Identification of Phox2b-regulated genes by expression profiling of cranial motoneuron precursors.
BACKGROUND
Branchiomotor neurons comprise an important class of cranial motor neurons that innervate the branchial-arch-derived muscles of the face, jaw and neck. They arise in the ventralmost progenitor domain of the rhombencephalon characterized by expression of the homeodomain transcription factors Nkx2.2 and Phox2b. Phox2b in particular plays a key role in the specification of branchiomotor neurons. In its absence, generic neuronal differentiation is defective in the progenitor domain and no branchiomotor neurons are produced. Conversely, ectopic expression of Phox2b in spinal regions of the neural tube promotes cell cycle exit and neuronal differentiation and, at the same time, induces genes and an axonal phenotype characteristic for branchiomotor neurons. How Phox2b exerts its pleiotropic functions, both as a proneural gene and a neuronal subtype determinant, has remained unknown.


RESULTS
To gain further insights into the genetic program downstream of Phox2b, we searched for novel Phox2b-regulated genes by cDNA microarray analysis of facial branchiomotor neuron precursors from heterozygous and homozygous Phox2b mutant embryos. We selected for functional studies the genes encoding the axonal growth promoter Gap43, the Wnt antagonist Sfrp1 and the transcriptional regulator Sox13, which were not previously suspected to play roles downstream of Phox2b and whose expression was affected by Phox2b misexpression in the spinal cord. While Gap43 did not produce an obvious phenotype when overexpressed in the neural tube, Sfrp1 induced the interneuron marker Lhx1,5 and Sox13 inhibited neuronal differentiation. We then tested whether Sfrp1 and Sox13, which are down-regulated by Phox2b in the facial neuron precursors, would antagonize some aspects of Phox2b activity. Co-expression of Sfrp1 prevented Phox2b from repressing Lhx1,5 and alleviated the commissural axonal phenotype. When expressed together with Sox13, Phox2b was still able to promote cell cycle exit and neuronal differentiation, but the cells failed to relocate to the mantle layer and to extinguish the neural stem cell marker Sox2.


CONCLUSIONS
Our results suggest novel roles for Sfrp1 and Sox13 in neuronal subtype specification and generic neuronal differentiation, respectively, and indicate that down-regulation of Sfrp1 and Sox13 are essential aspects of the genetic program controlled by Phox2b in cranial motoneurons.
Pla
Patrick
P
Ecole normale supérieure, Département de Biologie, 75005 Paris, France. Patrick.Pla@curie.u-psud.fr
Hirsch
Marie-Rose
MR
Le Crom
Stéphane
S
Reiprich
Simone
S
Harley
Vincent R
VR
Goridis
Christo
C
D000818:Animals; D001324:Autoantigens / Q000235:genetics; D001934:Branchial Region / Q000166:cytology / Q000196:embryology / Q000502:physiology; D002642:Chick Embryo; D002645:Chickens; D003391:Cranial Nerves / Q000166:cytology / Q000196:embryology / Q000502:physiology; D005260:Female; D019922:GAP-43 Protein / Q000235:genetics; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins / Q000235:genetics; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins / Q000235:genetics*; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D009046:Motor Neurons / Q000166:cytology / Q000502:physiology*; D054259:Neural Tube / Q000166:cytology / Q000196:embryology / Q000502:physiology; D011247:Pregnancy; D013116:Spinal Cord / Q000166:cytology / Q000196:embryology / Q000502:physiology; D013234:Stem Cells / Q000166:cytology / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics*; D000094742:Secreted Frizzled-Related Proteins
D001324:Autoantigens; D019922:GAP-43 Protein; D049452:Green Fluorescent Proteins; D000090322:Homeobox Protein Nkx-2.2; D018398:Homeodomain Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D014157:Transcription Factors; C109220:NBPhox protein; C000715627:Nkx2-2 protein, mouse; C485539:Sfrp1 protein, mouse; D000094742:Secreted Frizzled-Related Proteins; C110455:Sox13 protein, mouse