Follicular cells of the thyroid gland require Pax8 gene function.

The thyroid gland develops from two distinct embryonic lineages: follicular cells (which produce thyroxine) and parafollicular C-cells (which produce calcitonin) are of endodermal and neural crest origin, respectively. Little is known about the molecular mechanisms governing the generation of these different cell types. Mice lacking the transcription factor Ttf1 lack both cell types and thus are unable to develop a thyroid gland. By analysis of Pax8-/- mice, we demonstrate that Pax8 is required for the formation of the follicular cells in the thyroid. We present evidence that Pax8 is necessary for providing cues for the differentiation of competent endoderm primordia into thyroxin-producing follicular cells.

Prox1 function is crucial for mouse lens-fibre elongation.

Although insights have emerged regarding genes controlling the early stages of eye formation, little is known about lens-fibre differentiation and elongation. The expression pattern of the Prox1 homeobox gene suggests it has a role in a variety of embryonic tissues, including lens. To analyse the requirement for Prox1 during mammalian development, we inactivated the locus in mice. Homozygous Prox1-null mice die at mid-gestation from multiple developmental defects; here we describe the specific effect on lens development. Prox1 inactivation causes abnormal cellular proliferation, downregulated expression of the cell-cycle inhibitors Cdkn1b (also known as p27KIP1) and Cdkn1c (also known as p57KIP2), misexpression of E-cadherin and inappropriate apoptosis. Consequently, mutant lens cells fail to polarize and elongate properly, resulting in a hollow lens. Our data provide evidence that the progression of terminal fibre differentiation and elongation is dependent on Prox1 activity during lens development.

Mutations in the PAX3 gene causing Waardenburg syndrome type 1 and type 2.

Waardenburg syndrome (WS) is a combination of deafness and pigmentary disturbances, normally inherited as an autosomal dominant trait. The pathology involves neural crest derivatives, but WS is heterogeneous clinically and genetically. Some type I WS families show linkage with markers on distal 2q and in three cases the disease has been attributed to mutations in the PAX3 gene. PAX3 encodes a paired domain, a highly conserved octapeptide and probably also a paired-type homeodomain. Here we describe a further three PAX3 mutations which cause WS; one alters the octapeptide motif plus the presumed homeodomain; a second alters all three elements and the third alters the paired box alone. The latter occurs in a family with probable type 2 WS, a clinical variant usually considered not to be allelic with type 1 WS.

Pax6 lights-up the way for eye development.

Recent reports have exposed the temporal and spatial functions of the transcription factor Pax6 in the developing vertebrate eye. Pax6 is demonstrated to play essential roles in successive steps triggering lens differentiation while in the retina it functions to maintain multipotency and proliferation of retinal progenitor cells. These findings, together with the identification of Pax6 protein partners and downstream targets, pave the way for future work aimed to understand the molecular mechanism of eye development.

Pax genes and their roles in cell differentiation and development.

Members of the Pax gene family are expressed in various tissues during ontogenesis. Evidence for their crucial role in morphogenesis, organogenesis, cell differentiation and oncogenesis is provided by rodent mutants and human diseases. Additionally, recent experimental in vivo and in vitro approaches have led to the identification of molecules that interact with Pax proteins.

Differential induction of Pax genes by NGF and BDNF in cerebellar primary cultures.

The Pax genes encode sequence-specific DNA binding transcription factors that are expressed in embryonic development of the nervous system. Primary neuronal cell cultures derived from the cerebellar cortex of embryonic day 14, newborn and 7-d old mice, were used to investigate the cell-type specific expression patterns of three members of the murine paired box containing gene family (Pax gene family), in vitro. Cell types which express Pax-2, Pax-3, and Pax-6 RNA in primary cultures correspond to those found in regions of the cerebellum which show RNA signals in sections of the developing mouse brain. To find mechanisms regulating Pax gene expression during cerebellar development, the differential regulation of Pax-2, Pax-3, and Pax-6 by NGF and BDNF, two structurally related neurotrophins, was studied in such primary cultures. Pax-2 and Pax-6 RNA increased slightly by 1 h and remained elevated throughout a 24-h treatment with BDNF and NGF. Pax-3 RNA was not detected in newborn cultures, but underwent a rapid (1 h) and transient (2 h) induction upon treatment with either BDNF or NGF. No response was seen with EGF or FGF. Cycloheximide treatment amplified Pax-3 induction and prolonged the signal. Thus, Pax-3 induction resembles that of the immediate-early gene c-fos, which transduces growth factor signals during the development of particular neuronal/glial cell types. The changes in Pax expression were inductive rather than trophic.

Murine developmental control genes.

Various strategies have been used to isolate genes that participate in the regulation of mouse development. Gene families that have been identified on the basis of their homology to motifs within Drosophila control genes or human transcription factor genes, namely homeobox (Hox), paired-box (Pax), and POU genes, can be compared with respect to gene organization, structure, and expression patterns. The functions of these genes can be analyzed molecularly in vitro and in vivo with the use of available mouse mutants or transgenic mice. In addition, it has been possible to generate gain- or loss-of-function mutations by random or targeted introduction of transgenes. Models derived from these studies can reveal the successive steps of developmental control on a genetic level.

Multiple point mutations affecting the simian virus 40 enhancer.

Enhancers, or activators, dramatically increase the transcriptional activity of certain eukaryotic genes. A series of multiple point mutations affecting the simian virus 40 (SV40) enhancer-activator region were generated in order to define the nucleotide sequence required for this function. Three independent assays provided information leading to the identification of nucleotides essential for enhancer function. One class leads to a decrease in gene expression, while the second completely abolishes functional activity. One critical replacement appears to be the first G (guanine) in a sequence TGGAAAG (T, thymine, A, adenine) located in the 5' region of the 72 base-pair repeat of SV40. Comparison of this sequence with nucleotide sequences in other known enhancers leads to the identification of potential related core elements.

BK viral enhancer element and a human cellular homolog.

Comparison of two closely related primate papovaviruses, simian virus 40 (SV40) and human BK virus (BKV), reveals that the only region of extensive divergence, the tandem sequences adjacent to the origins of DNA replication, is responsible in SV40 for enhancing early gene expression. This study demonstrates a similar enhancer function for the analogous repeated region in BKV. The dissimilarity in sequence of the BKV and SV40 enhancer elements suggests that they may have been acquired since SV40 and BKV diverged. A locus cloned from the human genome homologous to the BKV tandem repeats has been shown to function as low level enhancer element in mammalian cells. These data support the hypothesis that viral enhancer sequences may be evolutionarily related to host cell sequences.

Pax6 controls radial glia differentiation in the cerebral cortex.

Radial glia cells perform a dual function in the developing nervous system as precursor cells and guides for migrating neurons. We show here that during forebrain neurogenesis, the transcription factor Pax6 is specifically localized in radial glia cells of the cortex but not of the basal telencephalon. In Pax6-deficient mice, cortical radial glia cells were altered in their morphology, number, tenascin-C (TN-C) expression, and cell cycle. We show that some of these alterations are cell-autonomous, whereas others were rescued by coculturing with wild-type cortical cells. Our results suggest that Pax6 plays an essential role in the differentiation of cortical radial glia. Thus, despite their widespread distribution, radial glia cells are regionally specified in the developing CNS.

Pax3: a paired domain gene as a regulator in PNS myelination.

Pax3 RNA is expressed in neural crest when Schwann cell (SC) precursors migrate to the PNS. Pax3 RNA and SC markers were monitored in sciatic nerves of mice during development and nerve repair. An inverse correlation was observed between expression of Pax3 RNA and myelin basic protein (MBP). Inverse correlation was also observed in SC primary cultures. Treating cultures with forskolin, an adenylate cyclase agonist, repressed Pax3 RNA, GFAP, NGFR, N-CAM, and L1 and elevated MBP. Subsequent microinjection with Pax3 expression vector elevated Pax3 RNA, GFAP, NGFR, N-CAM, and L1 and repressed MBP. Thus, Pax3 is likely involved in the differentiation pathway to myelinating SCs. Pax3 repressed a 1.3 kb MBP promoter fragment in cotransfection assays, suggesting that it represses MBP transcription.

Mini-Oct and Oct-2c: two novel, functionally diverse murine Oct-2 gene products are differentially expressed in the CNS.

We report that two novel alternatively spliced products of the murine Oct-2 gene encode Mini-Oct (Oct-2d), a protein consisting of almost only the POU domain, and Oct-2c, a protein lacking the last 12 amino acids of Oct-2a. Ectopic expression in HeLa cells shows that Oct-2c is a transactivator, whereas Mini-Oct fails to transactivate if the octamer motif is in a promoter position next to TATA box. Mini-Oct can repress the transcriptional signal generated by endogenous octamer factors in F9 cells. It seems that Mini-Oct has the potential to serve as a transcriptional modulator for genes regulated by different octamer-binding factors. In situ hybridization reveals that Mini-Oct expression follows the general pattern of other known Oct-2 transcripts. However, it is absent from the Purkinje cell layer in the cerebellum of adult mice, and strong expression is observed in the developing nasal neuroepithelium and primary spermatids. Differential expression patterns of the Oct-2 transcripts with different transactivation/repression capacities of the encoded proteins may have a specific role in gene expression in the developing nervous system and in adult brain.

Axial specification in higher vertebrates.

The specification of the body axis in the embryo is reflected in the structure of the vertebral column. Expression patterns of Hox genes in the prevertebrae suggest their involvement in this specification process and in the maintenance of vertebral identities. A single, ectopically expressed Hox gene can reprogramme the rostral part of the vertebral column and induce phenotypic alterations interpretable as homeotic transformations.

Pancreas development and diabetes.

The past few years have seen an increase in interest about the molecular and genetic events regulating pancreas development. Transcription factors such as Pdx1, p48 and Nkx2.2 have been shown to be essential for the proper differentiation of exocrine and endocrine tissue; however, pancreas development also involves intricate interactions between the pancreatic epithelium and its surrounding mesenchyme. Signalling factors emanating from the notochord have been shown to repress Sonic hedgehog expression in the endoderm whereas signals originating from the pancreatic mesenchyme determine the proportion of exocrine to endocrine tissue. Understanding the molecular and genetic events underlying pancreas development also opens the door for devising new therapeutic strategies against pancreatic diseases such as diabetes and cancer.

Interdigital cell death can occur through a necrotic and caspase-independent pathway.

Programmed cell death in animals is usually associated with apoptotic morphology and requires caspase activation. Necrosis and caspase-independent cell death have been reported, but mostly in experimental conditions that lead some to question their existence it in vivo. Loss of interdigital cells in the mouse embryo, a paradigm of cell death during development [1], is known to include an apoptotic [2] and caspase-dependent [3] [4] mechanism. Here, we report that, when caspase activity was inhibited using drugs or when apoptosis was prevented genetically (using Hammertoe mutant mice, or mice homozygous for a mutation in the gene encoding APAF-1, a caspase-activating adaptor protein), interdigital cell death still occurred. This cell death was negative for the terminal-deoxynucleotidyl-mediated dUTP nick end-labelling (TUNEL) assay and there was no overall cell condensation. At the electron microscopy level, peculiar 'mottled' chromatin alterations and marked mitochondrial and membrane lesions, suggestive of classical necrotic cell death, were observed with no detectable phagocytosis and no local inflammatory response. Thus, in this developmental context, although caspase activity confers cell death with an apoptotic morphotype, in the absence of caspase activity an underlying mechanism independent of known caspases can also confer cell death, but with a necrotic morphotype. This cell death can go undetected when using apoptosis-specific methodology, and cannot be blocked by agents that act on caspases.

Interaction of distinct nuclear proteins with sequences controlling the expression of polyomavirus early genes.

The interaction between cellular factors and polyoma virus (Py) DNA was investigated by using a gel retention assay. Nuclear extracts from various cell lines (NIH 3T3, NIH 3T6, LTK-, F9) contained proteins that formed specific and distinct complexes with Py B enhancer fragments of either wild-type or F9-1 mutant origin. The presence of an excess amount of other well-characterized DNA sequences, including the Py A enhancer, the murine sarcoma virus enhancer, and the simian virus 40 enhancer-promoter region, did not interfere with this protein-DNA interaction. However, a fragment previously defined as containing the lymphotropic papovavirus enhancer shares the binding of some common factor. This observation, in combination with the results of retention gel assays at different Mg2+ concentrations, indicates the interaction of several nuclear factors and Py DNA. The assay systems that were used allowed a distinction between some factors on the basis of their different biochemical and sequence requirements. The contact sites of these complexes were mapped to the B enhancer region of Py with Bal 31-derived mutant restriction fragments and ExoIII nuclease and are compatible with the functional domains determined in vivo.

Functional analysis of mouse Hoxa-7 in Saccharomyces cerevisiae: sequences outside the homeodomain base contact zone influence binding and activation.

The murine developmental control gene product, Hoxa-7, was shown to function as a DNA-binding transactivator in Saccharomyces cerevisiae. The importance of the ATTA core, the preference for antp class flanking nucleotides, the importance of Asn-51 of the homeodomain (HD), and the synergism of multiple binding sites all reflect properties that have previously been described for HOM or Hox proteins in tissue culture systems. A comparison of contact positions among genes of paralog groups and classes of mammalian HDs points to a lack of diversity in positions that make base contact, suggesting that besides the combination of HD amino acid-base pair contacts, another means of recognizing differences between targets must exist if Hox genes select different targets. The HD of antennapedia is identical to the Hoxa-7 HD. The interaction of Hoxa-7 with the exact sequence used in the nuclear magnetic resonance three-dimensional structural analysis on the antennapedia HD was studied. Hoxa-7 binding and transactivation was influenced by sequences outside of the known base contact zone of this site. We conclude that Hoxa-7 protein has a second means to interact with DNA or/and that the sequences flanking the base contact zone influence HD interactions by distorting DNA within the contact zone (base or backbone). This result is discussed in terms of DNA flexure and two modes of transcription used in S. cerevisiae.

A transcription factor interacting with the class I gene enhancer is inactive in tumorigenic cell lines which suppress major histocompatibility complex class I genes.

AKR leukemias display different amounts of major histocompatibility complex class I antigens on the cell surface. The absence of H-2Kk molecules correlates with the ability of these cell lines to form tumors in vivo as well as to escape lysis by cytotoxic T lymphocytes in vitro. In this report it is shown that the 5' regulatory area of the H-2Kk gene failed to activate transcription in H-2Kk-negative cells. Examination of the proteins interacting with the H-2Kk enhancer in expressing and nonexpressing cells revealed clear differences. In particular, the level of a nuclear protein interacting at position -166 was greatly reduced in the negative cell lines. A transcription factor, known as H2TF1 or KBF1, has been shown previously to interact with this binding site and to be essential for the expression of certain class I genes as well as the expression of beta 2-microglobulin. These results demonstrate that the molecular mechanism of class I gene suppression in malignant tumor cells is at the level of transcription and is most probably modulated by H2TF1/KBFI. In addition, it is shown that the same transcription factor is only present in mouse tissues expressing class I antigens.

Bovine papilloma virus deoxyribonucleic acid: a novel eucaryotic cloning vector.

A novel eucaryotic vector derived from the transforming region of bovine papilloma virus was established and demonstrated to be highly effective for introducing foreign genes into animal cells. The foreign deoxyribonucleic acid (DNA) is replicated and actively transcribed as an episome, and the transcripts are translated into an authentic gene product. We have constructed a DNA hybrid molecule, BPV69T-rI1, containing the transforming region of bovine papilloma virus DNA and the rat preproinsulin gene I (rI1), and used it to transform susceptible mouse cells. DNA hybridization analysis has demonstrated the presence of multiple unintegrated copies of hybrid DNA molecules, with the bovine papilloma virus 1 DNA segment and the rI1 gene covalently linked in selected transformed cell lines. S1 nuclease analysis revealed the presence of a correctly spliced coding segment of the preproinsulin transcript similar or identical in its electrophoretic mobility to that of messenger ribonucleic acid produced in rat insulinoma cells. Significant levels of a protein immunoreactive with anti-insulin serum were detected by radioimmunoassay in the culture medium of transformed cells. Immunoprecipitation analysis in conjunction with competitive binding to bovine proinsulin established the identity of the protein as that of rat proinsulin.

Current views on eye development.

Several genes involved in the regulation of eye development in different species have been identified. Structural and functional conservation have been found between some of these genes in organisms as diverse as Drosophila and mouse. One notable example is the relationship between the mouse Pax6 gene and eyeless of Drosophila. Ectopic expression of eyeless or mouse Pax6 in Drosophila results in the formation of additional eyes. Recently, another homeobox gene, Six3, was found to promote ectopic lens formation in fish embryos. The next step will be to unravel the associated regulatory pathways of these genes and assess the degree to which they display evolutionary conservation. This will be important in order to assimilate these findings with current anatomical and embryological models. It seems reasonable to believe that in the near future the characterization of the whole framework required for vertebrate eye development will be accomplished.