Heme metabolism enzymes are dynamically expressed during Xenopus embryonic development

As the key component of many hemoproteins (heme-containing proteins), heme is involved in a broad range of biological processes. Enzymes required for heme biosynthesis and degradation pathways are evolutionarily conserved. While heme metabolism has been studied extensively, the expression of heme metabolism enzymes during development has not been described. Here, we report that all heme biosynthases and two heme oxygenases, which initiate heme degradation, are dynamically expressed during Xenopus embryonic development. All heme synthases, with the exception of aminolevulinic acid synthase 2, are maternally expressed. At neurula stage, heme synthases are expressed in the developing neural tissue and in migrating neural crest cells. At the swimming tadpole stage, expression of heme synthases can be detected in multiple lineages, including eyes, neural crest cells, developing central nervous system, ventral blood island, pronephron, and pronephric tubule. Similar to heme synthases, heme oxygenases are expressed maternally. Zygotic expression of heme oxygenases is mainly restricted to the developing neural and neural crest lineages. Unlike heme synthases, heme oxygenases are not expressed in the ventral blood island and are expressed at a very low level in the pronephron and pronephric tubule. This indicates that heme metabolism may play important roles during development. BIOCELL 2008, 32(3): 259-263 ISSN 0327 9545 PRINTED IN ARGENTINA


Introduction
Heme is a prosthetic group with an iron in the center of a porphyrin.Many proteins contain heme and are called hemoproteins.Hemoproteins have diverse functions, ranging from electron transfer to signal transduction regulation (Hou et al., 2006;Mense and Zhang, 2006).A recent study has indicated that heme is involved in the maturation of microRNA as well (Faller et al., 2007).The heme biosynthetic pathway is evolutionarily conserved and consists of eight enzymes.(Kaplan et al., 1974;Sassa, 1976Sassa, , 1990;;Woodard and Dailey, 2000).The first reaction of heme biosynthesis, which is the rate-limiting step, occurs in the mitochondrial where succinyl-CoA and glycine are converted into δaminolevulinic acid (ALA).Enzymes catalyzing this reaction are aminolevulinic acid synthases (ALASs).
There are two ALAS isoenzymes encoded by two different genes, ALAS-1 and ALAS-2.ALAS-2 is specifically expressed in erythroid cells, whereas ALAS-1, which is considered as the "nonspecific ALAS", is expressed ubiquitously in adult animals.Interestingly, heme suppresses ALAS1 through a number of negative feedback loops.This results in a precise regulation of ALAS1 expression and, consequently, the level of heme biosysthesis.After δ-ALA is produced, the rest seven enzymes work sequentially to catalyze a series of chemical reactions, which ultimately leads to the formation of heme.These enzymes are ALA dehydratase (ALAD), PBG deaminase (PBGD), uroporphyrinogen III synthase (URO3S), uroporphyrinogen decarboxylase (UROD), coproporphyrinogen oxidase (CPO), protoporphyrinogen oxidase (PPO), and ferrochelatase (FECH).Once synthesized, heme is incorporated into heme binding proteins to form hemoproteins and regulates a variety of biological processes (Ajioka et al., 2006).The heme biosynthetic pathway has been extensively studied.All heme synthases have been cloned and the three-dimensional crystal structures of these enzymes have been determined (Ajioka et al., 2006).
While heme is essential for the functions of hemoprotein, free heme is toxic.The level of intracellular heme is precisely controlled by heme degrading enzymes.Two heme oxygenases (HO1 and HO2), the rate-limiting enzymes for heme degradation, catalyze the first reaction of heme turnover, leading to the production of biliverdin, iron, and carbon monoxide.Once this happens, biliverdin reductase reduces the central methene bridge of biliverdin, producing bilirubin.(Snyder and Baranano, 2001;Unno et al., 2007).HOs are involved in cell cycle and cell survival regulation.Recent studies have indicated that HOs may be involved in human diseases, for example, colon cancer (Oates and West, 2006).
Heme regulates the erythroid differentiation (Igarashi and Sun, 2006;Keel et al., 2008).A recent work has indicated that heme metabolism may be im-portant for in vitro neuronal differentiation (Shinjyo and Kita, 2006).These observations promoted us to investigate the expression pattern of heme biosynthetic enzymes during Xenopus embryonic development.And also, since free heme is toxic to cells, we further investigated the expression pattern of the enzymes initiating heme degradation (HO1 and HO2).

Embryos
Xenopus laevis were obtained from Nasco.Eggs were obtained from hCG-stimulated female X. laevis and in vitro fertilized.Fertilized eggs were dejellied in 2% cystein solution according to standard protocols.Staging of embryos was according to Nieuwkoop and Faber (1967).
Whole-mount in situ hybridization was carried out as described by Sive et al. (2000).Embryos were hybridized with either anti-sense probes or with sense probes as negative controls.

Expression of heme biosynthetic enzymes
Xenopus heme biosynthetic enzymes were identified from the GenBank database based on their homologies to human heme biosynthetic enzymes.As shown in Table 1, Xenopus heme synthases are highly similar to heme synthases in other vertebrates.RT-PCR was performed to investigate the temporal expression profile of heme synthases during Xenopus embryonic development.As shown in figure 1B, most heme synthases, with the exception of ALAS2, are maternally expressed.The expression level of these enzymes remains grossly

Percent identity of amino acid sequences between
Xenopus heme metabolism enzymes and their homologues in human, mouse, and zebrafish.
We next examined the spatial expression patterns of heme synthases by whole-mount in situ hybridization (Fig. 2).Heme synthases are ubiquitously expressed from the cleavage stage to gastrula stage (not shown).
Asymmetric expression of heme synthases was first detected at the beginning of the neurula stage.At this stage, expression of heme synthases is slightly stronger on the dorsal side of the embryo (data not shown).The expression of heme biosynthetic enzymes is gradually diminished on the ventral side of the embryo.By late neurula stage, heme synthases become clearly localized on the dorsal side.All enzymes are accumulated in the neural tube, the eye fields, and the migrating neural crest cells.Weak and diffused expression of all eight enzymes can be detected in some epidermis cells adjacent to the neural tube.It is worth to mention that in situ hybridization with ALAS1 or ALAS2 probes resulted in identical staining patterns.Since ALAS1 and ALAS2 share 73% identities, it is likely that these probes cross-react with both ALAS isoforms.At the tadpole stage, the highest level of heme biosynthetic enzymes was observed in the ventral blood island, the site of primitive erythropoiesis.In addition, multiple tissues express heme synthases at fairly high levels.These include head, eyes, central nervous system, migrating neural crest cells, developing pronephron, and pronephric tubule (Fig. 2).Given that all 8 heme synthases are expressed in an identical pattern, it is likely that heme biosynthesis occurs in these tissues during development.

Expression of heme oxygenases
Xenopus HO1 and HO2 were identified by blasting the GenBank database with their human homologues.Xenopus HO1 and HO2 share significant identities with HOs in other vertebrates (Table 1).Similar to most heme biosynthetic enzymes, both HO1 and HO2 are maternally expressed (Fig. 1B).Transcripts of HO1 and HO2 are distributed ubiquitously prior to the neurula stage (not shown).Expression of HO1 and HO2  becomes dorsally localized when embryos reach the neurula stage (Fig. 3).At this stage, the expression pattern of HO1 and HO2 is very similar, if not identical, to that of heme synthases.Strong expression of both enzymes were observed in the neural tube, developing eye field, and migrating neural crest cells.Weaker expression was detected in some epidermis cells adjacent to the neural tube.The expression of HO1 and HO2 persists in the neural and neural crest lineages by the tadpole stage.Unlike heme synthases, however, HO1 and HO2 are not expressed in the ventral blood island at this stage.In addition, they are expressed only very weakly in the developing pronephron and pronephric tubule (Fig. 3).

Discussion
Heme is an essential compound of hemoproteins and is expected to play important roles in many biological processes, including development.Previous studies have indicated that heme metabolism is critical for erythroid differentiation (Igarashi and Sun, 2006;Keel et al., 2008).Recently, it has been reported that the expression level of heme biosynthetic enzymes changes dynamically during in vitro neuronal differentiation (Shinjyo and Kita, 2006).This suggests that heme metabolism may have a broad impact on development.In agreement with this view, here we show for the first time that enzymes involved in heme metabolism are dynamically expressed during Xenopus embryonic development.All heme synthases share identical expression pattern and are expressed in the neural tissue, neural crest cells, developing pronephron, pronephric tubule, and ventral blood island.This raises the possibility that heme biosynthesis is indeed happening in these tissues and may be important for the development of these lineages.Heme synthases and heme oxygenases play opposite roles in heme metabolism.Interestingly, we observed that HO1 and HO2, which are responsible for heme degradation, were mainly restricted to the developing neural and neural crest lineages during early development.While further experiments are needed, above observations suggest that precise regulation of heme metabolism may be important for Xenopus early development.

FIGURE 1 .
FIGURE 1. Temporal expression profile of heme metabolism enzymes during Xenopus embryonic development.A. Schematic presentation of the heme biosynthetic pathway.B. RT-PCR result showing the temporal expression of all heme synthases and heme oxygenases at the one-cell stage (egg), late blastula stage (stage 9), gastrula stages (stage 10 and stage 12), neurula stage (stage 15), tailbud stages (stage 20 and stage 25), and tadpole stage (stage 35).

FIGURE 2 .
FIGURE 2. Whole mount in situ hybridization to show the expression of heme synthases at the late neurula stage and the tadpole stage.From left to right are: anterior views of neurula stage embryos (left column), dorsal views of neurula stage embryos (middle column), and lateral views of tadpole stage embryos (right column).Note that the expression patterns of heme synthases are identical to each other.Arrows point to the migrating neural crest cells.Arrowheads indicate the pronephron and pronephric tubule.* indicates the ventral blood island.

FIGURE 3 .
FIGURE 3. Whole mount in situ hybridization to show the expression of heme oxygenases (HO1 and HO2) at the neurula stage and the tadpole stage.From left to right are: anterior views of neurula stage embryos (left column), dorsal views of neurula stage embryos (middle column), and lateral views of tadpole stage embryos (right column).