FGFs control the patterning of the inner ear but are not able to induce the full ear program.

FGF2 or FGF8 applied ectopically, close to the developing otic placode enhances transcription of a subset of ear marker genes such as Nkx5-1, SOHo1 and Pax2. Other ear expressed genes (Dlx5 and BMP4) are not up-regulated by FGFs. Ectopic FGFs lead to an increase in size of the vestibulo-cochlear ganglion. This phenotypic change is due to an increased recruitment of epithelial cells to the neuronal fate rather than to an enhanced proliferation. We also observed an induction of additional, vesicle-like structures upon ectopic FGF treatment, but this induction never led to enrolment of a full ear program. We further demonstrate that FGF8 is expressed in two separate, short waves, first at the otic placode stage and later at the vesicle stage. Both activities correspond to critical morphogenetic events in ear development. We propose that FGF8 is an important regulator of otocyst patterning.

NKX2 gene expression in neuroectoderm but not in mesendodermally derived structures depends on sonic hedgehog in mouse embryos.

NKX2 genes in vertebrates encode a sub- family of homeodomain-containing transcription factors which regulate morphogenetic events and cell differentiation during embryogenesis. In mouse embryos several NKX2 genes are expressed in the ventral midline domains of the neuroectoderm, while other NKX2 genes are primarily expressed in the mesendoderm and mesendodermally derived organs, such as heart and gut. Within several patterning centers for tissue organization sonic hedgehog (Shh) is an important signal in the formation of ventral midline structures in vertebrate embryos. Here, we investigated the role of Shh in the embryonic expression of six different but closely related NKX2 genes in Shh null mutant mice. We found that expression of NKX2.1, NKX2.2, and NKX2.9 in neural domains requires Shh signaling, whereas NKX2.3, NKX2.5 and NKX2.6 expression in endoderm and mesoderm is independent of Shh.

Nkx2-9 is a novel homeobox transcription factor which demarcates ventral domains in the developing mouse CNS.

Nkx homeobox transcription factors are expressed in diverse embryonic cells and presumably control cell-type specification and morphogenetic events. Nkx2-9 is a novel family member of NK2 genes which lacks the conserved TN-domain found in all hitherto known murine Nkx2 genes. The prominent expression of Nkx2-9 in ventral brain and neural tube structures defines a subset of neuronal cells along the entire neuraxis. During embryonic development, Nkx2-9-expressing cells shift from the presumptive floor plate into a more dorsolateral position of the neuroectoderm and later become limited to the ventricular zone. Nkx2-9 expression overlaps with that of Nkx2-2 but is generally broader. While initially Nkx2-9 is expressed in close proximity to sonic hedgehog, its expression domain clearly segregates from sonic hedgehog at later developmental stages. The dynamic expression pattern of Nkx2-9 in ventral domains of the CNS is consistent with a possible role in the specification of a distinct subset of neurons.

Gene structure, expression in Escherichia coli and biochemical properties of the NAD+ -dependent glyceraldehyde-3-phosphate dehydrogenase from Pinus sylvestris chloroplasts.

Photosynthetic eukaryotes typically possess two distinct glyceraldehyde-3-phosphate dehydrogenases, an NAD+ -specific enzyme in the cytosol (GapC: EC 1.2.1.12) and an NADP+ -dependent enzyme in the chloroplast (GapAB: EC 1.2.1.13). The gymnosperm Pinus sylvestris is an exception in that it is known to express a gene encoding a transit peptide-bearing GapC-like subunit that is imported into chloroplasts (GapCp), but the enzymatic properties of this novel GAPDH have not been described from any source. We have expressed the mature GapCp unit from Pinus in Escherichia coli and have characterized the active enzyme. GapCp has a specific activity of 89 units per milligram and is strictly NAD+ -dependent, showing no detectable activity with NADP+. Values of the apparent Km for NAD+ and glyceraldehyde-3-phosphate were determined as 62 and 344 microM, respectively. The Pinus GapCpl gene possesses 12 introns, two in the region encoding the transit peptide and ten in the region encoding the mature subunit, all of which are found at positions strictly conserved across genes for higher plant GapC. A cDNA encoding a homologue of GapCp was isolated from the heterosporous fern Marsilea quadrifolia, indicating that NAD+ -dependent chloroplast GAPDH also occurs in other higher plants.

Two regulatory genes, cNkx5-1 and cPax2, show different responses to local signals during otic placode and vesicle formation in the chick embryo.

The early stages of otic placode development depend on signals from neighbouring tissues including the hindbrain. The identity of these signals and of the responding placodal genes, however, is not known. We have identified a chick homeobox gene cNkx5-1, which is expressed in the otic placode beginning at stage 10 and exhibits a dynamic expression pattern during formation and further differentiation of the otic vesicle. In a series of heterotopic transplantation experiments, we demonstrate that cNkx5-1 can be activated in ectopic positions. However, significant differences in otic development and cNkx5-1 gene activity were observed when placodes were transplanted into the more rostral positions within the head mesenchyme or into the wing buds of older hosts. These results indicate that only the rostral tissues were able to induce and/or maintain ear development. Ectopically induced cNkx5-1 expression always reproduced the endogenous pattern within the lateral wall of the otocyst that is destined to form vestibular structures. In contrast, cPax2 which is expressed in the medial wall of the early otic vesicle later forming the cochlea never resumed its correct expression pattern after transplantation. Our experiments illustrate that only some aspects of gene expression and presumably pattern formation during inner ear development can be established and maintained ectopically. In particular, the dorsal vestibular structures seem to be programmed earlier and differently from the ventral cochlear part.