single-minded


EVOLUTIONARY HOMOLOGS

The Drosophila (Dm) similar (sima) gene was isolated using a low-stringency hybridization screen employing a Dm single-minded gene basic helix-loop-helix (bHLH) DNA probe. sima is a member of the bHLH-PAS gene family and the conceptual protein shares a number of structural features, including a bHLH domain, PAS domain, and homopolymeric amino acid stretches. Sima is most closely related to the human hypoxia-inducible factor 1 alpha bHLH-PAS protein. In situ hybridization experiments reveal that sima is transcribed in most or all cells throughout embryogenesis. It has been cytologically mapped to position 99D on the third chromosome, and is not closely linked to other known bHLH-PAS genes (Nambu, 1996).

Juvenile hormone analog (JHA) insecticides are relatively nontoxic to vertebrates and offer effective control of certain insect pests. Recent reports of resistance in whiteflies and mosquitoes demonstrate the need to identify and understand genes for resistance to this class of insect growth regulators. Mutants of the Methoprene-tolerant (Met) gene in Drosophila melanogaster show resistance to both JHAs and JH, and previous biochemical studies have demonstrated a mechanism of resistance involving an intracellular JH binding-protein that has reduced ligand affinity in Met flies. Met flies are resistant to the toxic and morphogenetic effects of JH and several JHAs, but not to other classes of insecticide. Biochemical studies reveal a target-site resistance mechanism, that of reduced JH binding in cytosolic extracts from either of two JH target tissues in Met flies. This property of reduced JH binding was cytogenetically localized to the Met region on the X chromosome and can account for the resistance. Possible identities for this binding protein include either an accessory JH-binding protein in the cytoplasm, similar to the cellular retinoic acid-binding protein in vertebrates, or a JH receptor protein involved in the action of JH (Ashok, 1998).

The Met+ gene has been cloned by transposable P-element tagging and reduced transcript level has been found in several mutant alleles, showing that underproduction of the normal gene product can lead to insecticide resistance. Transformation of Met flies with a Met+ cDNA results in susceptibility to methoprene, indicating that the cDNA encodes a functional Met+ protein. Met shows homology to the basic helix-loop-helix (bHLH)-PAS family of transcriptional regulators, implicating Met in the action of JH at the gene level in insects. This family also includes the vertebrate dioxin receptor, a transcriptional regulator known to bind a variety of environmental toxicants. Met shows three regions of homology to members of a family of transcriptional activators known as bHLH-PAS proteins. Met generally has higher homology to the vertebrate bHLH-PAS proteins than to those identified in D. melanogaster. A D. melanogaster ARNT-like gene has recently been cloned, and DARNT has higher homology to vertebrate ARNT than does Met, suggesting that DARNT, not Met, may function like ARNT in flies. Met homology to these proteins includes the bHLH region that is involved in DNA binding (30-38% identity), the PAS-A region (28-40%), and the PAS-B region (22-35%). The arrangement of these domains in the Met gene is the same as for other bHLH-PAS genes (Ashok, 1998).

In insects and crustaceans, ventral midline cells are present that subdivide the CNS into bilateral symmetric halves. In both arthropod groups unpaired midline neurons and glial cells have been identified that contribute to the embryonic patterning mechanisms. In Drosophila, for example, the midline cells are involved in neural cell fate specification along the dorso-ventral axis but also in axonal pathfinding and organisation of the axonal scaffold. Both in insects and malacostracan crustaceans, the bHLH-PAS transcription factor single-minded is the master regulator of ventral midline development and homology has been suggested for individual midline precursors in these groups. The conserved arrangement of the axonal scaffold as well as the regular pattern of neural precursors in all euarthropod groups raises the question whether the ventral midline system is conserved in this phylum. In the remaining euarthropod groups, the chelicerates and myriapods, a single-minded homologue has been identified in the spider Achaearanea tepidariorum (chelicerate), however, the gene is not expressed in the ventral midline but in the median area of the ventral neuroectoderm. This study shows that At-sim is not required for ventral midline development. Furthermore, sim homologues were identified in representatives of arthropods that have not yet been analysed: the myriapod Strigamia maritima and a representative of an outgroup to the euarthropods, the onychophoran Euperipatoides kanangrensis. The expression patterns were compared to the A. tepidariorum sim homologue expression and furthermore the nature of the arthropod midline cells were analyzed. The data suggest that in arthropods unpaired midline precursors evolved from the bilateral median domain of the ventral neuroectoderm in the last common ancestor of Mandibulata (insects, crustaceans, myriapods). It is hypothesized that sim was expressed in this domain and recruited to ventral midline development. Subsequently, sim function has evolved in parallel to the evolution of midline cell function in the individual Mandibulata lineages (Linne, 2012).

xSim has been isolated from Xenopus. It encodes a protein of 760 amino acids containing a basic helix-loop-helix (bHLH) motif contiguous to a PAS domain characteristic of an emerging family of transcriptional regulators -- so called bHLH/PAS. xSim shares a strong amino acid sequence identity with the Drosophila Single-minded and with the murine Sim1 and Sim2 proteins. Phylogenetic analysis reveals that xSim gene is an ortholog gene of the mSim2 gene. Spatio-temporal analysis shows a maternal and a zygotic expression of xSim throughout early Xenopus development. In situ hybridization assays reveal that the transcripts are enriched in the animal hemisphere until blastula stage and extend to the marginal zone at early gastrula stage. As development proceeds, xSim is mainly restricted to the central nervous system (Coumailleau, 2000).

At segmentation stages, mRNA is first found in the animal hemisphere and later on in both the animal cap and marginal zone cells of blastula and early gastrula stages. At neurula stages expression is prominently detected in the neurectoderm and ectoderm. At this stage some weak expression level is also detected in the paraxial mesoderm. At late neurula stage, xSim expression is mainly seen in the neural tube. In tailbud embryos (stage 35), xSim transcripts are clearly distributed in the brain, optic vesicles, cement gland, branchial arches and somites. Transverse and sagittal sections of tail bud stage embryos (stage 30) clearly emphasizes the strong expression detected in the brain, optic vesicles and in the spinal cord. This expression pattern is similar to those observed in fruit fly, mouse and chicken where expression of Sim family genes has been observed in the central nervous system and somites. However it is noteworthy that this xSim expression pattern is wider than in Drosophila, where it is restricted to the midline of the central nervous system (Coumailleau, 2000).

There are two murine homologs of sim, Sim1 and Sim2, whose products show a high degree of sequence with SIM in their amino-terminal halves, with each containing a basic helix-loop-helix domain as well as a PAS domain. Sim2 maps to a portion of the distal end of mouse chromosome 16 that is syntenic to the Down's syndrome critical region of human chromosome 21. A human sim homolog appears to be located in this region. It is possible that increased dosage of this sim homolog in cases of trisomy 21 might be a causal factor in the pathogenesis of Down's syndrome. Both murine sim homologs are expressed in compartments of the developing forebrain, and the expression pattern of Sim2 provides evidence for early regionalization of the diencephalon prior to any overt morphological differentiation in this region. Outside the CNS, Sim1 is expressed in mesodermal and endodermal tissues, including developing somites, mesonephric duct, and foregut. Sim2 is expressed in facial and trunk cartilage, as well as trunk muscles. Both murine Sim genes are also expressed in the developing kidney (Fan, 1996).

The neuroendocrine system consists of two sets of hypothalamic neurons: the magnocellular and the parvocellular neurons. The magnocellular neurosecretory system projects to the posterior pituitary where it releases vasopressin (AVP) and oxytocin (OT) directly into the general circulation. Vasopressin participates in the control of blood volume, osmolality, and pressure, whereas OT promotes parturition and lactation. The magnocellular neurons are located in two nuclei of the anterior hypothalamus, the paraventricular (PVN) and the supraoptic (SON) nuclei. Within the PVN and the SON, AVP and OT are produced by mutually exclusive sets of neurons. The sum of AVP- and OT-producing cells corresponds to the total number of magnocellular neurons, indicating that AVP and OT define the two cell types of this neurosecretory system. The PVN, which contains both magnocellular and parvocellular neurons, and the SON, which is mainly composed of magnocellular neurons, originate from a small patch of neuroepithelium located at the level of the ventral diencephalic sulcus. Cells that form the PVN remain near the ventricular zone, whereas those that form the SON migrate laterally to reach the surface of the hypothalamus. The bHLH-PAS transcription factor SIM1 is expressed during the development of the hypothalamic-pituitary axis in three hypothalamic nuclei: the PVN, the anterior PVN (aPV), and the SON. To investigate Sim1 function in the hypothalamus, mice were produced carrying a null allele of Sim1 by gene targeting. Homozygous mutant mice die shortly after birth. Histological analysis shows that the PVN and the SON of these mice are hypocellular. At least five distinct types of secretory neurons, identified by the expression of oxytocin, vasopressin, thyrotropin-releasing hormone, corticotropin-releasing hormone, and somatostatin, are absent in the mutant PVN, aPV, and SON. Moreover, SIM1 controls the development of these secretory neurons at the final stages of their differentiation. A subset of these neuronal lineages in the PVN/SON are also missing in mice bearing a mutation in the POU transcription factor BRN2. Evidence is provided that, during development of the Sim1 mutant hypothalamus, the prospective PVN/SON region fails to express Brn2. These results strongly indicate that SIM1 functions upstream to maintain Brn2 expression, which in turn directs the terminal differentiation of specific neuroendocrine lineages within the PVN/SON (Michaud, 1998).

Drosophila Sim is a master regulator of the CNS midline. Loss of sim function results in the complete absence of midline development. Drifter, a POU domain transcription factor that binds the same DNA sequence as does BRN2, has also been implicated in controlling the development of CNS midline cells in the fly. Expression and phenotypic analysis have shown that Sim acts upstream of Drifter. In mice, SIM1 likewise acts upstream of a POU domain transcription factor BRN2. Specifically, Brn2 is down-regulated in a region of the prospective PVN/SON that continues to express the Sim1 mutant transcript, indicating that SIM1 and BRN2 function along the same pathway. The fact that Brn2 expression in the prospective hypothalamus of Sim1 mutant embryos is not altered until E12.5 suggests that Sim1 is not involved in initiating but in maintaining Brn2 expression. Whether SIM1 controls BRN2 transcription directly or indirectly remains an open question. Consistent with the conclusion that Sim1 functions upstream to maintain Brn2 expression, all the hypothalamic lineages that are reported to be affected by the loss of Brn2 function are also affected in Sim1-deficient mice; the loss of Brn2 function affects the development of vasopressin -, oxytocin-, and corticotropin-releasing hormone-producing cells, and the same cell types are affected by the loss of Sim1 function. In contrast, thyrotropin-releasing hormone- and somatostatin-producing cells are missing in Sim1 mutant but are present in Brn2 mutant PVN and aPV. This is consistent with the observation that thyrotropin-releasing hormone and BRN2 expression share minimal overlap in the PVN. Similarily, Brn2 is not expressed in the aPV, where somatostatin is produced abundantly (Michaud, 1998).

The loss of the five cell types studied here in Sim1 mutant mice raises the possibility that most, if not all, of the neuronal lineages constituting the PVN, SON, and aPV originate from the dorsal aspect of the prospective anterior hypothalamic Sim1 domain. This domain can be divided into an anterior region only expressing Sim1 and a posterior region expressing both Sim1 and Brn2. It is tempting to speculate that the corticotropin-releasing hormone (CRH), AVP, and OT lineages, which are affected in both Sim1 and Brn2 mutant mice, are derived from the posterior region, whereas the Thyrotropin-releasing hormone and somatostatin lineages, which are only affected in Sim1 mutant mice, are derived from the anterior region. This is consistent with the observation that in the newborn hypothalamus, Brn2 is not expressed in the aPV or in the anterior end of the PVN, where SS- and TRH-producing cells, respectively, are found. In Brn2 mutant mice, precursors of the PVN and SON survive up to E15.5 but fail to express the secreted neuropeptides. BRN2 binds and activates the CRH promotor, supporting a role for BRN2 in controlling the terminal stage of differentiation. The survival of the PVN/SON precursors up to E15.5 in both Sim1 and Brn2 mutant embryos and the down-regulation of Brn2 in Sim1 mutant embryos would suggest that the loss of Brn2 expression mediates the effect of the Sim1 mutant allele on the development of CRH, AVP, and OT neuroendocrine lineages. Whether Sim1 controls the differentiation of TRH- and SS-expressing cells directly or indirectly, through activation of another POU domain transcription factor, remains to be determined (Michaud, 1998).

Development of the neuroendocrine hypothalamus is characterized by a precise series of morphogenetic milestones culminating in terminal differentiation of neurosecretory cell lineages. The homeobox-containing gene Orthopedia (Otp), is expressed in neurons giving rise to the paraventricular (PVN), supraoptic (SON), anterior periventricular (aPV), and arcuate (ARN) nuclei throughout their development. Homozygous Otp-/- mice die soon after birth and display progressive impairment of crucial neuroendocrine developmental events such as reduced cell proliferation, abnormal cell migration, and failure in terminal differentiation of the parvocellular and magnocellular neurons of the aPV, PVN, SON, and ARN. Moreover, the data provide evidence that two proteins, Otp and Sim1 (the latter a bHLH-PAS transcription factor that directs terminal differentiation of the PVN, SON, and aPV), act in parallel and are both required to maintain Brn2 expression, which, in turn, is required for neuronal cell lineages secreting oxytocin (OT), arginine vasopressin (AVP), and corticotropin-releasing hormone (CRH) (Acampora, 1999).

Analysis of Brn2 mutant mice reveals that it acts relatively late in neuroendocrine development, being required for terminal differentiation events of CRH, AVP, and OT cell lineages. Sim1 mutant mice show a more general effect, because they are impaired in terminal differentiation events leading to the activation of neuropeptides of the PVN and SON as well as the activation of SS in the aPV. Interestingly, from E12.5 onward, Sim1 minus mutants gradually lack Brn2 expression in the dorsal supraoptic/paraventricular (spv) primordium, indicating that Sim1 acts upstream of Brn2 and is required for maintenance of its expression. There is a striking similarity with the Sim1 mutant phenotype. Except in the ARN, Otp is fully coexpressed in time and space with Sim1, and is required for both terminal differentiation of parvocellular and magnocellular neurons of aPV, PVN, and SON and for maintenance of Brn2 expression. Noteworthy, at E11.5, Brn2 expression is slightly toned down and, at E12.5, disappears from the entire spv and adjacent territory in which it is coexpressed with Otp, thus suggesting that as compared with Sim1 minus phenotype, Otp may have a more generalized role in controlling Brn2 expression in post-mitotic neurons and may open the question as to whether Sim1 and Otp act in parallel, or is one downstream of the other with regard to the control of Brn2 expression? Interestingly, in Otp minus embryos, Sim1 expression is maintained in lacZ-positive cells in which Brn2 is lost and, in Sim1 minus embryos, Otp is expressed in the territory in which Brn2 disappears. These findings provide strong in vivo evidence that Otp and Sim1 act in parallel and are both required for proper expression of Brn2 in the spv and its derivatives, the PVN and SON (Acampora, 1999).

Hypothalamic nuclei, including the anterior periventricular (aPV), paraventricular (PVN), and supraoptic (SON) nuclei strongly express the homeobox gene Orthopedia (Otp) during embryogenesis. Targeted inactivation of Otp in the mouse results in the loss of these nuclei in the homozygous null neonates. The Otp null hypothalamus fails to secrete the neuropeptides somatostatin, arginine vasopressin, oxytocin, corticotropin-releasing hormone, and thyrotropin-releasing hormone in an appropriate spatial and temporal fashion, and leads to the death of Otp null pups shortly after birth. Failure to produce these neuropeptide hormones is evident prior to E15.5, indicating a failure in terminal differentiation of the aPV/PVN/SON neurons. Absence of elevated apoptotic activity, but reduced cell proliferation together with the ectopic activation of Six3 expression in the presumptive PVN, indicates a critical role for Otp in terminal differentiation and maturation of these neuroendocrine cell lineages. Otp employs distinct regulatory mechanisms to modulate the expression of specific molecular markers in the developing hypothalamus. At early embryonic stages, expression of Sim2 is immediately downregulated as a result of the absence of Otp, indicating a potential role for Otp as an upstream regulator of Sim2. In contrast, the regulation of Brn4, which is also expressed in the SON and PVN is independent of Otp function. Hence no strong evidence links Otp and Brn4 in the same regulatory pathway. The involvement of Otp and Sim1 in specifying specific hypothalamic neurosecretory cell lineages has been shown to operate via distinct signaling pathways that partially overlap with Brn2 (Wang, 2000).

Several POU domain and bHLH-PAS transcription factors have been shown genetically to play crucial roles in the development of the hypothalamic-pituitary axis. For example, Brn2 and Sim1 are indispensable for the terminal differentiation of the endocrine neurons in the aPV, PVN, and SON. Some homeobox genes, such as Six3, Hmx2 and Lhx3, are expressed in discrete regions in the hypothalamus and pituitary, suggesting prospective roles in the maturation of these structures. In the wild-type hypothalamus, Brn2 is abundantly expressed in the PVN, SON, and lateral hypothalamic area. Another POU domain transcription factor, Brn4, is also positive in a subset of cells in the PVN and SON. Interestingly, the bHLH-PAS transcription factor Sim1 shows an identical expression pattern relative to Otp in all regions in the hypothalamus and amygdala. In the PVN, Sim2 is preferentially expressed in a subset of cells close to the third ventricle, even though at a low level. In the Otp lacZ null brain, Brn4 and Sim2 are absent in the hypothalamus. Similarly, Brn2 and Sim1 transcripts disappear from the presumptive PVN and are found distributed in the ventrolateral hypothalamic area. In the presumptive SON, both Brn2 and Sim1 show a reduced level of expression, apparently in a disorganized manner. Sim1 expression in the amygdaloid nuclei is unaffected in the Otp lacZ mutant. Losing the expression of both somatostatin (SS) and Sim1 in the aPV indicates that parvocellular neurons in the aPV are equally affected in the OtplacZ null brain (Wang, 2000).

Where does Otp fall in the hierarchy of gene activation and cellular development in the hypothalamus? Neonates lacking Otp function fail to develop anterior periventricular, paraventricular, and supraoptic nuclei. The arcuate nucleus is also affected, as indicated by its failure to express the neuropeptide somatostatin, even though no morphological defects are discernable. Two well-documented genes involved in the development of the neurons of the PVN and SON are the POU domain factor Brn2 and the bHLH-PAS transcription factor Sim1. Brn2 null mice lose neurosecretory neurons of the paraventricular and supraoptic nuclei as demonstrated by the failure to initiate neuropeptide gene expression, successful projection of axons to targets, and survival of these neurons. Brn2 has no effect on the early stage events leading to terminal differentiation of the PVN/SON neurons. Sim1 is a potential upstream regulator of Brn2 , since Sim1 is required to maintain Brn2 expression. Additional structures, including the aPV, PVN, and SON nuclei are affected in response to the loss of Sim1, as compared with Brn2 null mice. Sim1-expressing cells of the presumptive PVN/SON develop and survive up to the stage of E15.5 when the PVN/SON neurons have migrated to their correct location. This suggests that Sim1 is involved in the terminal differentiation of the hypothalamic neurons at the stages of neuropeptide gene expression and axonal outgrowth. Loss of Sim1 leads to the failure of postmitotic neurons to transverse these events. Cell death has been suggested as a consequence of the loss of Sim1. In contrast to Brn2 and Sim1, the homeobox gene Otp clearly engages different developmental pathways leading to terminal differentiation of the aPV/PVN/SON neurons. Structures expressing Otp, such as the aPV, PVN, and SON as well as ARN, are affected at different levels. Mice deficient in Otp fail to develop the aPV, PVN, and SON nuclei. Also, Otp null mice lose the capability to produce somatostatin by the arcuate nucleus. These defects are closely associated with reduced cell proliferation of neuroblasts and abnormal migration of postmitotic neurons. Thus Otp initiates its functions in terminal differentiation of neuroblasts at stages preceding those of either Brn2 or Sim1 (Wang, 2000).

In the supraoptic/paraventricular area of the wild-type animals, at early embryonic stages, Sim2 is confined to a subarea of the Otp-expression domain. Lack of functional Otp immediately extinguishes expression of Sim2, suggesting that Sim2 could be a downstream target gene of Otp. The function of Sim2 in the development of the hypothalamic endocrine neuron is not known yet, but it is possible that Otp affects cell identity, at least in a subset of the PVN/SON neurons, partially by regulating Sim2 at the transcriptional level. Similarly, Otp seems to be an upstream regulator of Brn2. Brn2 shows a delayed response to Otp as compared with Sim2. At E11.5, expression of this gene is not severely affected. But at the later stage of E13.0, Brn2 is absent in the developing PVN and SON even though relatively significant amounts of Otp-expressing cells are still present in the above regions. Unlike the genes mentioned above, the alteration of Sim1 expression appears to be closely associated with the progressive disappearance of the Otp-expressing cells. Sim1 colocalizes with Otp in both the wild-type and the Otp mutants. Therefore, in contrast to Sim2, there is no direct regulatory relationship linking Otp and Sim1 at the level of gene expression. Alteration of the Sim1 expression profile in the mice lacking Otp is more likely to be related to cellular identity. Since ectopic expression of Sim1 cannot induce neurons with PVN/SON identities, this suggests that Otp and Sim1 may need to work cooperatively to direct the terminal differentiation of the PVN/SON cells. In conclusion, the Sim1 and Otp genes may produce factors that are essential, but not sufficient, to determine hypothalamic endocrine and possibly other factors that eventually determine the successful maturation of hypothalamic endocrine cell lineages (Wang, 2000).

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).

Many features of Down's syndrome might result from the overdosage of only a few genes located in a critical region of chromosome 21. One exonic sequence is 93 % similar to part of the Drosophila single-minded (sim) gene, consisting of the PAS domain. No significant similarity is found to human aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT), both of which contain PAS domains. The sequence is present only in the Down's syndrome-critical region in the human genome. Hybridization of the exonic sequence with human poly(A)+ RNA reveals two transcripts of 6 and 4.3 kb. The corresponding gene is expressed during early fetal life in the central nervous system and in other tissues, including the facial, skull, palate, and vertebra primordia. The expression pattern suggests that it might be involved in the pathogenesis of some of the morphological features and brain anomalies observed in Down's syndrome (Dahmane, 1995).

The PAS motif, found in Single-minded and Period, is also found in two subunits of the mammalian dioxin receptor (Huang, 1993), and the aryl hydrocarbon nuclear translocator (ARNT) (Nambu, 1991). Information is given below on SIM structural homologs because of the insight this information provides about SIM. With the possible exception of mSIM, these homologs are not considered to be functional homologs of SIM (Dahmane, 1995).

A mouse gene (mSim) with homology to sim has been isolated. MSim heterodimerizes with Arnt (Ah receptor nuclear translocator), even more efficiently than AhR (Ah receptor) does with Arnt. MSim transcript is expressed in several limited tissues such as muscle, kidney and lung of adult animals. Distribution of MSim mRNA is always accompanied with that of Arnt. All the results suggest a regulatory role of mSim in partnership with Arnt. MSim mRNA is expressed in the ventral diencephalon, branchial arches and limbs (Ema, 1996).

The secreted protein sonic hedgehog is required to establish patterns of cellular growth and differentiation within ventral regions of the developing CNS. The expression of Shh in the two tissue sources responsible for this activity, the axial mesoderm and the ventral midline of the neural tube, is regulated along the anteroposterior neuraxis. Separate cis-acting regulatory sequences have been identified that direct Shh expression to distinct regions of the neural tube, supporting the view that multiple genes are involved in activating Shh transcription along the length of the CNS. The activity of one Shh enhancer, which directs reporter expression to portions of the ventral midbrain and diencephalon, overlaps both temporally and spatially with the expression of Sim2. Sim2 encodes a basic helix-loop-helix (bHLH-PAS) PAS domain containing transcriptional regulator whose Drosophila homolog, single-minded, is a master regulator of ventral midline development. Both vertebrate and invertebrate Sim family members were found sufficient for the activation of the Shh reporter as well as endogenous Shh mRNA. Although Shh expression is maintained in Sim2-/-embryos, it is absent from the rostral midbrain and caudal diencephalon of embryos carrying a dominant-negative transgene that disrupts the function of bHLH-PAS proteins. Together, these results suggest that bHLH-PAS family members are required for the regulation of Shh transcription within aspects of the ventral midbrain and diencephalon (Epstein, 2000).

Significant differences have been identified between Drosophila and mammals in the use of pathways that mediate ventral midline induction and downstream signaling properties. For instance in Drosophila, sim is expressed in cells fated to make up the ventral midline and is required for their formation. Sim functions by regulating a number of midline-specific genes including spitz, a secreted TGFalpha-like molecule that operates in a graded distribution in the ectoderm to establish distinct cell fates. This contrasts with floor plate induction within the spinal cord of higher vertebrates, which appears to be independent of Sim function and reliant on graded Shh signaling for the specification of distinct neuronal fates. hedgehog is expressed in the Drosophila neurectoderm, however it is localized to transverse stripes and does not play a role in signaling from the ventral midline. Although, the genes involved in ventral midline induction differ between the two organisms, the employment of a patterning strategy that relies on the graded response to a factor secreted from the ventral midline is a feature common to both. Given the many similarities between ventral midline cells of the CNS in Drosophila and mouse, it is rather intriguing that a role for Sim2 in ventral midline determination has re-emerged in vertebrates through its ability to regulate Shh expression in the ventral diencephalon. Whether this points to a conserved role for Sim2 or an example of convergent evolution remains to be determined (Epstein, 2000).

A wide range of physiological and behavioral processes, such as social, sexual, and maternal behaviors, learning and memory, and osmotic homeostasis are influenced by the neurohypophysial peptides oxytocin and vasopressin. Disruptions of these hormone systems have been linked to several neurobehavioral disorders, including autism, Prader-Willi syndrome, affective disorders, and obsessive-compulsive disorder. Studies in zebrafish promise to reveal the complex network of regulatory genes and signaling pathways that direct the development of oxytocin- and vasopressin-like neurons, and provide insight into factors involved in brain disorders associated with disruption of these systems. Isotocin, which is homologous to oxytocin, is expressed early, in a simple pattern in the developing zebrafish brain. Single-minded 1 (sim1), a member of the bHLH-PAS family of transcriptional regulatory genes, is required for terminal differentiation of mammalian oxytocin cells and is a master regulator of neurogenesis in Drosophila. this study shows that sim1 is expressed in the zebrafish forebrain and is required for isotocin cell development. The expression pattern of sim1 mRNA in the embryonic forebrain is dynamic and complex, and overlaps with isotocin expression in the preoptic area. Evidence is provided that the role of sim1 in zebrafish neuroendocrine cell development is evolutionarily conserved with that of mammals (Eaton, 2006).

SIM proteins and their dimerization partner ARNT

Two murine homologs of the Drosophila Single-minded protein interact with the mouse aryl hydrocarbon receptor nuclear translocator (ARNT) protein. Since ARNT and the two mouse SIM proteins are always coexpressed, this implies that the interaction is likely to be physiologically relevant and that SIM1 and SIM2 are tissue-specific modulators of ARNT activity. Both the helix-loop-helix and the PAS regions of SIM1 and of ARNT are required for effecient heterodimerization. SIM1 associates with the 90-kDa heat shock protein and inhibits binding of the aryl hydrocarbon receptor-ARNT dimer to the xenobiotic response element. Introduction of SIM1 in hepatoma cells inhibits transcriptional transactivation by the endogenous aryl hydrocarbon receptor-ARNT dimer. In adult mice, mRNA for SIM1 is expressed in lung, skeletal muscle and kidney, whereas the mRNA for SIM2 is found only in skeletal muscle and kidney. ARNT is also expressed in these organs. Thus mouse SIM1 and SIM2 are heterodimerization partners for ARNT in vitro, and they may function both as positive and negative transcriptional regulators in vivo, during embryogenesis and in the adult organism (Probst, 1997).

The HLH and PAS motifs of both ARNT and mSIM-2 proteins are required for optimal association. Forced expression of GAL4/mSIM-2 fusion constructs in mammalian cells demonstrate the presence of two separable repression domains within the carboxy terminus of mSIM-2. mSIM-2 is capable of repressing ARNT-mediated transcriptional activation in a mammalian two-hybrid system. This effect is (1) dependent on the ability of mSIM-2 and ARNT to heterodimerize, (2) dependent on the presence of the mSIM-2 carboxy-terminal repression domain, and (3) not specific to the ARNT activation domain. These results suggest that mSIM-2 repression activity can dominantly override the activation potential of adjacent transcription factors. mSIM-2 can functionally interfere with hypoxia-inducible factor 1alpha (HIF-1alpha)/ARNT transcription complexes, providing a second mechanism by which mSIM-2 may inhibit transcription (Moffett, 1997).

Arnt (Ah receptor nuclear translocator) is a member of a transcription factor family having characteristic motifs designated bHLH (basic helix-loop-helix) and PAS and was originally found as a factor forming a complex with Ah receptor (AhR) to bind the specific xenobiotic responsive element (XRE) sequence for induction of drug-metabolizing P4501A1. Interaction of Arnt with other PAS proteins (Drosophila Per, Sim, and AhR) has been studied by the coimmunoprecipitation method. Arnt forms a homodimer with itself as well as heterodimers with the others by means of the PAS and HLH domains in a cooperative way. The Arnt homodimer binds the sequence of adenovirus major late promoter (MLP) with the E box core sequence CACGTG, suggesting that the CAC half of the XRE, CACGCN(A/T), recognized by the AhR-Arnt heterodimer is a target for Arnt. Arnt markedly activates expression via the E box, indicative of a newly discovered regulatory role of Arnt (Sogawa, 1995).

The mammalian Ah receptor (AHR), the Ah receptor nuclear translocator protein (ARNT), and Drosophila Single-minded are members of the basic helix-loop-helix-PAS (bHLH-PAS) family of regulatory proteins. The DNA half-site recognition and pairing rules for these proteins were examined using oligonucleotide selection-amplification and coprecipitation protocols. Oligonucleotide selection-amplification reveals that a variety of bHLH-PAS protein combinations could interact, with each generating a unique DNA binding specificity. The AHR-ARNT complex shows a preference for the sequence commonly found in dioxin-responsive enhancers in vivo (TNGCGTG). The ARNT protein is capable of forming a homodimer with a binding preference for the palindromic E-box sequence: CACGTG. Further examination indicates that ARNT may have a relaxed partner specificity, since it was also capable of forming a heterodimer with SIM and recognizing the sequence GT(G/A)CGTG. Coprecipitation experiments using various PAS proteins and ARNT are consistent with the idea that the ARNT protein has a broad range of interactions among the bHLH-PAS proteins, while the other members appear more restricted in their interactions. Comparison of this in vitro data with sites known to be bound in vivo suggests that the high affinity half-site recognition sequences for the AHR, SIM, and ARNT are, respectively, T(C/T)GC, GT(G/A)C (5'-half-sites), and GTG (3'-half-sites) (Swanson, 1995).

Arnt2 is highly similar to, but distinct from, the aryl hydrocarbon receptor (AhR) nuclear translocator (Arnt). The predicted Arnt2 polypeptide carries a characteristic basic helix-loop-helix (bHLH)/PAS motif in its N-terminal region with close similarity (81% identity) to that of mouse Arnt and has an overall sequence identity of 57% with Arnt. Arnt2 interacts with AhR and mouse Sim as efficiently as Arnt, and the Arnt2-AhR complex recognizes and binds specifically the xenobiotic responsive element (XRE) sequence. Expression of Arnt2 mRNA is restricted to the brains and kidneys of adult mice, while Arnt mRNA is expressed ubiquitously. Arnt2 mRNA is expressed in 9.5-day mouse embryos in the dorsal neural tube and branchial arch 1, while Arnt transcripts are detected broadly in various tissues of mesodermal and endodermal origins. These results suggest that Arnt2 may play different roles from Arnt both in adult mice and in developing embryos. Sequence comparison of the currently known bHLH/PAS proteins indicates a division into two phylogenetic groups: the Arnt group, containing Arnt, Arnt2, and Per, and the AhR group, consisting of AhR, Sim, and Hif-1alpha (Hirose, 1996).

Hypoxia-inducible factor-1 (HIF-1), a DNA-binding complex implicated in the regulation of gene expression by oxygen, has been shown to consist of a heterodimer of two basic helix-loop-helix Per-AHR-ARNT-Sim (PAS) proteins, HIF-1alpha, and HIF-1beta. One partner, HIF-1beta, had been recognized previously as the aryl hydrocarbon receptor nuclear translocator (ARNT), an essential component of the xenobiotic response. ARNT-deficient mutant cells have been used to analyze the role of ARNT/HIF-1beta in oxygen-regulated gene expression. Induction of the DNA binding and transcriptional activity of HIF-1 is absent in the mutant cells, indicating an essential role for ARNT/HIF-1beta. Analysis of deleted ARNT/HIF-1beta genes indicated that the basic, helix-loop-helix, and PAS domains, but not the amino or carboxyl termini, are necessary for function in the response to hypoxia. Comparison of gene expression in wild type and mutant cells demonstrated the critical importance of ARNT/HIF-1beta in the hypoxic induction of a wide variety of genes. Nevertheless, for some genes a reduced response to hypoxia persists in these mutant cells, clearly distinguishing ARNT/HIF-1beta-dependent and ARNT/HIF-1beta-independent mechanisms of gene activation (Wood, 1996).


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Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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