BIOLOGICAL OVERVIEW

Is trachealess the master gene for trachea induction? Probably not. No tracheal pits are seen in trh knockouts. Nevertheless, other genes expressed in trachea, the respiratory system of the fly, are not affected by trachealess mutation during early stages of development (for example, crumbs, an integral membrane protein and drifter/ventral veinless, a transcription factor expressed in and required by tracheal cells). Thus the defect in trh mutant embryos lies not in the identity of tracheal cells, because they continue to express other tracheal genes, but rather a defect in the ability of the tracheal precursors to organize tubes, the same defect seen in salivary glands.

Breathless, a Drosophila FGF-receptor gene is required for tubulogenesis, but in breathless mutants, initial tracheal invagination appears normal (Isaac, 1996). trachealess expression in trachea development is likely to be controlled by segment polarity genes because of the segment-to-segment iterated pattern of expression. In salivary gland trachealess espression is under the control of Sex combs reduced and forkhead. trachealess is thus the first potential downstream gene of SCR, with a distinct role in morphological changes involved in organ formation (Isaac, 1996).

Genetic analyses have identified several genes that are specifically expressed in the tracheal cells and are required for tracheal development. Among them, trachealess (trh) is able to induce ectopic tracheal pits and therefore it has been suggested that it would act as an inducer of tracheal cell fates; however, this capacity appears to be spatially restricted. The expression of the tracheal specific genes in the early steps of tracheal development and their crossinteractions have been examined. There is a set of primary genes including trh and ventral veinless (vvl) whose expression does not depend on any other tracheal gene and a set of downstream genes whose expression requires different combinations of the primary genes. The combined expression of primary genes is sufficient to induce some downstream genes but not others. These results indicate that there is not a single master gene responsible for the appropriate expression of the tracheal genes and support a model where tracheal cell fates are induced by the cooperation of several factors rather than by the activity of a single tracheal inducer (Boube, 2000).

trh and vvl appear to initiate or to act very early in the genetic hierarchy specifying tracheal development. vvl expression in the tracheal cells is independent of trh function. It is also found that trh expression in the tracheal cells is independent of vvl function indicating that the two genes act in parallel in the control of tracheal cell development. btl, a gene encoding an FGF receptor homolog required for tracheal migration, is a target of trh: btl requires vvl for the maintenance of its transcription. Transduction of the FGF signalling also requires the Downstream of FGF (Dof) protein, which is specifically expressed in the tracheal cells. However, dof is not a target gene activated as a result of FGF signaling as its expression is not affected in btl mutant embryos. Conversely, the results show that the specific expression of dof in the tracheal cells is dependent on trh and vvl activity. Thus, trh and vvl enable the tracheal cells to be competent to FGF signaling by regulating the expression of at least two elements (btl and dof) acting at different steps in the Btl pathway (Boube, 2000).

In contrast to the general requirement of the Btl pathway, the Dpp and EGF pathways are required for migration of certain branches of the tracheal system; competence of the tracheal cells to those signals depends on the specific tracheal expression in the tracheal cells of tkv and rho, respectively. Similarly to the btl pathway, rho expression in the tracheal cells depends both on trh and vvl function. However, while tracheal expression of tkv also depends on vvl, it appears to be independent of trh. The opposite appears to be the case for two other tracheal genes, tracheal defective (tdf) and pebbled (peb) [also known as hindsight (hnt)], which code for two putative transcription factors. Both genes appear to be targets of trh but they are present in the tracheal cells of vvl mutant embryos. Thus, some tracheal genes seem to be common targets of vvl and trh but others seem to depend only on one of them (Boube, 2000).

Whether knirps (kni) and knirps related (knrl) would fit in this genetic hierarchy was also analyzed. Both genes code for putative transcription factors that are expressed in overlapping patterns and share redundant functions during tracheal development; both genes have an early expression in the tracheal placodes and a later expression in a particular subset of tracheal branches. Quite surprisingly, it was found that the early tracheal expression of kni is not abolished in either trh or vvl mutant embryos and also that all the tracheal genes mentioned above are expressed in the tracheal cells of embryos that are mutant for a deficiency that uncovers both kni and knrl. In summary, there are some primary tracheal genes whose expression appears not to be regulated by any other tracheal gene. Subsequently, one or more of those primary genes are necessary for the appropriate expression of other downstream genes in the tracheal cells (Boube, 2000).

The complexity of regulatory interactions described above indicates that more than one gene can act as an inducer of the expression of downstream tracheal genes. This seems to contrast with earlier results suggesting that the trh gene acts as the master gene for tracheal fate. Evidence for this comes from experiments of ubiquitous expression of trh, which generates new tracheal pits at the correct position in anterior and posterior segments that normally do not form pits. The same result is observed when an UAS-trh construct is specifically expressed in the embryonic terminal regions by means of a sal-Gal4 line. In both cases, induction of extra tracheal pits can be visualized very early in development by the appearance of additional clusters of cells that express btl in more anterior and posterior segments. However, the capacity of trh to induce btl expression appears restricted to specific positions in the embryo. The restricted activation of btl by trh is not due to low levels of trh since the use of the Gal4/UAS system induces high levels of trh transcripts. Conversely, the alternative conclusion is favored: the activity of trh alone is not sufficient to induce btl expression, probably because other factors are also required in combination with trh. vvl is a good candidate for such a factor. vvl is required to induce some of the tracheal genes. In addition, vvl is expressed independent of trh in the tracheal placodes and in the analogous location within the segments that do not form tracheal pits. These are precisely the positions where trh can induce additional tracheal pits. Thus, it was asked whether both trh and vvl are required to instruct those cells to adopt a tracheal fate. On inducing an UAS-trh construct in the terminal regions of vvl mutant embryos (with the same sal-Gal4 line as above), ectopic induction of btl in new patches of cells is suppressed. This result indicates that it is the localized expression of vvl that accounts for the restricted induction of btl in a particular set of cells upon general expression of trh. The situation is different in the normal tracheal placodes where vvl is dispensable for induction of btl expression and is only required for its maintenance (Boube, 2000).

The above experiments indicate that vvl is required for the induction of extra tracheal pits in additional segments. However, general expression of vvl with an UAS-vvl construct does not induce additional tracheal pits or ectopic expression of btl. It was asked whether the co-expression of vvl and trh would be sufficient to induce tracheal fates, as monitored by induction of btl expression. Indeed, simultaneous expression of an UAS-vvl and an UAS-trh construct in the embryonic terminal regions under the common control of the sal-Gal4 line induces btl expression throughout both regions. Also, co-expression of vvl and trh in unrelated regions such as the distal leg primordia (directed by a Dll-Gal4 line) is sufficient to induce btl expression. Thus, vvl and trh are both required and their co-expression is sufficient to ectopically induce btl expression (Boube, 2000).

On the contrary, vvl and trh appear not to be sufficient for the expression of the remaining tracheal genes. While tracheal expression of dof, tdf, peb, tkv and rho require either vvl or trh, or both, no induction of any of these genes is observed upon ectopic expression of vvl and/or trh. Therefore, these results raise the possibility that full induction of tracheal fates requires one or more additional factors. In this regard it is worth noting that the tracheal branches generated from ectopic trh in vvl expressing cells are abnormal and do not fuse with the normal tracheal tree (Boube, 2000).

Expression of trh is repressed by sal in the terminal regions leading to the suggestion that this is the mechanism that accounts for the confinement of tracheal placodes to the central segments of the embryo. In contrast, vvl is expressed at the correct positions in segments that normally do not form tracheal placodes, although its expression in those sites is much weaker. Whether sal could also regulate vvl expression was investigated. Indeed, vvl expression is strongly increased in those sites in sal mutant embryos suggesting that vvl is downregulated by sal in the segments that do not form tracheal pits. Similarly, kni expression is also upregulated in the same sites in sal mutant embryos. Repression of vvl and kni by sal could in principle be attributed to the downregulation of trh by sal. However, this seems not to be the case because expression of vvl and early expression of kni in the tracheal placodes does not depend on trh. Therefore, sal seems to independently downregulate trh, vvl and kni in the most anterior and posterior embryonic regions (Boube, 2000).

Because trh and vvl are sufficient to activate btl, additional patches of btl expression were found in sal mutant embryos. dof and rho are expressed in additional patches of cells in sal mutant embryos. Repression of dof and rho by sal could also be attributed to the downregulation of trh and vvl by sal. However, this seems not to be the case because co-expression of trh and vvl in the sal domain is not sufficient to induce either dof or rho expression. Instead, sal could directly repress dof and rho or, alternatively, it could repress an additional factor necessary for their induction. In summary, many tracheal genes appear to be independently downregulated by sal in the terminal regions. Besides, the lack of sal expression does not have the same effect on the tracheal genes. In particular, some of the additional patches of trh expression are much weaker than the normal ones. This difference is not so pronounced in the case of vvl expression in sal mutant embryos. Also, one additional anterior pair of cell clusters for rho and dof expression is observed. Therefore, not all the tracheal placodes are equivalent in sal mutant embryos (Boube, 2000).

Salivary eyegone expression is regulated positively by Sex combs reduced and trachealess (trh) but is regulated negatively by forkhead. Scr, the homeotic gene responsible for patterning parasegment 2, is responsible for the activation of every salivary gene that has been tested. As expected, eyg is not expressed in the salivary primordium of Scr-mutant embryos. The trh gene product is necessary for invagination of all salivary duct cells and it is required for expression of downstream duct markers. Because eyg is also expressed in part of the salivary duct primordium, the relationship between trh and eyg was tested in the pathway for duct determination. In wild-type embryos, both trh and eyg expression in the salivary primordium begin early during stage 10. At this stage, eyg expression in trh-mutant embryos is indistinguishable from expression in wild-type embryos. Therefore, initiation of eyg expression in the salivary primordium is independent of trh. In early stage 12, however, eyg expression becomes dependent on trh. Although eyg is expressed strongly in the posterior preduct cells of wild-type embryos, this expression is completely absent in trh-mutant embryos. Therefore it is eyg maintenance, and not its initiation, that depends on trh. Whether trh expression depends on eyg was also tested and it was found that trh expression is unaffected in eyg null-mutant embryos (Jones, 1998).

forkhead plays an important role in establishing the pregland/preduct border by dorsally limiting duct-specific gene expression. trh, like eyg, is also initially expressed throughout the gland primordium. In fkh-mutant embryos, trh transcript never disappears from the pregland cells (Isaac, 1996). Does fkh play a similar negative regulatory role in eyg transcription? When the wild-type eyg expression pattern is compared to that of fkh-mutant embryos, it becomes clear that fkh indeed negatively regulates eyg. eyg expression persists in gland precursors in fkh-mutant embryos. Thus, fkh represses expression of trh and eyg, both of whose expression disappears from the pregland cells at approximately the same time. eyg plays no role in the regulation of fkh expression (Jones, 1998).

Armed with the knowledge that (1) fkh is responsible for the exclusion of both trh and eyg from the pregland cells and (2) trh is necessary for maintenance of eyg expression in the duct cells, it is possible to ask whether fkh represses eyg in the pregland cells simply by repressing trh or if fkh downregulates trh and eyg independently. To address this question, embryos were generated that were doubly mutant for trh and fkh. If the reason for eyg disappearance from the pregland cells in wild-type embryos is disappearance of trh, then it would be predicted that eyg expression would not persist in trh;fkh-mutant embryos. eyg expression, however, does persist in pregland cells in trh;fkh-mutant embryos, suggesting that trh plays no role in eyg repression by fkh. Thus, after the initial establishment of the salivary primordium by Sex combs reduced, forkhead excludes eyegone expression from the pregland cells so that eyegone's salivary expression is restricted to the posterior preduct cells. trachealess, in contrast, activates eyegone expression in the posterior preduct cells (Jones, 1998).

GENE STRUCTURE

Exons - 10

Base pairs in 3' UTR - 980

PROTEIN STRUCTURE

There are two instances of alternative splicing in the region between the two PAS domains (Wilk, 1996 and Isaac, 1996).

Amino Acids - 949, 925 and 896

Structural Domains

The Trachealess protein has an N-terminal bHLH domain and two central PAS domains. The translational activation domain in the C-terminal portion of the molecule is rich in proline glutamine and serine (Wilk, 1996 and Isaac, 1996).

EVOLUTIONARY HOMOLOGS

The protein displays the greatest degree of homology to Single-minded (Wilk, 1996). TRH is homologous to human hypoxia inducible factor-1 alpha. Distal to the C-terminal of the PAS domain there is a region of homology with HIF-1 alpha and Single minded (Isaac, 1996).

Embryonic development of the Bombyx silk gland is described in this study. To extend the analysis further, a Bombyx counterpart gene of the Drosophila trachealess gene has been isolated. Bombyx trh encodes a protein of 849 amino acids. When compared with the amino acid sequence of Drosophila trh, the identity of Bombyx bHLH, PAS-A and PAS-B domains is 100%, 97%, and 80%, respectively. Northern blot analysis reveals the presence of a single Bombyx trh transcript of 5.4 kb. The expression pattern of the Bombyx trh transcript during embryogenesis was examined by in situ hybridization. Bombyx trh mRNA is first detected in the tracheal primordial cells at around embryonic stage 18. Thereafter levels of Bombyx trh mRNA increases, and the high expression level is maintained until hatching. At embryonic stage 19 the transcript is also detected in the posterior basal region of the labial segment from where the silk gland invaginates. By the blastokinesis stage (around stage 23), the silk gland is lengthened, and, interestingly, the Bombyx trh transcript is restricted to the anterior silk gland. These results suggest that Bombyx trh plays a role in the formation of the trachea and the anterior silk glands (Matsunami, 1999).

It has been proposed that the Bombyx mori silk gland is a modified salivary gland because of its origin from the labial segment. Indeed, although silk moth larvae also possess a pair of salivary glands that is derived from the mandibular segment, the silk gland shares many similarities with the larval salivary gland of Drosophila in its function and postembryonic development. For instance, when the PSG specific Bombyx silk gene P 25 was introduced into Drosophila eggs, P 25 was specifically but enigmatically expressed in the anterior portion of the salivary gland of the transformed Drosophila larvae. This finding suggests that there are similar mechanisms of transcriptional regulation in the Drosophila salivary gland and the Bombyx silk gland. These two species are evolutionarily quite close, although 240 million years separation is probably a minimum estimate. Thus it is believed that the studies of the Drosophila salivary gland gene homologs would bring useful hints for understanding the mechanism of Bombyx silk gland gene regulation (Matsunami, 1999).

The silk glands of the silkworm Bombyx mori produce vast amounts of several silk proteins. The L-chain fibroin gene is expressed in the posterior part of the silk glands (PSG) whereas several serine-rich proteins, products of the sericin-1 gene, are expressed in the middle part of the silk glands (MSG). Both the fibroin and sericin-1 genes are actively expressed in the feeding stages but are repressed during molting stages. Several silk gland factors (SGF-1-4) involved in controlling the silk genes have been identified. SGF-1 interacts with the SA site of the sericin- 1 promoter and with A and B sites of the fibroin promoter. SGF-1 has been identified to be a homolog of Drosophila Fork head (Fkh), a member of the Fork head/HNF-3 family of transcriptional regulators. SGF-2 interacts with the C, D, and E sites of the fibroin promoter. SGF-2 has recently been identified as a complex including homologs of Lim, nuclear Lim interactors and other proteins. SGF-3 interacts strongly with the SC site and weakly with the SB site of the sericin-1 promoter, and weakly with the C, D, and E sites of the fibroin promoter. SGF-3 has been identified as a homolog of Drosophila Cf1a (Ventral veins lacking) (Matsunami, 1999 and references therein).

The silk gland starts to invaginate at embryonic stage 19. An initial tubular structure of the silk gland is observed at stage 19-20, and by this stage the tracheal pits are observed. The two pits of the silk gland gradually moved toward the midline and finally fuse to form one spinneret during head development. The silk gland continues to elongate in contact with branches of the visceral trachea. At around embryonic stage 23, the distal end of the silk gland reaches to the probable muscle originated from the sixth abdominal segment, and the MSG and PSG are held entirely by the tracheal branches. Since the distal end of the silk gland is fixed in the sixth abdominal segment by stage 23, the silk gland is probably forced to curve as the cells grow and divide thereafter, and the MSG finally becomes thick and shaped as the letter 'S' due to pulling tension by the two abdominal transverse tracheae and the characteristic morphological structure of the silk gland becomes apparent. The cells gradually rearrange from the beginning of silk gland development, and by stage 23 the cells surrounding the lumen of the silk gland become arranged in two orderly rows. After stage 23 the cells continue to grow, but no cell division takes place. When the silk gland arrives at the maximum cell numbers, there are about 250 cells for the anterior silk gland (ASG), 200 cells for the MSG, and 450 cells for the PSG. Thus by around stage 25 the silk gland has completed morphological change and differentiated into three specific parts. The function of the ASG is thought to be as a duct for silk proteins, and MSG and PSG are secretory tissues of silk proteins. These functional differences suggest that there is at least one regulatory gene that is differently expressed and determines the specific cell fate between the duct cells part, ASG, and the secretory parts, MSG and PSG. It is hypothesized that the Bombyx counterpart of Drosophila trachealess is expressed and plays a role in silk gland development (Matsunami, 1999).

A model for silk gland development is presented. In Drosophila embryos Drosophila Scr appears to direct salivary gland formation by regulating the transcription of downstream genes whose products uniquely define the salivary gland. Drosophila trh and fkh expression in the salivary gland is activated by Drosophila Scr. The Bombyx Nc mutant lacks the homeobox and its downstream region of Bombyx Antennapedia (Antp). The loss of function of Bombyx Antp causes an ectopic expression of Bombyx Scr, and as a result additional silk gland formation is induced. Therefore the Bombyx Scr gene is essential for silk gland induction and belongs to an upper part of the cascade required for silk gland formation. In the wild-type embryo Bombyx Scr is first expressed at stage 16 in the labial segment where the silk gland invaginates. Interestingly Bombyx Scr expression in the silk gland primordia is diminished at stage 18. Alternatively, SGF-1/Bombyx Fkh, Bombyx trh, and SGF-3/POU-M1 start to be expressed at stages 18-19. These findings show that Bombyx Scr might directly or indirectly regulate the SGF-1/Bombyx Fkh, Bombyx trh, and SGF-3/POU-M1 expression. These expressions continue during silk gland development, and finally Bombyx trh expression is restricted to ASG, SGF-1/Bombyx Fkh to MSG and PSG, and SGF-3/POU-M1 to ASG and MSG. These results suggest that these factors are important for ASG, MSG and PSG, and ASG and MSG development, respectively. It is suggested that Bombyx trh, SGF-1/Bombyx Fkh and SGF-3/POU-M1 play roles for the silk gland development probably in the downstream of Bombyx Scr (Matsunami, 1999 and references therein).

In the Drosophila salivary gland, Drosophila Fkh is limited to the secretory cells, whereas Drosophila trh is initially expressed in the entire salivary primordia but is shut off in the secretory cells during stage 12 through repression by Drosophila Fkh in the secretory cells. Thereafter Drosophila trh expression is limited to the duct cells. Drosophila fkh and trh are expressed distinctly and required for the differentiation of secretory cells and duct cells, respectively. In contrast, in the Bombyx silk gland, SGF-1/Bombyx Fkh is expressed in MSG and PSG, and Bombyx trh is expressed in nearly all cells of the silk gland initially but disappears from the posterior region and finally localizes in the ASG. The cells of the MSG and PSG are specialized for the production and secretion of silk proteins, and the ASG cells probably play a role as transporter duct for silk proteins to the spinneret. These expression and functional similarities suggest that Drosophila salivary gland and Bombyx silk gland are homologous organs, and that similar genetic cascades exist. The limited expression of Bombyx trh in the ASG might be through the repression by Bombyx Fkh, as in the case of Drosophila (Matsunami, 1999 and references therein).

Hypoxia-inducible factor, a heterodimeric transcription complex, regulates cellular and systemic responses to low oxygen levels (hypoxia) during normal mammalian development or tumor progression. Evidence is presented that a similar complex mediates response to hypoxia in C. elegans. This complex consists of HIF-1 and AHA-1, which are encoded by C. elegans homologs of the hypoxia-inducible factor (HIF) alpha and ß subunits, respectively. hif-1 mutants exhibit no severe defects under standard laboratory conditions, but they are unable to adapt to hypoxia. Although wild-type animals can survive and reproduce in 1% oxygen, the majority of hif-1-defective animals die in these conditions. The expression of an HIF-1:green fluorescent protein fusion protein is induced by hypoxia and is subsequently reduced upon reoxygenation. Both hif-1 and aha-1 are expressed in most cell types, and the gene products can be coimmunoprecipitated. It is concluded that the mechanisms of hypoxia signaling are likely conserved among metazoans. Additionally, it is found that nuclear localization of AHA-1 is disrupted in an hif-1 mutant. This finding suggests that heterodimerization may be a prerequisite for efficient nuclear translocation of AHA-1 (Jiang, 2001).

Although mammalian HIF-1alpha has an essential role in embryonic development, C. elegans hif-1 mutants are viable and fertile when cultured in standard laboratory conditions. This reflects the relatively simple physiology of C. elegans. A mammalian embryo relies on hypoxia-induced angiogenesis to oxygenate tissues. Hif-1alpha -/- mice die by E9.0 with severe vascular defects. In contrast, an adult C. elegans hermaphrodite has no apparent need for specialized respiratory structures or a complex circulatory system. Any cell in the organism is only a few cell widths from the outer surface of the worm or the intestinal lumen. Oxygen sensing and hif-1 function is likely to be very important in the soil environment inhabited naturally by C. elegans. In the laboratory, C. elegans are cultured on top of an agar-based medium, and the ambient oxygen concentrations are relatively high. However, high concentrations of bacteria, the C. elegans food source, can deplete oxygen in a soil microenvironment. The nematodes must be able to sense and adapt to these hypoxic conditions (Jiang, 2001).

AHA-1 translocation to the nuclei of intestinal cells is inefficient in hif-1 mutants. This result was not predicted by the prevalent models for hypoxia signaling. In the mammalian cell lines commonly used to study hypoxia-inducible factor or aryl hydrocarbon receptor (AHR) signaling, ARNT is localized to the nucleus constitutively. After HIF-1alpha or activated AHR translocates to the nucleus, it forms a dimer with ARNT. However, in the Drosophila embryo, Drosophila ARNT (encoded by the tango gene) apparently remains cytoplasmic until a bHLH-PAS dimerization partner is expressed. It is concluded that the role of AHA-1 in the formation and nuclear localization of an active transcriptional complex may depend on cell type-specific factors, such as the expression of other bHLH-PAS proteins. This hypothesis will be explored by examining the expression of other bHLH-PAS genes in those cells that localize AHA-1 to the nucleus in the absence of hif-1 (Jiang, 2001).

Mammalian Hypoxia-inducible factor

Transcription factors of the bHLH-PAS protein family are important regulators of developmental processes such as neurogenesis and tracheal development in invertebrates. Hypoxia-inducible factor (HIF-1 alpha) is a vertebrate relative of Drosophila trachealess that is likely to be involved in the growth of blood vessels by the induction of vascular endothelial growth factor (VEGF) in response to hypoxia. Mouse HIF-related factor (HRF), a novel close relative of HIF-1 alpha, is expressed most prominently in brain capillary endothelial cells and other blood vessels as well as in bronchial epithelium in the embryo and the adult. In addition, smooth muscle cells of the uterus, neurons, brown adipose tissue and various epithelial tissues express HRF mRNA as well. High expression levels of HRF mRNA in embryonic choroid plexus and kidney glomeruli, places where VEGF is highly expressed, suggest a role for this factor in VEGF gene activation similar to that of HIF-1 alpha. Given the similarity between morphogenesis of the tracheal system and the vertebrate vascular system, the expression pattern of HRF in the vasculature and the bronchial tree raises the possibility that this family of transcription factors may be involved in tubulogenesis. HRF's bHLH and PAS domains are more closely related to those of HIF-1alpha than to those of trachealess (Flamme, 1997).

A novel Per-Arnt/AhR-Sim basic helix-loop-helix (bHLH-PAS) factor has been isolated that interacts with the Ah receptor nuclear translocator (Arnt). Its predicted amino acid sequence exhibits significant similarity to the hypoxia-inducible factor 1alpha (HIF1alpha) and Drosophila Trachealess (dTrh) gene product. The HIF1alpha-like factor (HLF) encoded by the isolated cDNA binds the hypoxia-response element (HRE) found in enhancers of genes for erythropoietin, vascular endothelial growth factor (VEGF), and various glycolytic enzymes, and activates transcription of a reporter gene harboring the HRE. Although transcription-activating properties of HLF are very similar to those reported for HIF1alpha, their expression patterns are quite different; HLF mRNA is most abundantly expressed in lung, followed by heart, liver, and various other organs under conditions where oxygen level is normal, whereas HIF1alpha mRNA is ubiquitously expressed at much lower oxygen levels. In lung development around the time of parturition, HLF mRNA expression is markedly enhanced, whereas that of HIF1alpha mRNA remains apparently unchanged, continuing at a much lower level. HLF mRNA expression is closely correlated with that of VEGF mRNA. Whole mount in situ hybridization experiments demonstrated that HLF mRNA is expressed in vascular endothelial cells at the middle stages (9.5 and 10.5 days postcoitus) of mouse embryo development, where HIF1alpha mRNA is almost undetectable. The high expression level of HLF mRNA in the O2 delivery system of developing embryos and adult organs suggests that at normal oxygen levels, HLF regulates gene expression of VEGF, various glycolytic enzymes, and others driven by the HRE sequence, and may be involved in development of blood vessels and the tubular system of lung (Ema, 1997).

The basic helix-loop-helix-PAS (bHLH-PAS) protein ARNT is a dimeric partner of the Ah receptor (AHR) and hypoxia inducible factor 1 alpha (HIF1 alpha). These dimers mediate biological responses to xenobiotic exposure and low oxygen tension. The recent cloning of ARNT and HIF1 (homologs ARNT2 and HIF2 alpha) indicates that at least six distinct bHLH-PAS heterodimeric combinations can occur in response to a number of environmental stimuli. In an effort to understand the biological relevance of this combinatorial complexity, their relative expression at a number of developmental time points was characterized by parallel in situ hybridization of adjacent tissue sections. In general there is limited redundancy in the expression of these six transcription factors, and each of these bHLH-PAS members displays a unique pattern of developmental expression emerging as early as embryonic day 9.5 (Jain, 1998).

Hypoxia inducible factors (HIF1, 2 and 3), consisting of alpha and beta subunits, play an essential role in various responses to hypoxia. Nuclear entry of alpha subunits is a necessary step for the formation of DNA-binding complex with beta subunit, which is constitutively localized in the nucleus. The nuclear accumulation of HIF2alpha induced by hypoxia is mediated through a novel variant of bipartite-type nuclear localization signal (NLS) in the C-terminus of the protein, which has an unusual length of spacer sequence between two adjacent basic domains. When the ubiquitin-proteasome system is deficient or inhibited, HIF2alpha accumulates in the nucleus even under normoxia, also mediated through the bipartite NLS. These findings indicate that the protein stability is critical for the nuclear localization of HIF2alpha and hypoxia is not a necessary factor for the process. Importantly, the NLS of HIF2alpha is also conserved in the other HIF family members, HIF1alpha and HIF3alpha. Mutational analyses prove that the NLS mediating the nuclear localization of HIF1alpha is indeed bipartite-, but not monopartite-type as previously thought. These results suggest that the newly identified NLS is crucial for the functional regulation of HIF family (Luo, 2001).

Hypoxia-inducible factor 1alpha (HIF1alpha) plays a pivotal role in embryogenesis, angiogenesis, and tumorigenesis. HIF1alpha-mediated transcription requires the coactivator p300, at least in part, through interaction with the cysteine- and histidine-rich 1 domain of p300. To understand the molecular basis of this interaction, a random mutagenesis screen in yeast has been employed for efficient identification of residues that are functionally critical for protein interactions. As a result, four residues (Leu-795, Cys-800, Leu-818, and Leu-822) in the C-terminal activation domain of HIF1alpha have been identified as crucial for HIF1 transactivation in mammalian systems. Moreover, data from residue substitution experiments indicate the stringent necessity of leucine and hydrophobic cysteine for C-terminal activation domain function. Likewise, Leu-344, Leu-345, Cys-388, and Cys-393 in the cysteine- and histidine-rich 1 domain of p300 have also been shown to be essential for the functional interaction. It is proposed that hypoxia-induced HIF1alpha-p300 interaction relies upon a leucine-rich hydrophobic interface that is regulated by the hydrophilic and hydrophobic sulfhydryls of HIF1alpha Cys-800 (Gu, 2001).

The ubiquitously expressed basic helix-loop-helix (bHLH)-PAS protein ARNT forms transcriptionally active heterodimers with a variety of other bHLH-PAS proteins, including HIF-1alpha and arylhydrocarbon receptor (AHR). These complexes regulate gene expression in response to hypoxia and xenobiotics, respectively, and mutation of the murine Arnt locus results in embryonic death by day 10.5 associated with placental, vascular, and hematopoietic defects. The closely related protein ARNT2 is highly expressed in the central nervous system and kidney and also forms complexes with HIF-1alpha and AHR. To assess unique roles for ARNT2 in development, and reveal potential functional overlap with ARNT, a targeted null mutation of the murine Arnt2 locus was generated. Arnt2(-/-) embryos die perinatally and exhibit impaired hypothalamic development, phenotypes previously observed for a targeted mutation in the murine bHLH-PAS gene Sim1, and consistent with the recent proposal that ARNT2 and SIM1 form an essential heterodimer in vivo. In addition, cultured Arnt2(-/-) neurons display decreased hypoxic induction of HIF-1 target genes, demonstrating formally that ARNT2/HIF-1alpha complexes regulate oxygen-responsive genes. Finally, a strong genetic interaction between Arnt and Arnt2 mutations is observed, indicating that either gene can fulfill essential functions in a dose-dependent manner before embryonic day 8.5. These results demonstrate that Arnt and Arnt2 have both unique and overlapping essential functions in embryonic development (Keith, 2001).

The von Hippel-Lindau tumor-suppressor protein (pVHL: Drosophila homolog von Hippel-Lindau, [Adryan, 2001]) regulates the stability of HIF1 alpha and HIF2 alpha and thus is pivotal in cellular responses to changes in oxygen tension. Paradoxically, human cytotrophoblasts proliferate under hypoxic conditions comparable to those measured in the early gestation placenta (2% O₂), but differentiate into tumorlike invasive cells under well-oxygenated conditions such as those found in the uterus. Attempts have been made to explain this phenomenon in terms of pVHL expression. In situ, pVHL immunolocalized to villous cytotrophoblast stem cells, and expression is enhanced at sites of cell column initiation; in both of these relatively hypoxic locations, cytoplasmic staining for HIF2 alpha is also detected. As cytotrophoblasts attach to and invade the uterus (which results in their increased exposure to oxygen), pVHL staining is abruptly downregulated concordant with localization of HIF2 alpha to the nucleus. In vitro, hypoxia (2% O₂) upregulates cytotrophoblast pVHL expression together with HIF2 alpha, which localizes to the cytoplasm; culture under well-oxygenated conditions greatly reduces levels of both molecules. These results, together with the placental defects previously observed in VHL(-/-) mice, suggest that pVHL is a component of the mechanism that transduces local differences in oxygen tension at the maternal-fetal interface to changes in the biological behavior of cytotrophoblasts. Furthermore, these data provide the first example of oxygen-dependent changes in pVHL abundance (Genbacev, 1991).

The phosphatidylinositol 3-kinase signaling pathway has inherent oncogenic potential. It is up-regulated in diverse human cancers by either a gain of function in PI3K itself or in its downstream target Akt, or by a loss of function in the negative regulator PTEN. However, the complete consequences of this up-regulation are not known. Insulin and epidermal growth factor or an inactivating mutation in the tumor suppressor PTEN specifically increase the protein levels of hypoxia-inducible factor (HIF) 1alpha but not of HIF-1beta in human cancer cell lines. This specific elevation of HIF-1alpha protein expression requires PI3K signaling. In the prostate carcinoma-derived cell lines PC-3 and DU145, insulin- and epidermal growth factor-induced expression of HIF-1alpha is inhibited by the PI3K-specific inhibitors LY294002 and wortmannin in a dose-dependent manner. HIF-1beta expression is not affected by these inhibitors. Introduction of wild-type PTEN into the PTEN-negative PC-3 cell line specifically inhibits the expression of an HIF-1alpha but not that of HIF-1beta. In contrast to the HIF-1alpha protein, the level of HIF-1alpha mRNA is not significantly affected by PI3K signaling. Vascular endothelial growth factor reporter gene activity is induced by insulin in PC-3 cells and is inhibited by the PI3K inhibitor LY294002 and by the coexpression of a HIF-1 dominant negative construct. Vascular endothelial growth factor reporter gene activity is also inhibited by expression of a dominant negative PI3K construct and by the tumor suppressor PTEN (Jiang, 2001).

One major function of the hypothalamus is to maintain homeostasis by modulating the secretion of pituitary hormones. The paraventricular (PVN) and supraoptic (SON) nuclei are major integration centers for the output of the hypothalamus to the pituitary. The bHLH-PAS transcription factor SIM1 is crucial for the development of several neuroendocrine lineages within the PVN and SON. bHLH-PAS proteins require heterodimerization for their function. ARNT, ARNT2, and BMAL1 are the three known general heterodimerization partners for bHLH-PAS proteins. Evidence is provided that Sim1 and Arnt2 form dimers in vitro, that they are co-expressed in the PVN and SON, and that their loss of function affects the development of the same sets of neuroendocrine cell types within the PVN and SON. Together, these results implicate ARNT2 as the in vivo dimerization partner of SIM1 in controlling the development of these neuroendocrine lineages (Michaud, 2000).

Hypoxia-selective macroautophagy and cell survival signaled by autocrine PDGFR activity

The selective regulation of macroautophagy remains poorly defined. This study reports that PDGFR signaling is an essential selective promoter of hypoxia-induced macroautophagy. Hypoxia-induced macroautophagy in tumor cells is also HIF1alpha-dependent, with HIF1alpha integrating signals from PDGFRs and oxygen tension. Inhibition of PDGFR signaling reduces HIF1alpha half-life, despite buffering of steady-state protein levels by a compensatory increase in HIF1alpha mRNA. This markedly changes HIF1alpha protein pool dynamics, and consequently reduces the HIF1alpha transcriptome. As autocrine growth factor signaling is a hallmark of many cancers, cell-autonomous enhancement of HIF1alpha-mediated macroautophagy may represent a mechanism for augmenting tumor cell survival under hypoxic conditions (Wilkinson, 2009).

date revised: 5 August 2021

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