InteractiveFly: GeneBrief

orthopedia: Biological Overview | References


Gene name - orthopedia

Synonyms -

Cytological map position - 57B4-57B4

Function - homeodomain transcription factor

Keywords - involved hindgut development of Drosophila, downstream factor of branchyenteron

Symbol - otp

FlyBase ID: FBgn0015524

Genetic map position - chr2R:20,883,625-20,897,584

Classification - Homeobox domain

Cellular location - nuclear



NCBI links: EntrezGene, Nucleotide, Protein

orthopedia orthologs: Biolitmine
BIOLOGICAL OVERVIEW

The Drosophila hindgut is commonly used model for studying various aspects of organogenesis like primordium establishment, further specification, patterning, and morphogenesis. During embryonic development of Drosophila, many transcriptional activators are involved in the formation of the hindgut. The transcription factor Orthopedia (Otp), a member of the 57B homeobox gene cluster, is expressed in the hindgut and nervous system of developing Drosophila embryos, but due to the lack of mutants no functional analysis has been conducted yet. This study shows that two different otp transcripts, a hindgut-specific and a nervous system-specific form, are present in the Drosophila embryo. Using an otp antibody, a detailed expression analysis during hindgut development was carried out. otp was not only expressed in the embryonic hindgut, but also in the larval and adult hindgut. To analyse the function of otp, the mutant otp allele otpGT was generated by ends-out gene targeting. In addition, two EMS-induced otp alleles were isolated in a genetic screen for mutants of the 57B region. All three otp alleles showed embryonic lethality with a severe hindgut phenotype. Anal pads were reduced and the large intestine was completely missing. This phenotype is due to apoptosis in the hindgut primordium and the developing hindgut. These data suggest that otp is another important factor for hindgut development of Drosophila. As a downstream factor of byn otp is most likely present only in differentiated hindgut cells during all stages of development rather than in stem cells (Hildebrandt, 2020).

The Drosophila embryonic hindgut is a single-layered ectodermally derived epithelium surrounded by visceral musculature. It arises from a group of cells at the posterior part of the blastoderm stage embryo referred to as the hindgut primordium. The hindgut primordium is a ring of about 200 blastoderm cells that is internalised during gastrulation to form a short, wide sac. In a relatively short time this epithelium sac is transformed into a long tube containing approximately 700 cells. The growth of the hindgut starting at stage 12 is not due to cell divisions, but a twofold endoreplication that leads to an increase in cell size, and as a consequence total length of the hindgut. During this process, the developing hindgut becomes subdivided along the anterior posterior (AP) axis and the dorsoventral (DV) axis. Along the AP axis, the hindgut forms three morphologically distinct regions: the small intestine, large intestine, and rectum. The small intestine is the most anterior part of the hindgut and is connected to the posterior midgut, whereas the large intestine is the central part of the hindgut and forms three distinct regions along the DV axis. The dorsal and ventral regions constitute the outer and inner portions of the hindgut loop, respectively. Two rows of boundary cells are organised between these two regions and as two rings at the anterior and posterior borders of the large intestine. The most posterior-most portion of the hindgut is the rectum, which connects to the anal pads (Hildebrandt, 2020).

Several genes are required to establish the hindgut primordium and to pattern the hindgut along the AP axis. At the blastoderm stage a group of posterior cells, called the proctodeal primordium, will later on give rise to the hindgut. In these cells the transcription factor Tailless (Tll) is expressed and subsequently activates other transcription factors like Brachyenteron (Byn), Fork head (Fkh) and Bowel (Bowl) as well as the signalling protein Wingless (Wg), which are all necessary for hindgut development. The transcription factor Caudal (Cad) is also expressed in the proctodeal ring, but independently of Tailless. Tll and Wg are necessary to establish the primordium, whereas Cad is necessary for the internalisation of the hindgut primordium later on. Proper gene expression in and maintenance of the large intestine requires byn, Dichaete (D), raw, lines (lin) and mummy (mmy), while bowl and drumstick (drm) are required for gene expression in and maintenance of the small intestine (Hildebrandt, 2020).

The Drosophila T-box gene brachyenteron (byn) is expressed in the ring of cells that internalise to form the embryonic hindgut and expression is maintained in the hindgut throughout embryogenesis. In byn mutants the hindgut is shortened due to apoptosis and the large intestine is missing. The Drosophila homeobox gene orthopedia (otp) is also expressed in the hindgut, anal pads and along the central nervous system (Simeone, 1994). It is located in 57B region of the second chromosome in close vicinity to the other homeobox genes Drosophila retinal homeobox (Drx) and homeobrain (hbn). In the hindgut, otp is directly activated by byn in a dose-dependent manner via multiple binding sites present in a regulatory element of otp (Hildebrandt, 2020).

Otp is highly conserved through evolution and has been identified in most multicellular organisms. Among these are several invertebrates such as sea urchins, the mollusc Patella vulgata, the annelid Platynereis dumerilii and several vertebrates such as zebrafish, that have two genes namely otp1 and otp2, chicken, mouse and human. otp genes of vertebrates have a major function in the development of the hypothalamic neuroendocrine system (see Del Giaccom 2008 for review) (Hildebrandt, 2020).

The function of otp during Drosophila development has been unknown so far as no mutants have been described. The present study shows that otp is required for proper hindgut development in Drosophila. One otp allele was generated by ends-out gene targeting and two additional otp alleles were isolated in an EMS-mutagenesis screen. All three otp alleles are characterised by a dramatically reduced hindgut lacking the complete large intestine. This reduction in hindgut length is due to apoptosis in the hindgut primordium and the developing hindgut (Hildebrandt, 2020).

This paper analysed the function of the transcription factor Orthopedia during hindgut development. In the embryo, otp is expressed in the hindgut primordium, the developing hindgut, and the anal pads. This expression is dependent on several upstream regulators, such as lines and byn. Byn directly activates otp through modular binding sites upstream of hindgut specific promoter of otp in a cooperative fashion. otp is then expressed in the large intestine, rectum and anal pads, unlike Byn, which is also expressed in the small intestine. Byn alone is therefore not sufficient to activate otp; lines expression might also be needed. In the small intestine where byn is expressed, lines is repressed by drumstick preventing otp activation. If lines is overexpressed in the small intestine, the repressive effect of drumstick can be overruled and otp expression can take place, supporting the proposed model that lines and byn are necessary for otp expression in the hindgut. Once otp is activated in the embryonic hindgut, its expression continues until the adult stage. The only region where otp in contrast to byn is not expressed is the larval hindgut proliferation zone in the anteriorly located pylorus which supposedly generates hindgut stem cells capable of replacing the larval hindgut cells undergoing apoptosis and building the adult hindgut. The presence of adult hindgut stem cells has been questioned when it was shown that proliferation only takes place in response to tissue damage. The current view is that all parts of the Drosophila intestinal tract maintain stem cells that could migrate across organ boundaries. Since otp expression was never shown in areas where intestinal stem cells are present, otp is rather expressed in and a marker for differentiated hindgut cells (Hildebrandt, 2020).

To analyse the function of otp, a mutant allele was generated by gene targeting via homologous recombination and using this targeting strain, two additional mutant alleles were identified by complementation among an EMS-induced collection of mutants from the 57B region. In the gene targeting mutant, part of exon 4 and exon 5 were missing resulting in an N-terminal deletion of the otp-PC protein form including most of the homeodomain. In the otp1024 mutant, the N-terminus and most of the homeodomain were present, but the C-terminal part of the protein was missing. In both cases no otp protein expression was detected since the anti-Otp antibody was directed against the C-terminal part of Otp. In the case of otp13064, no protein was detected with anti-Otp antibody nor was a sequence alteration in the coding region detected. The splice sites were intact, but it cannot be ruled out that a cryptic splice site might be generated. The generation of cryptic splice sites by mutations is often the case in human genetic disorders like Neurofibromatosis type I (NFI) or Cystic fibrosis where such mutations generate pseudo-exons. Another possibility might be a mutation in a regulatory region of the gene. All otp alleles showed embryonic lethality with a strong hindgut phenotype. The loss of the large intestine led to a dramatic reduction in hindgut length to about one third of the wildtype length. This phenotype is comparable to the byn phenotype, since byn is directly regulating otp. The three transcriptional regulators drm, bowl and lin are required for patterning and cell rearrangements during elongation in the hindgut, but when compared to otp, showed only a reduction to 40% (drm and bowl) or 50% (lin) in the mutant phenotype, suggesting that the loss of otp function is much more severe compared to these genes. A gut specific function of otp like seen in Drosophila is not known for otp genes in higher organisms, where an expression in the nervous system and a function in various aspects of nervous system development is known. In Drosophila, otp is also expressed in the ventral nerve cord and the brain. Expression in the nerve cord seems to be post-transcriptionally regulated, since, in contrast to the mRNA expression posterior to segment A2, the otp protein is not expressed there. This might be due to the regulation via a miRNA as it was seen for other developmental processes (Hildebrandt, 2020).

The nervous system function of otp in higher organisms has been mainly analysed in various model organisms like zebrafish and mice. It was shown that otp is expressed in the hypothalamus that exerts influence on physiological function in various processes like blood pressure, circadian rhythmicity, energy balance and homeostasis. In zebrafish otp in the hypothalamus is required in the preoptic area for the production of the neurohypophysial peptide arginine vasotocin, for dopaminergic and neuroendocrine cell specification, regulation of stress response and through neuropeptide switching that impacts social behavior. In mice, a loss of otp leads to a progressive impairment of neuroendocrine cells in the hypothalamus, and homozygous mutant animals die soon after birth with a failure of terminal differentiation of neurosecretory cells. Recently, it was shown that a mutation in otp is associated with obesity and anxiety in mice. The otp gene from humans was cloned some time ago, but only during the last few years, it could be shown that otp has a high diagnostic value concerning pulmonary neuroendocrine tumours. Even if these tumours accounted for only 1%-2% of all lung tumours, their occurrence increased over the last decades. People with poor survival rates showed a strong downregulation of otp. otp that is normally located in the nucleus (nOTP) could also be detected in the cytoplasm (cOTP) or not be present at all. Patients with nOTP have a favourable disease outcome, those with cOTP have an intermediate outcome and those with no otp expression have the worst disease outcome demonstrating the diagnostic value of OTP. Due to these very interesting aspects of otp function in the nervous system of higher organisms, it would be interesting to analyse the function of otp during embryonic brain development of Drosophila, as well as later functions of otp during larval development and in the adult, using the newly generated otp alleles in the future (Hildebrandt, 2020).

Using gene expression analysis and newly generated mutant otp alleles, this study has shown that the Drosophila homeodomain transcription factor Orthopedia is an important factor for hindgut development. These findings demonstrate a requirement of otp to build the large intestine of the hindgut and also in the correct formation of the anal pads. This phenotype is caused by apoptosis at the beginning of hindgut development. otp as a downstream factor of byn is most likely present only in differentiated hindgut cells during all stages of development rather than in stem cells (Hildebrandt, 2020).

Brachyury proteins regulate target genes through modular binding sites in a cooperative fashion

Brachyury proteins, a conserved subgroup of the T domain transcription factors, specify gut and posterior mesoderm derivatives throughout the animal kingdom. The T domain confers DNA-binding properties to Brachyury proteins, but little is known how these proteins regulate their target genes. A direct target gene of the Drosophila Brachyury-homolog Brachyenteron has been characterized. Brachyenteron activates the homeobox gene orthopedia (otp; Simeone, 1994) in a dose-dependent manner via multiple binding sites with the consensus (A/G)(A/T)(A/T)NTN(A/G)CAC(C/T)T. The sites and their A/T-rich flanking regions are conserved between D. melanogaster and Drosophila virilis. Reporter assays and site-directed mutagenesis demonstrate that Brachyenteron binding sites confer in part additive, in part synergistic effects on otp transcription levels. This suggests an interaction of Brachyenteron proteins on the DNA, that maps to a conserved motif within the T domain. Mouse Brachyury also interacts with Brachyenteron through this motif. The Xenopus and mouse Brachyury homologs activate orthopedia expression when expressed in Drosophila embryonic cells. It is proposed that the mechanisms to achieve target gene expression through variable binding sites and through defined protein-protein interactions might be conserved for Brachyury relatives (Kusch, 2002).

Based on its expression pattern, the homeobox gene otp is a likely candidate for a direct target of Byn. otp mRNA expression becomes detectable shortly after the onset of byn expression in the common primordium of hindgut and anal pads (in the following only referred to as the hindgut primordium). byn is necessary for the gut-specific otp expression and can activate ectopic otp expression when overexpressed by means of the GAL4/UAS system. The otp locus has been characterized and two transcription start sites were found, one for hindgut-specific transcripts, another ~5.8 kb further upstream for CNS-specific transcripts. During later stages of embryogenesis otp becomes expressed in the CNS, an aspect independent of byn (Kusch, 2002 and references therein).

To identify the hindgut-specific regulatory elements within the otp locus, various genomic fragments of the locus were fused to lacZ reporters and the transgenic embryos were monitored for ß-galactosidase expression in the hindgut. A 1.8-kb XhoI/EcoRI fragment ~950 bp upstream of the putative hindgut-specific transcription start site confers lacZ expression in the hindgut primordium in a pattern identical to endogenous otp. lacZ is strongly expressed throughout the hindgut including anal pads, at lower levels at the distal part of the hindgut tube close to the pads, and is not found in the small intestine of the hindgut. Thus, the element contains all the information for proper regulation of otp. A further subdivision of the 1.8-kb element yielded three regulatory fragments: a proximal, a central, and a distal. The proximal (P1310-1817) and the central (C872-1309 ) fragment were sufficient to direct hindgut-specific lacZ expression in an otp-specific pattern, indicating that the gut expression of otp is regulated by at least two independent Byn-responsive elements. The third, distal fragment (D1-871) did not give detectable lacZ expression in the embryo (Kusch, 2002).

Does Byn directly regulate otp expression via the C872-1309 and the P1310-1817 fragments and what do possible Byn binding sites look like? To address these questions, DNase I protection experiments were performed and three protected regions were found in the C872-1309 and two in the P1310-1817 fragment. Three of these regions consist of two binding sites, which are arranged in tandem repeats (viii/ix, x/xi) or as nested inverted repeats (xiva and xivb). DNA around the repeats (e.g., the entire region between the binding sites xi and xii) became more sensitive to DNase I in the presence of Byn protein. Such hypersensitive sites indicate a compression of the major groove due to DNA bending and suggest a loop formation between the sites (Kusch, 2002) .

The comparison of the protected regions in the fragments P1310-1817 and C872-1309 identifies a consensus sequence for Byn binding. This consensus of (A/G)(A/T)(A/T)NTN(A/G)CAC(C/T)T is similar to the consensus for Brachyury binding. Astonishingly, seven regions were also found within the D1-871 fragment that were protected by Byn from DNase I. Because D1-871 does not confer lacZ expression in embryos and each of these sites deviates from the consensus sequence of the sites in the P and C fragments in at least one position, it is concluded that they must be significantly less efficient for transcriptional activation by Byn. These sites probably have low affinity to Byn as suggested by their deviations from the consensus (Kusch, 2002).

Whether the Byn sites and their arrangement or spacing are conserved was investigated. For this purpose, the sequences of the otp loci of Drosophila melanogaster and Drosophila virilis were compared. Seven of the Byn binding sites and their flanking sequences are highly conserved between the two species; only their spacing is somewhat different. Instead of two Byn tandem sites (viii/ix, x/xi), in D. virilis only two individual sites with spacing similar to the tandems in D. melanogaster are found. Also most of the sites in fragment D are not strictly conserved, although the total number of Byn sites in D. virilis (14) is about the same as in D. Melanogaster (15). The large conserved regions flanking the Byn sites are A/T rich and presumably facilitate DNA-bending, but do not appear to contain obvious binding sites for other factors. Thus, the high number of binding sites and their embedding in flexible DNA stretches are a conserved feature of the otp regulatory region (Kusch, 2002).

All three high affinity (type A) Byn sites show a high homology to a half of the palindromic Brachyury binding site. Thus, it is very likely that vertebrate Brachyury proteins can recognize Byn sites and are able to activate otp transcription. To test this hypothesis, mouse Brachyury and Xenopus Xbra were tested in Drosophila embryos by means of the GAL4/UAS system. Of the two proteins, Brachyury is able to ectopically activate otp expression in the developing salivary glands of the late embryo. Both Brachyury homologs can activate transcription, although to a lesser extent than Byn, and therefore are apparently able to recognize the Byn sites in the otp gene (Kusch, 2002).

Byn binding sites of putatively low affinity (type B, on fragment D) exert synergistic effects on target gene expression when combined with putatively high affinity sites. Likewise, the synthetic combination of type A and type B sites reveals a strong synergism in transcriptional activation. Furthermore, extended regions between Byn sites become hypersensitive to DNase I suggesting an interaction between different Byn sites during transcriptional activation, possibly by the physical interaction of Byn itself. X-ray analysis of Xbra T domains shows that two Xbra molecules are close enough for a dimerization on the synthetic 24-bp palindrome of the Brachyury consensus. However, this contact in the crystal could be attributable to the two inverted binding half sites in the palindrome rather than being a biologically relevant dimerization. Therefore, a possible Byn-Byn interaction was tested in a cellular context, and the Byn open reading frame (ORF) was fused to the activation (GAD-Byn) and to the DNA binding domain (GBD-Byn) of yeast GAL4, respectively. The constructs were introduced into an appropriate yeast strain and, in case of a Byn-Byn interaction, should allow growth on a selective medium due to the formation of a functional GAL4 activator. In fact, combinations of constructs containing the Byn T domain (amino acids 84-276) were able to confer growth when combined in the cells, supporting the idea that Byn proteins interact via their DNA-binding domain. To narrow down the interaction region, N- and C-terminal deletions of the Byn T domain were generated. These truncations revealed that the central region of the T domain (amino acids 151-205) is sufficient for the interaction. This region contains a stretch of amino acids that forms most of the contact surface of the two Xbra T domains in the crystal, and therefore, most likely, is also being used for dimerization in vivo. The conservation of this region of the Brachyury-type T domain suggests that all members of the Brachyury subgroup can dimerize. Indeed, a murine Brachyury-GAL4 fusion construct can interact with the Drosophila Byn interaction domain, indicating that the ability to dimerize is a general feature of Brachyury proteins (Kusch, 2002).

Surprisingly, in the regulatory region of otp, no single Byn site was found that would be sufficient to activate transcription on its own. Also in other cases in which natural target sites for T domain transcription factors have been described, only single half-sites have been identified by virtue of their high similarity to the original Brachyury consensus. For Xbra, recognition motifs matching the Byn consensus have been found in the efgf locus. Also the T domain proteins Tbx-2, Mga, and VegT/Tpit recognize binding motifs similar to high affinity Brachyury half-sites. Thus, rather bind than to a palindrome Brachyury site, proteins seem to bind a 12-bp site in vivo. Possibly, the palindrome is even disadvantageous for the animal and might cause an exceedingly stable occupancy of the site by the protein (Kusch, 2002).

Within the regulatory region of otp an unusually high degree of tolerated variability for Byn binding sites is found. Within the 15 characterized sites two types were distinguished, A and B. Type A sites (to which the sites x, xii, and xiii belong) have a consensus, which differs only by two positions from the Brachyury consensus. This consensus has been selected from DNA molecules that bind optimally to Brachyury by binding site selection, a method that enriches for high-affinity binding sites. It is therefore assumed that the type A sites constitute high-affinity sites for Byn. More importantly, type A sites are essential for strong transcriptional up-regulation. Site x is required for 80% of the transcriptional activation by the C fragment, site xii is absolutely essential for C, and variants of the P fragment without site xiii have lost the ability to activate transcription. Finally, two type A sites, in tandem or in inverted orientation, are sufficient to activate Byn-dependent transcription (Kusch, 2002).

The Byn binding sites of type B yield a more relaxed consensus that encompasses the high-affinity consensus. They are significantly less efficient to activate Byn-dependent transcription than type A sites. For instance, the set of seven type B sites in the D fragment (i to vii) is not sufficient for transcription on its own, and the sites ix and xi contribute little to the activity of fragment C. Based on these observations it is proposed that the type B sites constitute low-affinity sites for Byn. In contrast to type A sites, a type B doublet is not able to activate transcription in the synthetic Byn response elements, neither in tandem nor in inverted orientation. Transcriptional activation is observed only in combination with a type A site, which is inverted to the type B site (Kusch, 2002).

What could be the basis for the ability of the Byn T domain to bind to such variable target sequences? Due to the high homology between Xbra and Byn and between their target sites, it is reasonable to assume that the crystal structure analysis of the Xbra T domain could serve as a model for Byn. The basic residues of the highly conserved motif CAC(C/T)T are contacted by amino acids of the T domain and constitute a sequence-specific binding interaction. Nucleotide exchanges within this stretch would disrupt the hydrogen bonds to the protein and lead to significantly lower binding efficiency. This is the case for the variant site Deltaxii-4, which is only marginally protected against DNase by Byn. A number of type A sites do not have the 3'-T of CAC(C/T)T, a feature which presumably contributes to their low binding efficiency. At the 5' end of the Byn consensus the RWW-motif defines a particular conformation of the double helix rather than a specific base sequence. At these positions the T domain contacts the phosphoribosyl backbone of the nucleotides, stabilizing the protein-DNA interaction, but allowing a higher variability of the bases. Thus, the structural data for the T domain give a good explanation how Brachyury proteins can accommodate the variability of their target sequences. It is reasonable to assume that T domain transcription factors, not belonging to the Brachyury-type subgroup, also recognize a wide range of target sites with variable affinities (Kusch, 2002).

This study shows that Byn binds to different types of target sites cooperatively and thereby activates a certain level of otp expression. Particular arrangements of such high- and low-affinity sites could also serve another purpose: the dose-dependent activation of target genes. There are several indications in Drosophila that the dose of Byn is relevant and informative. Byn is expressed in a dorso-ventral gradient in the blastoderm and allelic series of byn mutants exhibit graded defects in the embryos. This is fully consistent with the finding that above a certain threshold of Byn effector, the cis-regulatory region of otp strongly responds transcriptionally. In other species, Brachury-type transcription factors also give very clear dose-dependent responses. In vertebrates, Brachyury is distributed in an antero-posterior gradient in the embryo, and the formation of distinct embryonic structures depends on certain thresholds of Brachyury. It is conceivable that target gene responses depend on the levels of Brachyury expression as well as the amount and combination of high- and low-affinity sites within their regulatory regions (Kusch, 2002).

Homeobrain, a novel paired-like homeobox gene is expressed in the Drosophila brain

The homeobrain (hbn) gene is a new paired-like homeobox gene that is expressed in the embryonic brain and the ventral nerve cord. Expression of homeobrain initiates during the blastoderm stage in the anterior dorsal head primordia and the gene is persistently expressed in these cells that form parts of the brain during later embryonic stages. An additional weaker expression pattern is detected in cells of the ventral nerve cord from stage 11 on. The homeodomain in the Homeobrain protein is most similar to the Drosophila proteins Rx, Aristaless and Munster. In addition, the localized brain expression patterns of homeobrain and Rx resemble each other. Two other homeobox genes, orthopedia and Rx are clustered in the 57B region along with homeobrain. The current evidence indicates that homeobrain, Rx and orthopedia form a homeobox gene cluster in which all the members are expressed in specific embryonic brain subregions (Walldorf, 2000).

Drosophila brachyenteron regulates gene activity and morphogenesis in the gut

T-related gene expression is activated by tailless, but Trg does not regulate itself. Trg expression in the hindgut and anal pad primordia is required for the regulation of genes encoding transcription factors (even-skipped, engrailed, caudal, AbdominalB and orthopedia) and cell signaling molecules (wingless and decapentaplegic). In Trg mutant embryos, the defective program of gene activity in these primordia is followed by apoptosis (initiated by reaper expression and completed by macrophage engulfment), resulting in severely reduced hindgut and anal pads. Only anal pad expression of cad requires Trg; expression in Malpighian tubules and hindgut is not affected. dpp expression is absent in the hindgut. Females mutant for Trg are sterile, and no egg chambers in their ovaries progress beyond stage 7 of ovarian development (Singer, 1996).

Orthopedia, a novel homeobox-containing gene expressed in the developing CNS of both mouse and Drosophila

A novel homeobox-containing gene has been identified. Its name, Orthopedia (Otp), exemplifies the homology shared by both the orthodenticle and Antennapedia homeodomains. otp is highly conserved in evolution. In mouse, otp is expressed only in restricted domains of the developing forebrain, hindbrain, and spinal cord. In Drosophila, otp first appears at gastrulation in the ectodermal proctodeum and later in the hindgut, anal plate, and along the CNS. This study compared the Otp-, Distal-less homeobox 1-(DIx1-), Orthodenticle homolog 1-(Otx1-), Otx2-, and Empty spiracles homolog 2-expressing domains. The results indicate that otp is expressed along the CNS both in mouse and Drosophila; otp could specify regional identities in the development of the forebrain and spinal cord; transcription of otp and DIx1 takes place in alternating hypothalamic regions reminiscent of a segment-like pattern; and the structural and functional conservation could correspond to a conserved function maintained in evolution (Simeone, 1994).


Functions of Orthopedia orthologs in other species

Development and characterization of an otp conditional loss of function allele

Orthopedia (Otp) is a homeodomain transcription factor that plays an essential role in the development of hypothalamic neurosecretory systems. Loss of otp results in the failure of differentiation of key hypothalamic neuroendocrine cell types, and pups die soon after birth. Although the constitutive knockout otp mouse model (Otp (KO)) has significantly expanded understanding of Otp's function in vivo, a conditional loss of function otp allele that enables tissue or cell-type specific ablation of otp has not been developed. This study used CRISPR/Cas9 gene-editing technology to generate a conditional otp knockout mouse line in which exon 2 of the murine otp gene is flanked by LoxP sites (Otp (f/f)). Crossing the otp (f/f) mouse with Agrp-Ires-cre mouse, this study demonstrated the requirement for otp in the continuous differentiation of AgRP neurons after cell fate determination. The residual AgRP neurons in Agrp-Ires-cre;Otp (f/f) mice project to similar downstream target regions. This newly developed otp (f/f) mouse can be used to explore the functions of otp with cell-type or temporal specificity (Hu, 2020).

Homeodomain protein otp affects developmental neuropeptide switching in oxytocin neurons associated with a long-term effect on social behavior

Proper response to stress and social stimuli depends on orchestrated development of hypothalamic neuronal circuits. This study addresses the effects of the developmental transcription factor Orthopedia (Otp) on hypothalamic development and function. Developmental mutations in the zebrafish paralogous gene otpa but not otpb affect both stress response and social preference. These behavioral phenotypes were associated with developmental alterations in oxytocinergic (OXT) neurons. Thus, otpa and otpb differentially regulate neuropeptide switching in a newly identified subset of OXT neurons that co-express the corticotropin-releasing hormone (CRH). Single-cell analysis revealed that these neurons project mostly to the hindbrain and spinal cord. Ablation of this neuronal subset specifically reduced adult social preference without affecting stress behavior, thereby uncoupling the contribution of a specific OXT cluster to social behavior from the general otpa(-/-) deficits. These findings reveal a new role for otp in controlling developmental neuropeptide balance in a discrete OXT circuit whose disrupted development affects social behavior (Wircer, 2017).

Disruption of the homeodomain transcription factor orthopedia homeobox (Otp) is associated with obesity and anxiety

Genetic studies in obese rodents and humans can provide novel insights into the mechanisms involved in energy homeostasis. This study genetically mapped the chromosomal region underlying the development of severe obesity in a mouse line identified as part of a dominant N-ethyl-N-nitrosourea (ENU) mutagenesis screen. The metabolic and behavioral phenotype of obese mutant mice were characterized and changes were examined in hypothalamic gene expression. In humans, genetic data was examined from people with severe early onset obesity. An obese mouse heterozygous for a missense mutation (pR108W) in orthopedia homeobox (Otp), a homeodomain containing transcription factor required for the development of neuroendocrine cell lineages in the hypothalamus, a region of the brain important in the regulation of energy homeostasis. Otp(R108W/+) mice exhibit increased food intake, weight gain, and anxiety when in novel environments or singly housed, phenotypes that may be partially explained by reduced hypothalamic expression of oxytocin and arginine vasopressin. R108W affects the highly conserved homeodomain, impairs DNA binding, and alters transcriptional activity in cells. otp was sequenced in 2548 people with severe early-onset obesity and a rare heterozygous loss of function variant was identified in the homeodomain (Q153R) in a patient who also had features of attention deficit disorder. It is concluded that otp is involved in mammalian energy homeostasis and behavior and appears to be necessary for the development of hypothalamic neural circuits. Further studies will be needed to investigate the contribution of rare variants in otp to human energy homeostasis (Moir, 2017).

Homeodomain protein otp and activity-dependent splicing modulate neuronal adaptation to stress

Regulation of corticotropin-releasing hormone (CRH) activity is critical for the animal's adaptation to stressful challenges, and its dysregulation is associated with psychiatric disorders in humans. However, the molecular mechanism underlying this transcriptional response to stress is not well understood. Using various stress paradigms in mouse and zebrafish, this study shows that the hypothalamic transcription factor Orthopedia modulates the expression of CRH as well as the splicing factor Ataxin 2-Binding Protein-1 (A2BP1/Rbfox-1). It was further shown that the G protein coupled receptor PAC1, which is a known A2BP1/Rbfox-1 splicing target and an important mediator of CRH activity, is alternatively spliced in response to a stressful challenge. The generation of PAC1-hop messenger RNA isoform by alternative splicing is required for termination of CRH transcription, normal activation of the hypothalamic-pituitary-adrenal axis and adaptive anxiety-like behavior. This study identifies an evolutionarily conserved biochemical pathway that modulates the neuronal adaptation to stress through transcriptional activation and alternative splicing (Amir-Zilberstein, 2012).

Anteroposterior patterning in hemichordates and the origins of the chordate nervous system

The chordate central nervous system has been hypothesized to originate from either a dorsal centralized, or a ventral centralized, or a noncentralized nervous system of a deuterostome ancestor. In an effort to resolve these issues, the hemichordate Saccoglossus kowalevskii was examined and the expression of orthologs of genes that are involved in patterning the chordate central nervous system was examined. All 22 orthologs studied are expressed in the ectoderm in an anteroposterior arrangement nearly identical to that found in chordates. Domain topography is conserved between hemichordates and chordates despite the fact that hemichordates have a diffuse nerve net, whereas chordates have a centralized system. It is proposed that the deuterostome ancestor may have had a diffuse nervous system, which was later centralized during the evolution of the chordate lineage (Lowe, 2003).

The adult S. kowalevskii has tripartite, tricoelomic organization. At the anterior is the muscular proboscis or prosome, used for burrowing and collecting food particles. It contains the heart, kidney, a section of the dorsal nerve cord, and the protocoel. The middle region, which is the collar or mesosome, contains the mouth, a section of dorsal nerve cord formed by neurulation, the paired mesocoels, and the base of the stomochord, which projects forward into the prosome. The posterior region or metasome contains the gill slits, the remainder of the dorsal nerve cord, the entire ventral nerve cord, paired metacoels, gonads, a long through-gut, and terminal anus. At juvenile stages, a ventral post-anal extension (called a tail or sucker) is present (Lowe, 2003).

Gastrulation entails uniform and simultaneous inpocketing of the vegetal half of the hollow blastula. As the blastopore closes, a gumdrop-shaped gastrula is formed. As the embryo lengthens, two circumferential grooves indent and divide the length into prosome, mesosome, and metasome regions. Mesodermal coeloms outpouch from the gut anteriorly and laterally. The first gill slit pair appears externally by day 5, and the animal bends from the dorsal side. The hatched juvenile elongates and adds further pairs of gill slits successively. The animal is nearly bilaterally symmetric, except that the prosome excretory pore (the proboscis pore) from the kidney is reliably on the left, defining a left-right asymmetry (Lowe, 2003).

The hemichordate adult nervous system is not centralized but is a diffuse intraepidermal, basiepithelial nerve net. Nerve cells are interspersed with epidermal cells and account for 50% or more of the cells in the proboscis and collar ectoderm and a lower percentage in the metasome. Axons form a meshwork at the basal side of the epidermis. The two nerve cords are through-conduction tracts of bundled axons and are not enriched for neurogenesis. This general organizational feature of the nervous system has been largely underemphasized in recent literature that focuses on possible homologies between chordate and hemichordate nerve cords (Lowe, 2003).

Twenty-two full-length coding sequences of orthologs associated with neural patterning in chordates were isolated. These genes are probably present as single copies in S. kowalevskii because orthologs of most of them are present as single copies in lower chordates and echinoderms, and many of the genes were recovered multiple times in the EST analysis without finding any closely related sequences (Lowe, 2003).

Using full-length probes for in situ hybridization, all 22 genes were found to be expressed strongly in the ectoderm as single or multiple bands around the animal, in most cases without dorsal or ventral differences (rx, hox4, nkx2-1, en, barH, lim1/5, and otx are exceptions). Circumferential expression is consistent with diffuse neurogenesis in the ectoderm. The domains resemble the circumferential expression of orthologs in Drosophila embryos. In chordates, by contrast, most of these neural patterning genes are expressed in stripes or patches only within the dorsal neurectoderm and not in the epidermal ectoderm. Also, in chordates, the domains are often broader medially or laterally within the neurectoderm, and there are usually additional expression domains in the mesoderm and endoderm. In most of the 22 cases in S. kowalevskii, the ectodermal domain is the only expression domain (six3, otx, gbx, otp, nkx2-1, dbx, hox11/13, and irx are exceptions) (Lowe, 2003).

Although each of the 22 genes has a distinct expression domain along the anteroposterior dimension of the chordate body, attempts were made to divide them into three broad groups to facilitate the comparison with hemichordates: anterior, midlevel, and posterior genes. Anterior genes are those which in chordates are expressed either throughout or within a subdomain of the forebrain. Midlevel genes are those expressed at least in the chordate midbrain, having anterior boundaries of expression in the forebrain or midbrain, and posterior boundaries in the midbrain or anterior hindbrain. Posterior genes are those expressed entirely within the hindbrain and spinal cord of chordates. Many of the chordate genes have additional domains of expression elsewhere in the nervous system and in other germ layers, but comparisons were restricted to domains involved in specifying the neuraxis in the anteroposterior dimension. Taking these groups of genes one at a time, it was asked where the orthologous genes are expressed in S. kowalevskii. In all comparisons, no morphological homology is implied between the subregions of the chordate and hemichordate nervous systems (Lowe, 2003).

Ten genes expressed in midlevel neural domains were examined, namely tailless (tll), paired box homeobox 6 (pax6), emptyspiracles-like (emx), barH, orthopedia (otp), developing brain homeobox (dbx), lim domain homeobox 1/5 (lim1/5), iroquois (irx), orthodenticle-like (otx), and engrailed (en). These genes are all expressed in chordates at least in the midbrain of the central nervous system, and thus, as a group, their domains are more posteriorly located than the anterior set. Some have the anterior border of the domain in the forebrain (tll, pax6, emx, lim1/5, and otx), and some have their anterior border in the midbrain (otp, barH, dbx, irx, and en). Most have posterior borders in the midbrain, but two (en and irx) have posterior borders in the anterior hindbrain. Thus, while all are expressed in the midbrain, each differs in its anterior and posterior extent. Several of the chordate genes (pax6, dbx, en, and irx) have separate posterior expression domains running the length of the chordate hindbrain and spinal cord at different dorsoventral levels of the neural tube (Lowe, 2003).

In S. kowalevskii, these ten orthologs are expressed in circumferential bands in the ectoderm at least of the mesosome (collar) or anterior metasome, that is, more posteriorly than the anterior group. Each gene differs in the exact anteroposterior extent of its domain -- some are expressed in part or all of the prosome. The most broadly expressed orthologs of this group are pax6, otp, lim1/5, irx, and otx. All are expressed in the prosome (relatively weakly for otx), mesosome (weakly in the case of otp and lim1/5), and anterior metasome, all ceasing by the level of the first gill slit. pax6 is strongest at the base of the proboscis, and lim1/5 is expressed most strongly in a dorsal patch at the base of the proboscis. The most narrowly expressed orthologs are barH, tll, emx, and en. tll is detected in early stages in the anterior prosome, posterior prosome, and anterior mesosome and in later stages restricted to the anterior mesosome. The emx domain is a single ring in the anterior mesosome plus an additional domain in the ciliated band in the posterior metasome, the only gene of the 25 to be expressed in the band cells. barH and en are both expressed in narrow ectodermal bands; barH in the anterior mesosome and en in the anterior metasome. A dorsal view of both en and barH reveals a dorsal narrow gap in expression in the midline. Ventrally, no such gap is observed. Two additional spots of en expression are detected in the ectoderm on either side of the dorsal midline in the proboscis. In the most posterior ring of otx expression in the metasome, a similar gap in expression is observed. otp is expressed predominantly in a punctate pattern in the apical layer of prosome ectoderm and in a diffuse pattern in the basal layer of prosome ectoderm, similar to dlx. It is also expressed in a circumferential ring of intermittant ectodermal cells in the posterior mesosome and then in two parallel lines of cells bilateral to the dorsal axon tract of the anterior metasome. Early dbx expression is most strongly detected in an ectodermal ring in the developing mesosome overlapping the posterior domain of tll. dbx is also expressed in the prosome at low levels throughout the ectoderm and at high levels in scattered individual cells or groups of cells. Later expression is restricted to two ectodermal bands marking the anterior and posterior limits of the mesosome. An additional endodermal domain of expression is observed predominantly in the ventral anterior pharyngeal endoderm (Lowe, 2003).

otx, en, and irx deserve description in more detail because in chordates, especially vertebrates, the products of these regionally expressed genes are thought to interact in setting up the midbrain-hindbrain boundary and the isthmic organizer. Furthermore, the otx domain at the midbrain level is the site from which neural crest cells migrate ventrally to the first branchial arch. In S. kowalevskii, otx is expressed at low but readily detectable levels in the prosome ectoderm and at high levels in four closely spaced ectodermal rings: one at the base of the prosome, two in the mesosome, and one in the anterior metasome. This fourth stripe of otx expression crosses the site where the first gill slit perforates the ectoderm. As evidence, beyond morphology, that the hemichordate gill slit is homologous to the chordate gill slit/branchial arch, the pax1/9 ortholog, known to be expressed in chordate gill slits, is expressed in the endoderm of the developing S. kowalevskii gill slit. Gill slit expression of pax1/9 is observed in the adult of P. flava. Thus, chordates and hemichordates have in common the association of the posterior limit of the otx domain with the position of the first gill slit or branchial arch (Lowe, 2003).

In hemichordates, the en domain overlaps the posterior part of the otx domain, and the irx domain runs through both of these, as is also the case in chordates. However, otx expression in S. kowalevskii extends slightly more posteriorly than does en, whereas in chordates the en domain extends slightly more posteriorly (Lowe, 2003).

In summary, for this midlevel group of genes, the S. kowalevskii orthologs are expressed in the mesosome and anterior metasome (with some domains extending anteriorly into the prosome), that is, more posteriorly than those genes of the anterior group. In general, expression domains that end posteriorly near the midbrain-hindbrain boundary in chordates, end in the anterior metasome in hemichordates. Although the anterior metasome is not the site of an obvious morphological boundary, it is the site of the first gill slit. The first gill slit/branchial arch in chordates is at the same body level as the midbrain-hindbrain boundary (Lowe, 2003).

The 22 expression domains of orthologs of chordate neural patterning genes of S. kowalevskii correspond strikingly to those in chordates. There are differences such as the extent of overlap of edges of domains of otx, en, and gbx and other midlevel genes that are critical for forming boundaries within the chordate brain, but the relative domain locations are nonetheless very similar. This similar topography of domains is most parsimoniously explained by conservation in both lineages of a domain arrangement (a map) already present in the common ancestor, the ancestor of deuterostomes (Lowe, 2003).

At least 14 of the 22 conserved domains have similar locations in one or more protostome groups. Such similarities are most parsimoniously explained as a conservation of domains from the ancestral bilaterian. In the case of the hox genes, otx, emx, pax6, six3, gbx, and tll, there is strong evidence for such conservation, but less so for the others (barH and rx). At least four of the chordate-hemichordate conserved domains may not be shared by protostomes. Namely, three of these genes (dbx, vax, and hox11/13) are absent from the Drosophila genome and have not been cloned from other protostome groups. Also, one gene, engrailed, has no clear corresponding domain of expression known in protostomes. In Drosophila, en is expressed in the posterior compartments of 14 body segments and at three or more sites in the head that probably derive from ancient preoral segments. This pattern for en appears very different from the single ectodermal band in deuterostomes (Lowe, 2003).

The nerve net of hemichordates could represent the basal condition of the deuterostome ancestor, or it could represent the secondary loss of a central nervous system from an ancestor. Was the complex map of the ancestor associated with a complex diffuse nerve net or a central nervous system in the ancestor? It is suggested that the deuterostome ancestor may have had a diffuse basiepithelial nervous system with a complex map of expression domains, though not necessarily a diffuse net exactly like that of extant hemichordates. Hemichordates would then have retained a diffuse system in their lineage and early in the chordate lineage, centralization would have taken place. In this proposal, the domain map predates centralization and is carried into the nervous system. In this respect, the core questions of nervous system evolution would concern the modes of centralization utilized by the ancestor's various descendents rather than a dorsoventral inversion, per se. Thus, it is proposed that in chordates, especially vertebrates, the major innovation may have been the formation of a large contiguous nonneural (epidermogenic) region (Lowe, 2003).

Progressive impairment of developing neuroendocrine cell lineages in the hypothalamus of mice lacking the Orthopedia gene

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 otp and Sim1, 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).


REFERENCES

Search PubMed for articles about Drosophila Orthopedia

Acampora, D., et al. (1999). Progressive impairment of developing neuroendocrine cell lineages in the hypothalamus of mice lacking the Orthopedia gene. Genes Dev. 13: 2787-2800. PubMed ID: 10557207

Amir-Zilberstein, L., Blechman, J., Sztainberg, Y., Norton, W. H., Reuveny, A., Borodovsky, N., Tahor, M., Bonkowsky, J. L., Bally-Cuif, L., Chen, A. and Levkowitz, G. (2012). Homeodomain protein otp and activity-dependent splicing modulate neuronal adaptation to stress. Neuron 73(2): 279-291. PubMed ID: 22284183

Del Giacco, L., Pistocchi, A., Cotelli, F., Fortunato, A. E. and Sordino, P. (2008). A peek inside the neurosecretory brain through Orthopedia lenses. Dev Dyn 237(9): 2295-2303. PubMed ID: 18729222

Hildebrandt, K., Bach, N., Kolb, D. and Walldorf, U. (2020). The homeodomain transcription factor Orthopedia is involved in development of the Drosophila hindgut. Hereditas 157(1): 46. PubMed ID: 33213520

Hu, Y., Li, J., Zhu, Y., Li, M., Lin, J., Yang, L., Wang, C. and Lu, Z. (2020). Development and characterization of an otp conditional loss of function allele. Genesis 58(9): e23370. PubMed ID: 32468663

Kusch, T., Storck, T., Walldorf, U. and Reuter, R. (2002). Brachyury proteins regulate target genes through modular binding sites in a cooperative fashion. Genes Dev 16(4): 518-529. PubMed ID: 11850413

Lowe, C. J., Wu, M., Salic, A., Evans, L., Lander, E., Stange-Thomann, N., Gruber, C. E., Gerhart, J. and Kirschner, M. (2003). Anteroposterior patterning in hemichordates and the origins of the chordate nervous system. Cell 113(7): 853-865. PubMed ID: 12837244

Moir, L., Bochukova, E. G., Dumbell, R., Banks, G., Bains, R. S., Nolan, P. M., Scudamore, C., Simon, M., Watson, K. A., Keogh, J., Henning, E., Hendricks, A., O'Rahilly, S., Barroso, I., consortium, U. K., Sullivan, A. E., Bersten, D. C., Whitelaw, M. L., Kirsch, S., Bentley, E., Farooqi, I. S. and Cox, R. D. (2017). Disruption of the homeodomain transcription factor orthopedia homeobox (Otp) is associated with obesity and anxiety. Mol Metab 6(11): 1419-1428. PubMed ID: 29107289

Simeone, A., D'Apice, M. R., Nigro, V., Casanova, J., Graziani, F., Acampora, D. and Avantaggiato, V. (1994). Orthopedia, a novel homeobox-containing gene expressed in the developing CNS of both mouse and Drosophila. Neuron 13(1): 83-101. PubMed ID: 7913821

Singer, J. B., et al. (1996). Drosophila brachyenteron regulates gene activity and morphogenesis in the gut. Development 122: 3703-18 . PubMed Citation: 9012492

Walldorf, U., Kiewe, A., Wickert, M., Ronshaugen, M. and McGinnis, W. (2000). Homeobrain, a novel paired-like homeobox gene is expressed in the Drosophila brain. Mech Dev. 96(1): 141-4. 10940637

Wircer, E., Blechman, J., Borodovsky, N., Tsoory, M., Nunes, A. R., Oliveira, R. F. and Levkowitz, G. (2017). Homeodomain protein otp affects developmental neuropeptide switching in oxytocin neurons associated with a long-term effect on social behavior. Elife 6. PubMed ID: 28094761


Biological Overview

date revised: 10 February 2021

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