknown, and this gap in knowledge stems in part from a lack of mechanistic understanding of how these transcription factors function in neural development.
A possible clue to this puzzle comes from studies of Bhlhb5 in the dorsal telencephalon. In mice lacking Bhlhb5
, neurons of the dorsal telencephalon survive and send out axons, but these projections fail to reach their targets. For instance, corticospinal motor neurons terminate prematurely along the pyramidal tract in the ventral hindbrain and none extend into the spinal cord (Joshi et al., 2008
). Moreover, as we report here, Bhlhb5
mutants also show a complete absence of the three fiber tracts that connect the cerebral hemispheres, suggesting that axonal mistargeting in the absence of Bhlhb5
is a widespread phenomenon. Since mice lacking Bhlhb5
show axon targeting defects, we reasoned that one of the roles of Bhlhb5 in the dorsal telencephalon may be to regulate neuronal connectivity, perhaps by repressing specific genes until they are needed, thereby ensuring that genes are expressed at the right time and place for correct neural circuit assembly. To investigate this possibility, we identified the targets of the Bhlhb5 transcriptional repressor, hoping to elucidate how this transcription factor functions at a mechanistic level.
Here we show that Bhlhb5 functions by binding to specific DNA sequence elements and then recruiting the PR/SET domain-containing protein, Pr-domain 8 (Prdm8) to mediate the repression of target genes. Our observations suggest that Bhlhb5 and Prdm8 are obligate partners for key aspects of neuronal development and, consistent with this idea, we find that mice lacking either Bhlhb5 or Prdm8 have strikingly similar cellular and behavioral abnormalities. We use genetic rescue experiments to demonstrate that one important target of the Prdm8/Bhlhb5 repressor complex is Cadherin-11 (Cdh11), a cell-cell adhesion molecule involved in neural circuit assembly. Taken together these experiments have revealed how a bHLH transcription factor associates with a PR/SET-domain repressor protein to regulate genes involved in neural development.