Expression of avian Pax1 and Pax9 is intrinsically regulated in the pharyngeal endoderm, but depends on environmental influences in the paraxial mesoderm.

in Developmental Biology (1996), 178(2), 403-17

Pax1 and Pax9 represent a subfamily of paired-box-containing genes. In vertebrates, Pax1 and Pax9 transcripts have been found specifically in mesodermal tissues and the pharyngeal endoderm. Pax1 ... [more ▼]

Pax1 and Pax9 represent a subfamily of paired-box-containing genes. In vertebrates, Pax1 and Pax9 transcripts have been found specifically in mesodermal tissues and the pharyngeal endoderm. Pax1 expression in the sclerotomes has been shown to be indispensable for proper formation of the axial skeleton, but expression of Pax1 in the endoderm has not been studied in detail. We have cloned the chick homologue of the murine Pax9 gene. Our results show that transcripts of Pax1 and Pax9 are first detectable in the prospective foregut endoderm of headfold-stage avian embryos. Endodermal expression correlates with the highly proliferative zones of the folding foregut and evaginating pharyngeal pouches. In later stages, Pax1 and Pax9 are expressed in overlapping but distinct patterns within the developing sclerotomes and limb buds. From grafting experiments we conclude that activation of pharyngeal Pax1 and Pax9 expression is an intrinsic property of the endoderm, not requiring midline structures or head mesoderm. In contrast, notochord is required to induce Pax1 in competent sclerotomes. Here we show that in vitro there is a cranio-caudal gradient of inductive capacity in the notochord. This coincides with the graded expression of Pax1 and Pax9 along the cranio-caudal axis in 2- to 3-day-old embryos. Furthermore, paraxial head mesoderm shows no competence to express Pax1. Finally, in vitro we find counteracting influences on notochord signaling by lateral tissues (lateral plate, intermediate mesoderm), leading to an inhibition of Sonic hedgehog (Shh) expression in notochord and floor plate, as well as Pax1 and Pax9 expression in sclerotomes. Taken together, our results demonstrate that different mechanisms regulate expression of Pax1 and Pax9 in foregut and sclerotome, but suggest a common function for both genes in the two tissues that is promoting proliferation and preventing fusion of neighboring blastemas. [less ▲]

Pax-1, a regulator of sclerotome development is induced by notochord and floor plate signals in avian embryos.

in Anatomy & Embryology (1995), 191(4), 297-310

Pax-1 encodes for a DNA-binding transcriptional activator that was originally discovered in murine embryos using a probe from the Drosophila paired-box-containing gene, gooseberry-distal. We have cloned ... [more ▼]

Pax-1 encodes for a DNA-binding transcriptional activator that was originally discovered in murine embryos using a probe from the Drosophila paired-box-containing gene, gooseberry-distal. We have cloned the avian Pax-1 gene as a basis for experimental studies of the induction of Pax-1 in the paraxial mesoderm. The amino acid sequence of the paired-domain is exactly the same in the quail and mouse, whereas outside the paired-domain there is 61% homology. Starting at about the eight-somite stage, quail Pax-1 is expressed in the paraxial mesoderm in a craniocaudal sequence. The unsegmented paraxial mesoderm and the two most recently formed somites do not express Pax-1. In the epithelial somite, the somitocoele cells and the cells of the ventral two-thirds of the epithelial wall are positive. As soon as the sclerotome is formed, only a subset of sclerotome cells expresses Pax-1. These are the cells that migrate towards the notochord to form the perinotochordal tube. Expression then becomes restricted to the intervertebral discs, the perichondrium of the vertebral bodies and the connective tissue surrounding the spinal ganglia. Additional expression domains are found in the scapula and the pelvic region, distinct areas of the head, and the epithelium of the second to the fourth visceral pouch. In later stages the thymus is positive. In vitro and in vivo experiments show that the notochord induces Pax-1 in the paraxial mesoderm, but limb bud mesoderm is not competent to respond to notochordal signals. Floor plate is also capable of inducing Pax-1 expression in sclerotome cells. Our studies show that in competent cells of the paraxial mesoderm, Pax-1 is a mediator of signals emanating from the notochord and the floor plate. [less ▲]

The role of Pax-1 in axial skeleton development.

in Development (1994), 120(5), 1109-21

Previous studies have identified a single amino-acid substitution in the transcriptional regulator Pax-1 as the cause of the mouse skeletal mutant undulated (un). To evaluate the role of Pax-1 in the ... [more ▼]

Previous studies have identified a single amino-acid substitution in the transcriptional regulator Pax-1 as the cause of the mouse skeletal mutant undulated (un). To evaluate the role of Pax-1 in the formation of the axial skeleton we have studied Pax-1 protein expression in early sclerotome cells and during subsequent embryonic development, and we have characterized the phenotype of three different Pax-1 mouse mutants, un, undulated-extensive (unex) and Undulated short-tail (Uns). In the Uns mutation the whole Pax-1 locus is deleted, resulting in the complete absence of Pax-1 protein in these mice. The other two genotypes are interpreted as hypomorphs. We conclude that Pax-1 is necessary for normal vertebral column formation along the entire axis, although the severity of the phenotype is strongest in the lumbar region and the tail. Pax-1-deficient mice lack vertebral bodies and intervertebral discs. The proximal part of the ribs and the rib homologues are also missing or severely malformed, whereas neural arches are nearly normal. Pax-1 is thus required for the development of the ventral parts of vertebrae. Embryonic analyses reveal that although sclerotomes are formed in mutant embryos, abnormalities can be detected from day 10.5 p.c. onwards. The phenotypic analyses also suggest that the notochord still influences vertebral body formation some days after the sclerotomes are formed. Furthermore, the notochord diameter is larger in mutant embryos from day 12 p.c., due to increased cell proliferation. In the strongly affected genotypes the notochord persists as a rod-like structure and the nucleus pulposus is never properly formed. Since the notochord is Pax-1-negative these findings suggest a bidirectional interaction between notochord and paraxial mesoderm. The availability of these Pax-1 mutant alleles permitted us to define an early role for Pax-1 in sclerotome patterning as well as a late role in intervertebral disc development. Our observations suggest that Pax-1 function is required for essential steps in ventral sclerotome differentiation, i.e. for the transition from the mesenchymal stage to the onset of chondrogenesis. [less ▲]

A role for Pax-1 as a mediator of notochordal signals during the dorsoventral specification of vertebrae.

in Development (1993), 119(3), 649-60

The notochord plays an important role in the differentiation of the paraxial mesoderm and the neural tube. We have analyzed the role of the notochord in somite differentiation and subsequent formation of ... [more ▼]

The notochord plays an important role in the differentiation of the paraxial mesoderm and the neural tube. We have analyzed the role of the notochord in somite differentiation and subsequent formation of the vertebral column using a mouse mutant, Danforth's short-tail (Sd). In this mutant, the skeletal phenotype is most probably a result of degeneration and subsequent loss of the notochord. The Sd gene is known to interact with undulated (un), a sclerotome mutant. Double mutants between Sd and un alleles show an increase in the severity of the defects, mainly in the ventral parts of the vertebrae. We also show that part of the Sd phenotype is strikingly similar to that of the un alleles. As un is known to be caused by a mutation in the Pax-1 gene, we analyzed Pax-1 expression in Sd embryos. In Sd embryos, Pax-1 expression is reduced, providing a potential molecular basis for the genetic interaction observed. A complete loss of Pax-1 expression in morphologically intact mesenchyme was found in the lower thoracic-lumbar region, which is phenotypically very similar to the corresponding region in a Pax-1 null mutant, Undulated short-tail. The sclerotome developmental abnormalities in Sd coincide closely, both in time and space, with notochordal changes, as determined by whole-mount T antibody staining. These findings indicate that an intact notochord is necessary for normal Pax-1 expression in sclerotome cells, which is in turn required for the formation of the ventral parts of the vertebrae. The observed correlation among structural changes of the notochord, Pax-1 expression levels and skeletal phenotypes, suggests that Pax-1 might be an intrinsic mediator of notochordal signals during the dorsoventral specification of vertebrae. [less ▲]