Skip to main content

Function of the transcription factors Olig3 and Lbx1 in brainstem respiratory nuclei

Final Report Summary - OLIG3-LBX1 BREATHING (Function of the transcription factors Olig3 and Lbx1 in brainstem respiratory nuclei)

Publishable Summary

Congenital breathing disorders are life-threatening conditions for humans and a concern in public health. Although these disorders are rare, they are of a great burden to the affected individuals and their families, and their treatment by mechanical ventilation is expensive and laborious. Our understanding on congenital breathing disorders is limited. Neuronal populations that control respiration in the brainstem are small and difficult to access, which complicate the study of respiratory disorders in humans. Combining mouse genetics with anatomical, physiological and behavioral studies, we investigated in the funding project the function of transcription factors in the establishment of breathing centers and the role of such centers in health and disease (see Figure 1A for a schematic representation of breathing centers in mice).

Olig3 in breathing

Olig3 encodes a transcription factor of the bHLH family that is expressed in progenitor cells of the dorsal brainstem. It contributes to the formation of the dorsal respiratory column, i.e. the nucleus of the solitary tract (see Storm et al., 2009). The nucleus of the solitary tract receives information on the concentration of oxygen (O2) from the carotid bodies located at the periphery (Blessing, 1997). This information is subsequently relayed to the respiratory rhythm generators (i.e retrotrapezoid nucleus and preBötzinger complex).

We report here that mutation of Olig3 in mice results in cyanosis shortly after birth, but this can be partly rescued by mild cutaneous stimulation. Analysis of breathing parameters showed that Olig3 mutant mice breathe similarly as control littermates during the first three postnatal hours (Figure 1B). This is in agreement with the fact that both respiratory rhythm generators are anatomically and functionally preserved in Olig3 mutant mice. Nevertheless, the characteristic maturation of breathing in control animals, which takes place after the third hour of life, is not observed in Olig3 mutants. This lack of breathing maturation results in severe hypoventilation (Figure 1B). The only respiratory center affected anatomically by mutation of Olig3 is the nucleus of the solitary tract, which is not formed (Figure 1C). Given that O2 sensitivity develops during the first day of life, our data indicate that lack of breathing maturation in Olig3 mutant mice results from a deficit of the nucleus of the solitary tract to relay critical information on O2 levels to the respiratory rhythm generators.

To unambiguously assign a role in breathing maturation to the nucleus of the solitary tract, we studied Tlx3 null mutant mice in which this center is also lacking (see Figure 1C and Qian et al., 2001). We demonstrated that in Tlx3, like in Olig3, mutant mice postnatal breathing does not mature (Figure 1B). Altogether, our data revealed the pivotal role of the nucleus of the solitary tract - carotid body system in maturation of breathing.

Identification of a new gene that causes Congenital Central Hypoventilation Syndrome in humans and mice

Congenital Central Hypoventilation Syndrome (CCHS, also known as Ondine’s curse) is a pathological condition that becomes already apparent shortly after birth. It is characterized by deficiencies in the neuronal control of breathing (Weese-Mayer et al., 2005, 2008). Most CCHS patients can breathe normally while awake, but suffer from profound apneas and respiratory arrest during sleep. Severely affected children never breathe. To date, the only gene to which CCHS mutations have been assigned is PHOX2B; the mutations expand a short sequence repeat that encodes the amino acid alanine. Interestingly, in a genetic screen of CCHS patients, Professor Stephan Mundlos (Max-Planck-Institute of Genetics, Berlin) identified a frameshift mutation in a novel gene that encodes a transcription factor. This frameshift mutation alters the C-terminal sequence of the encoded protein.

To study the consequences of the frameshift mutation observed in CCHS patients, we engineered a mouse strain in such a manner that it carries the analogous frameshift mutation. In the frameshift mutant mice, we observed slower and shallower breathing when compared to control littermates (Figure 1D). Anatomical examination of brainstems showed that a single breathing center was lacking in these animals, the retrotrapezoid nucleus (Figure 1E). The retrotrapezoid nucleus consists of a small group of neurons and is located in the ventral brainstem. The retrotrapezoid nucleus senses CO2 and functions as a respiratory rhythm oscillator. We performed Ca+2 imaging analyses on brainstem preparations of the frameshift mutant mice and discovered no rhythmic neuronal activity in the ventral brainstem. Furthermore, we found no responsiveness towards CO2 (Figure 1D).

In conclusion, we demonstrated that the frameshift mutation identified in humans is responsible for CCHS. We are currently analyzing on a genome wide scale whether the proteins encoded by the wildtype and frameshift alleles bind differentially to chromatin. This will also help to identify direct target genes relevant for breathing disorders.

References

Blessing, (1997). Oxford University Press.
Qian et al., (2001) Genes Dev 15:2533-2545.
Storm et al., (2009) Development 136:295-305.
Weese-Mayer et al., (2005). Respir Physiol Neurobiol 149:73-82.
Weese-Mayer et al., (2008). Respir Physiol Neurobiol 164:38-48.

final1-figure-1-publishable.jpg