Traditionally, understanding the role of a specific gene product in a particular cell population, or region, of the brain relies on cell- or tissue-specific deletion of the gene through a conditional knock out approach (CKO). This conditional approach has been successfully utilized to understand the cell-autonomous role of MeCP2 (Fyffe et al., 2008
; Adachi et al., 2009
; Samaco et al., 2009
; Chao et al., 2010
) and has been invaluable towards determining the requirement for a gene’s function in a specific cell population or region. However, in the context of distributed neural circuits coordinating behavioral and physiological processes, removing a gene from any single component of the circuit may completely disrupt the overall circuit’s function. There may be many possible ways to disrupt the function of the circuit by removing the gene of interest from any number of cellular populations within that circuit. On the other hand, the ability to rescue a particular phenotype by restoring MeCP2 function within a select region or group of cells demonstrates conclusively that the entire neural circuit function has been restored. In the case of MeCP2 dysfunction, this has important clinical implications: the identification of an anatomically restricted region in which restoration of MeCP2 function causes dramatic changes in clinically relevant phenotypes implies that specific therapy, such as gene therapy, targeted towards this restricted anatomic area may have dramatic effects on clinical features.
MeCP2 function is required in the anatomical region defined by the HoxB1 lineage for normal lifespan. Furthermore, restoring MeCP2 function within this same region restores lifespan, demonstrating that MeCP2 function is both necessary and sufficient within the HoxB1 lineage for survival. In addition to the effect on lifespan, several autonomic and respiratory phenotypes are dependent on MeCP2 function within the HoxB1 domain. In particular, loss of MeCP2 in this region contributes to a decreased heart rate as well as a greater increase in respiratory rate during an acute hypoxic challenge. There is clear complementarity in these phenotypes as they result from loss of MeCP2 function within the HoxB1 domain and are reversed when MeCP2 function is restored within this domain (). Survival, weight, heart rate, basal hyperventilation, and hypoxic respiratory response all exhibited reciprocal phenotypes between the HoxB1 CKO and RESCUE models. The ability to identify such reciprocal phenotypes between the CKO and Rescue animals argues strongly for a tissue-autonomous function for MeCP2 in the hindbrain. Furthermore, the reciprocity of the weight phenotypes observed between the CKO and RESCUE mice (loss of MeCP2 within the HoxB1 domain causing an underweight phenotype, and loss of MeCP2 rostral to the HoxB1 domain causing an overweight phenotype) revealed that MeCP2 is required within two separate anatomical domains for competing drives affecting weight.
Comparison of phenotypes reproduced in regional knock out and rescued in regional re-expression of MeCP2 function.
Interestingly, some of the abnormalities observed in the NULL mice are not reproduced when MeCP2 function is removed from the HoxB1 domain and are not rescued when MeCP2 expression is restored in this domain (). This indicates that such phenotypes are clearly the result of MeCP2 dysfunction in the region outside the HoxB1 domain. Of the phenotypes not dependent on MeCP2 function within the HoxB1 domain, perhaps the most notable is the increased breathing rate observed in the NULL animals during baseline breathing. This breathing abnormality is not present in CKO animals but is present in the RESCUE animals suggesting that the phenotype has an origin outside the HoxB1 expression domain. The fact that some of the breathing abnormalities are dependent on MeCP2 function within the HoxB1 domain (i.e. response to hypoxia), whereas other breathing abnormalities such as the basal hyperventilation are not, demonstrates that MeCP2 function is important in multiple nodes of respiratory circuitry, and that the function within these anatomically distinct regions are separable. Furthermore, the increase in breathing rate, during basal conditions or during hypoxic challenge, is largely due to a reduction of the expiratory time per breath, a result that is consistent with models of increased net activation of respiratory pattern generating pre-inspiratory neurons of the pre-Botzinger complex (Smith et al., 2009
). Given the anatomical separation in the requirement of MeCP2 for normal baseline or hypoxic breathing rates, the requirement of MeCP2 rostral to the HoxB1 domain for normal baseline respiration suggests impaired signaling from nuclei rostral to the HoxB1 domain, possibly including the parabrachial nuclei and the Kölliker-Fuse nucleus, to the ventral respiratory group of the medulla (Stettner et al., 2007
; Song et al., 2010
). In contrast, the requirement within the HoxB1 domain for normal respiratory rate during hypoxia suggests impairments in the circuits responding to oxygen levels. One possibility is that signaling from the NTS relaying the sensation of hypoxia detected in the carotid body to the ventral respiratory group of the medulla is altered (Teppema and Dahan, 2010
); specifically, deficits in GABA signaling caused by loss of MeCP2 (Medrihan et al., 2008
; Chao et al., 2010
) may underlie the apparently impaired hypoxic ventilatory decline caused by loss of MeCP2 in the hindbrain, presenting as relative hyperventilation during an acute hypoxic challenge (Teppema and Dahan, 2010
). Alternatively, because the carotid body itself is targeted in the CKO and RESCUE animals by HoxB1Cre
, direct involvement of this oxygen sensor is also a possibility.
Additionally, the origin of the abnormal response to hypoxia within the HoxB1 domain is particularly important because it has been proposed that early exposure to hypoxic stress may be an inciting feature in the development of the disrupted breathing in the mouse models as well as in girls with RTT (Voituron et al., 2009
). However, the occurrence of the abnormal basal breathing rate in the RESCUE animals despite a normalized respiratory rate during hypoxia suggests that basal hyperventilation is not secondary to the chemosensory deficits. It remains to be seen whether the chemosensory deficits precede other respiratory deficits not explored in this study, or more specifically if exposure to hypoxic stress plays a causal role in the progression of other autonomic deficits.
A surprising finding of this study is that restoring MeCP2 protein to only 50% of the cells within the hindbrain results in rescue of lifespan and other autonomic and breathing abnormalities. Detailed stereological quantification of MeCP2 expression across several regions of the hindbrain suggests that the approximate 50% efficiency of rescue was uniform throughout the HoxB1 domain. Similar phenotypic and histological characterization using more regionally restricted Cre lines that subdivide the HoxB1 domain are likely to refine our understanding of the anatomical requirements of MeCP2 function. However, the apparent uniformity of the modest efficiency of rescue suggests that restoring MeCP2 function in some cells has non cell-autonomous effects. Restoring MeCP2 may result in production of secreted factors such as growth factors that improve the function of nearby cells. On the other hand, the effect may be at the circuit level, where only a fraction of the cells within a circuit that express MeCP2 are capable of restoring the entire circuit’s function. These possibilities are not mutually exclusive, and the overall positive effect of restoring MeCP2 function in a fraction of cells in a region may be due to both possibilities. However, there is a requirement to re-establish MeCP2 in particular anatomic regions to induce such a dramatic effect, because restoring MeCP2 function solely within the forebrain does not rescue lifespan (Alvarez-Saavedra et al., 2007
). In the context of developing therapies to treat RTT, aside from the technical challenge of effectively restoring MeCP2 function in specific cells within the human brain, an additional challenge will be to determine exactly which cellular populations have critical requirements for MeCP2 function to modulate specific phenotypes. The work outlined here provides a methodology for identifying these cellular populations which then could be useful in the development of novel regionally-targeted therapies.