Novel mechanisms that pattern and shape the midbrain-hindbrain boundary

Abstract

The midbrain-hindbrain boundary (MHB) is a highly conserved vertebrate signalling centre, acting to pattern and establish neural identities within the brain. While the core signalling pathways regulating MHB formation have been well defined, novel genetic and mechanistic processes that interact with these core components are being uncovered, helping to further elucidate the complicated networks governing MHB specification, patterning and shaping. Although formation of the MHB organiser is traditionally thought of as comprising three stages, namely positioning, induction and maintenance, we propose that a fourth stage, morphogenesis, should be considered as an additional stage in MHB formation. This review will examine evidence for novel factors regulating the first three stages of MHB development and will explore the evidence for regulation of MHB morphogenesis by non-classical MHB-patterning genes.

Fig. 1

Positioning of the presumptive MHB is regulated by the expression of Otx and Gbx genes. Following overall posteriorisation of the neuroectoderm by Wnt8, Otx2 is expressed within the presumptive midbrain region, and Gbx1/2 is expressed within the presumptive hindbrain territory. Otx2 expression in the midbrain is regulated partially by Meis2, whereas Gbx1/2 expression in the hindbrain is regulated by members of the Iroquois (iro) gene family. The tightly demarcated interface of Otx/Gbx gene expression is regulated by FGF8, which inhibits Gbx1/2 expression in the midbrain and Otx2 expression in the hindbrain, respectively, resulting in defined lineage restriction. Lrrn1-mediated suppression of FGF8 results in defective boundary demarcation, lineage-mixing, and defective organiser formation, indicating that the establishment of a sharp boundary is critical for further MHB development

Fig. 2

Induction of the core-MHB cascade is regulated by several factors. Once the presumptive MHB territory has been established, genes of the FGF, Wnt, Pax and Engrailed gene families (the core-MHB cascade) are rapidly induced within this region. FGF8 activity is critical, and expression and function of this molecule are regulated by miR-9 and members of the Notch pathway (Serrate, Hairy, Lunatic Fringe and Delta1). The interplay between members of the core-MHB cascade begins, and this is regulated by Lrrn1, Grg4 and XHR1 at multiple levels within the cascade. Specific interactions between members have also been described, notably RPTPλ-mediated activation of Wnt1 following FGF8 stimulation and the transcription of Engrailed 2a by FGF8-mediated activation of grhl2b

Fig. 3

Maintenance of MHB development relies on transcriptional interdependence between members of the core MHB cascade. During this stage of MHB development, functional interplay among FGF8, Wnt1, Pax2/5/8 and En1/2 is critical, as loss of any of these factors here leads to complete loss of isthmic stability. Several other factors are also postulated to regulate this cascade, namely Wnt3a, Autotaxin and Pou2 which seem to globally regulate the cascade, as well as Lmx1b, Pbx and grhl2b, which exert their effects on specific genes within the cascade. In addition to regulation of Wnt1, Lmx1b may also regulate Eng1/2 and Pax2/5/8 in this context. Which pathways are activated by the interdependence of the core MHB cascade remain largely unknown, although some candidates (such as Brn1, Sef, Tapp1 and Ncrms) are known to lie downstream of certain cascade members, in this case Pax2. Failure of MHB interdependence during this stage leads to a variety of pleiotropic defects with profound implications for organiser integrity, namely increased cell death, defective precursor cell proliferation, and impaired differentiation, migration and functional integration of more mature neurons within the midbrain and hindbrain territories

Fig. 4

MHB morphogenesis is genetically separable from the actions of genes of the core MHB cascade. The Wnt/PCP pathway plays critical roles in the establishment of planar polarity of the neural plate during neurulation and may also regulate correct apico-basal polarity of neuroepithelial cells during morphogenesis of the MHB. Formation of the MHB constriction is likely to involve regulation of cytoskeleton plasticity, regulated by cdc42, following grhl2b-mediated activation of spec1. Several classes of adhesion molecules, junctional complex and extracellular matrix proteins, such as laminin γ1, laminin β1, N-cadherin and Mpp5, as well as the transcription factor CREB, also play a role in shaping the MHB independently of the core-MHB cascade; precisely what active or instructive role the core-MHB cascade plays during this stage is not clear. Loss of Dicer leads to aberrant morphology of the neural tube, a defect that can be rescued by miR430. However, the identity of other mature miRNAs that are lost in Dicer mutants and direct genetic targets of miR430 (both indicated by an “X?” on the figure) remain to be elucidated. Many factors, both genetic (e.g. Atp1a1, mypt1) and cell-intrinsic (cytoskeleton fidelity, cell polarity), are likely to play critical roles during basal constriction of MHB cells during morphogenesis, and extrinsic forces, such as ventricle inflation and cerebrospinal fluid (CSF) pressure, may play a role in allowing full ventricular expansion and MHB morphogenesis