Gata3 mutant mice expire of noradrenergic deficiency by embryonic day (E) 11 and can be rescued pharmacologically or, as shown here, by restoring Gata3 function specifically in sympathoadrenal (SA) lineages using the human DBH promoter to direct Gata3 transgenic expression. In Gata3-null embryos, there was significant impairment of SA differentiation and increased apoptosis in adrenal chromaffin cells and sympathetic neurons. Additionally, mRNA analyses of purified chromaffin cells from Gata3 mutants show that levels of Mash1, Hand2 and Phox2b(postulated upstream regulators of Gata3) as well as terminally differentiated SA lineage products (tyrosine hydroxylase, Th, and dopamineβ-hydroxylase, Dbh) are markedly altered. However, SA lineage-specific restoration of Gata3 function in the Gata3 mutant background rescues the expression phenotypes of the downstream, as well as the putative upstream genes. These data not only underscore the hypothesis that Gata3 is essential for the differentiation and survival of SA cells, but also suggest that their differentiation is controlled by mutually reinforcing feedback transcriptional interactions between Gata3, Mash1, Hand2 and Phox2b in the SA lineage.

In response to instructive cues including bone morphogenetic protein (BMP)signaling from the wall of the dorsal aorta, trunkderived neural crest cells aggregate near the dorsal aorta to generate SA progenitor cells(Goridis and Rohrer, 2002; Howard, 2005). Those progenitors subsequently migrate again to their final destinations: the sympathetic ganglia (SG), the adrenal medulla (AM) and the extra-adrenal chromaffin tissue. Transcription factors that mediate BMP signaling in specifying the SA lineage have been identified and include the zinc-finger protein, Gata3, the bHLH proteins Mash1 (Ascl1 - Mouse Genome Informatics) and Hand2, as well as the closely related homeodomain transcription factors Phox2a and Phox2b.

Gata3 encodes a transcription factor containing two steroid hormone receptor-like zinc fingers that serve as its DNA-binding domain(Yamamoto et al., 1990) that binds most avidly to the consensus motif AGATCTTA(Ko and Engel, 1993) and is highly conserved among all six members (Gata1 to Gata6) of this multigene family (Patient and McGhee,2002). Gata3 is prominently expressed in the primary sympathetic chain and persists during the development of sympathetic neurons, adrenal chromaffin cells and para-aortic chromaffin cells [the organ of Zuckerkandl,OZ, which degenerates in adult animals(George et al., 1994; Lakshmanan et al., 1999; Lim et al., 2000)]. Gata3 homozygous mutants die at around E11(Pandolfi et al., 1995) but can be rescued by feeding heterozygous intercrossed dams with catecholamine intermediates, demonstrating a central role for Gata3 in catecholamine biosynthesis (Lim et al.,2000).

In the SG of Gata3 mutants, normal expression of Phox2 and other neuronal markers, but not Th or Dbh, was detected, suggesting that Gata3 acts genetically downstream of Phox2 and is required only for the expression of terminal noradrenergic traits. Loss- and gain-of-function experiments examining Mash1, Hand2, Phox2a and Phox2b indicated that they act before Gata3 in the SA regulatory cascade (Goridis and Rohrer,2002; Lim et al.,2000). More-recent studies have implied that Gata3 may also be essential for aspects of early sympathetic neuronal development(Tsarovina et al., 2004). However, the effect of Gata3 loss-of-function during adrenal medullary development is largely undetermined.

Here we have examined the consequences of Gata3 deficiency on sympathetic neuron and adrenal medullary chromaffin cell development, anticipating possible revelation of new insights into Gata3 neuroendocrine function(s). We show that Gata3 is essential not only for Th and Dbhactivation, but also for cell survival and differentiation of sympathetic neurons and adrenal chromaffin cells. A human DBH (hDBH)gene promoter was used to direct Gata3 expression specifically to the murine SA system (Mercer et al.,1991), and this significantly restores Th and Dbh deficiency in Gata3 mutant mice, thereby overcoming the Gata3mutation-induced embryonic lethality. In contrast to drug-rescued Gata3 mutants, SA tissue-specific Gata3 complementation restored nearly normal development of sympathetic neurons and neuroendocrine chromaffin cells as well as of Mash1, Hand2 and Phox2b expression. A host of previous studies suggested that Mash1, Hand2 and Phox2b function both genetically and biochemically earlier than Gata3 in SA differentiation(Tsarovina et al., 2004);however, the synthesis of previous observations with these new data indicates the more likely possibility that these SA lineage-restricted transcription factors collaborate in a mutually interdependent cell-autonomous manner to regulate the interactive genetic circuitry governing SA differentiation.

Mice

To generate SA lineage-specific transgenic mice, a murine Gata3 cDNA(Ko et al., 1991) or eGFP gene was inserted 3′ to the 5.8 kb hDBH promoter(Kapur et al., 1991; Mercer et al., 1991). Both expression constructs were co-microinjected into BDF1(C57BL/6×DBA/2)-fertilized ova, and two independent transgenic lines were recovered. Each line was mated with either heterozygotes of Gata3 germline knockout (Gata3+/-)(Pandolfi et al., 1995) or nlslacZ knock-in (Gata3Z/+)(van Doorninck et al., 1999)to generate Gata3 heterozygotes that were hemizygous for the transgene. Pharmacological rescue of Gata3 mutant embryos has been described (Lim et al., 2000). Animals were genotyped using PCR and/or Southern blotting. Primers used to detect the hDBH-Gata3 transgene are shown in Table 1.

Quantitative real time PCR (Q-PCR)

Total RNA was extracted from the caudal half of E10.5 embryos or lacZ+CD45- chromaffin cells of E18.5 adrenal glands (100∼200 cells/embryo) using ISOGEN (Nippon Gene). First-strand cDNA was synthesized from 0.5 μg of total RNA in a 20 μl reaction using Superscript II (Invitrogen). Q-PCR was performed using 2×SYBR Green PCR master mix (PE-Applied Biosystems), reverse-transcribed cDNA and gene-specific primers according to the manufacturer's protocol in an ABI PRISM 7000 sequence detector system (PE-Applied Biosystems). To quantify the amount of target mRNA in the samples, a standard curve was prepared using a cDNA containing the target transcript as the control. This procedure enabled standardization of the initial mRNA content of cells relative to the amount of GAPDH mRNA. The sequences of gene-specific primers used are listed in Table 1. The data were recorded as means±s.e.m. The statistical significance of differences among means of several groups was determined by Student's t-test.

TUNEL and immunofluorescent analyses

Embryos (E10.5, E12.5, E13.5, E15.5, E18.5) or neonates were fixed overnight in 4% PFA at 4°C and then processed for immunostaining as frozen or paraffin-embedded sections. For immunofluorescence analysis, rabbit anti-tyrosine hydroxylase (Pel-Freez Biological), rabbit anti-Phox2b (gift from C. Goridis), rabbit anti-chromogranin A (Santa Cruz Biochemicals), rabbit anti-SF1 (gift from G. Hammer), and rabbit anti-GFP (Molecular Probes)antibodies were used. For co-staining of Th and GFP, monoclonal anti-Th antibody (Sigma) was used. Co-localization of β-galactosidase and GFP was performed using a Cy3-conjugated rabbit anti-β-galactosidase antibody(K.C.L., unpublished) and rabbit anti-GFP antibody (Molecular Probes). Fluorescence was visualized using a Leica DM inverted microscope. Separate images were taken and merged using OpenLab software. Whole-mount X-gal staining of embryos was performed as previously described(Lakshmanan et al., 1999). For detection of apoptosis, TUNEL assays were performed on 10 μm tissue cryosections using an In Situ Cell Death Detection Kit (Roche Diagnostics)according to the manufacturer's instructions.

Morphometric analysis

Meta Imaging Series 6.1 (Molecular Device) was used to quantify morphometrically the surface area morphometrically and the anti-β-gal,anti-Th and anti-Phox2b immunoreactive areas, as well as the TUNEL-positive cells in SA tissues. Measurements from at least four sections of bilateral sympathetic ganglia or adrenal glands from at least two different wild-type embryos of each genotype were arbitrarily set at 100%.

Electron microscopy

For electron microscopy, E18.5 adrenal glands were fixed overnight in 2.5%gluteraldehyde in 0.1 M Sorenson's buffer (pH 7.4), washed with 0.1 M Sorenson's buffer and then post fixed in 1.0% OsO4. After more rinsing with 0.1 M Sorenson's buffer, tissues were stained `en-block' using 3%uranyl acetate and processed for embedding in Epon. Ultrathin sections (75 nm)were examined using a Philips CM-100 transmission electron microscope.

Flow cytometry

E18.5 adrenal glands were treated with 0.25% collagenase type B (Roche Diagnostics) for 1 h at 37°C, and cells were then dissociated using finetipped pipettes. After the cells had been filtered through a 35 μm nylon mesh, they were resuspended in PBS containing 4% FCS. Determination ofβ-gal activity using FDG and subsequent incubations with mAb have been described previously (Hendriks et al.,1999). FACS analysis and sorting was carried out using FACS VantageSE and CellQuest software (Becton Dickinson, San Jose, CA). Phycoerythrin (PE)-conjugated CD45 antibody was purchased from BD pharmingen(San Diego, CA). The isolated cells were resuspended in ISOGEN (Nippon Gene)for RT-PCR analysis.

Sympathoadrenal-specific Gata3 expression rescues Gata3mutant embryos from embryonic lethality

Previous studies demonstrated that the expression of a reporter transgene directed by the hDBH promoter begins at around E10.5 in the SG and transiently in other neural tissues including the ventral neural tube. This hDBH-directed reporter expression persists throughout later development in all adult SA tissues (Kapur et al., 1991; Mercer et al.,1991). In order to generate SA lineage-specific forced co-expression of Gata3 and eGFP in transgenic animals, the hDBHpromoter was cloned upstream of both mouse Gata3 and eGFP DNAs(Fig. 1A). Both constructs were co-microinjected into fertilized ova to generate double transgenic animals. The hDBH/Gata3 and hDBH/eGFP transgenes cosegregated through multiple generations in two independent TghDBH-G3 lines,demonstrating co-integration of the two expression constructs.

Immunofluorescent studies examining tissues from E18.5 Gata3Z/+:TghDBH-G3 embryos confirmed that transgenic eGFP (Fig. 1D,G,J)and lacZ expression [from a Gata3/lacZ knock-in allele(Gata3 KI) (van Doorninck et al.,1999); Fig. 1C,F,I]were completely coincident (Fig. 1E,H,K) in the AM, the SG and the OZ. These data demonstrate that the hDBH-directed transgenes accurately recapitulate endogenous Gata3 SA expression.

To determine whether TghDBH-G3 could rescue the embryonic lethality conferred by the Gata3 germline mutation, Gata3Z/+:TghDBH-G3 and Gata3+/- animals were intercrossed(Fig. 1B). Only 15.3% (21/254)of the Gata3Z/-:TghDBH-G3 mutants survived to E18.5 and both transgenic lines did not differ in their ability to rescue Gata3 mutation-induced midgestational lethality(Fig. 1B and Table 2), so only one line was used for all subsequent experiments. We conclude that transgenic expression of Gata3, when placed under the control of the hDBH promoter, rescued Gata3-deficient mice from mid-embryonic demise. As with the drug-rescued mutants (Lim et al.,2000), Tg-rescued mutant embryos were similar in size to their wild-type littermates, but additionally exhibited hypoplastic mandibles and glandular aplasia (the parathyroid glands and thymuses) as well as inner ear and kidney deficiencies (Fig. 2A-H; data not shown). These phenotypes are representative of many of the same affected tissues and organs in individuals with GATA3haploinsufficient HDR (hypoparathyroidism, sensorineural deafness and renal dysplasia) syndrome (Van Esch et al.,2000), underscoring the central role Gata3 plays in the development of each of these organ systems.

Th, Dbh and Gata3 mutant embryos suffer in common from severe cardiac wall atrophy, as noradrenalin is crucial for maintaining cardiac homeostasis, and alterations in sympathetic activity have been directly linked to heart failure (Lim et al., 2000; Thomas et al.,1995; Zhou and Palmiter,1995). The cardiac wall thickness of the Tg-rescued Gata3mutants was fully restored, suggesting that SA system-specific Gata3 complementation reconstituted the noradrenalin biosynthesis pathway, which in turn leads to fully restored myocardial compaction in the mutants(Fig. 2I,J).

TghDBH-G3expression restores sympathetic neuronal differentiation in Gata3mutant mice

We next assessed individual E10.5 embryos for Th, Dbh and Gata3 mRNA synthesis by real time-quantitative RT-PCR (Q-PCR). We found, as anticipated,markedly diminished levels of Gata3, Th and Dbh in the trunk region (which normally expresses the highest level of noradrenalin) of Gata3-null embryos. Furthermore, these transcript species were restored to 70-80% of wild-type levels in the Tg-rescued mutants(Fig. 3A).

When the expression pattern of transgenic eGFP and Th immunoreactivity was assessed on transverse sections of E10.5 embryos of each genotype, we observed strong Th expression in the SG as well as weaker Th immunoreactivity in the ventrolateral neural tube (NT) (Fig. 3B) (Son et al.,1996). In E10.5 Gata3 mutant embryos, Th expression is significantly weaker in both the SG and NT(Fig. 3C)(Lim et al., 2000), suggesting that both SG- and NT-specific Th expression may be under the regulatory influence of Gata3. The hDBH promoter directs transgenic eGFP expression in the SG and in the NT in E10.5 embryos(Fig. 3D)(Kapur et al., 1991; Mercer et al., 1991). As anticipated, Th immunoreactivity was significantly restored both in the SG and NT of E10.5 Tg-rescued Gata3 null mutants(Fig. 3E).

By stark contrast, Th expression was observed only in the SG of E12.5 wild-type embryos, and not in the NT (Fig. 3F) (Son et al.,1996), whereas Th was fully suppressed in the E12.5 Gata3mutant SG (Fig. 3G). Transgenic eGFP expression was still observed weakly in the NT at E12.5(Fig. 3H), although that also gradually extinguished and was undetectable by E18.5 (data not shown)(Kapur et al., 1991; Mercer et al., 1991). As anticipated, Th expression was restored in the SG, but not in the NT, of the E12.5 Tg-rescued Gata3 mutant(Fig. 3I). These results demonstrate that SG-specific stable and NT-specific transient Gata3 complementation restored Th expression in both tissues, thus leading to restoration of the catecholamine deficiency in the E10.5 Gata3mutants. From E12.5 onwards, SA lineage-restricted restoration allows survival of Gata3 mutant embryos to birth.

To evaluate SA development in older embryos, we examined whole-mount X-gal-stained E18.5 Gata3 knock-in embryos using the lacZgene as a marker for the SA lineage. We observed that E18.5 drug-rescued Gata3Z/- mutants had significantly smaller thoracic paravertebral SG in comparison with Gata3Z/+ littermates(Fig. 4A,C). This conclusion was further substantiated when Hematoxylin and Eosin (HE)-stained histological sections of E18.5 embryos were examined. In drug-rescued Gata3mutants, the thoracic paravertebral SG were reduced by 65% in area, with few visible neurons, when compared with that of wild-type littermates(Fig. 4D,F).

Phox2b is a paired/homeodomain transcription factor that has been shown to play a key regulatory role in early SA differentiation in a common genetic pathway upstream of Gata3 (Goridis and Rohrer, 2002; Huber et al.,2005; Pattyn et al.,1999; Tsarovina et al.,2004). As anticipated, in Gata3-deficient embryos, Phox2b staining was evident in only a few surviving sympathetic neurons, and the Phox2b-immunoreactive area was significantly reduced (by over 80%) in comparison with that in wild-type littermates(Fig. 4G,I,M). At the same time, Th expression was virtually extinguished in the Gata3 mutant sympathetic neurons (Fig. 4J,L,M).

In Tg-rescued Gata3 embryos, the size as well as anti-Phox2b and anti-Th immunoreactivities of the paravertebral SG were concomitantly restored to 70-80% of wild-type levels by E18.5(Fig. 4B,E,H,K,M,N). These data underscore the previous observations concluding that Gata3 is indispensable for the differentiation of sympathetic neurons(Tsarovina et al., 2004) and that Th and Phox2b gene expression is suppressed in the (few) remaining viable sympathetic neurons after Gata3 ablation. Restoration of Gata3 function in the mutants by transgenic complementation results in restoration of reasonably normal differentiation of sympathetic neurons accompanied by the recovery of Phox2b and Th expression.

TghDBH-G3 expression restores adrenal chromaffin cell development in Gata3 mutant animals

We next examined the development and differentiation of adrenal medullary chromaffin cells in E18.5 Gata3 mutant embryos. Whole-mount X-gal staining showed that drug-rescued Gata3Z/- mutants exhibit fewer lacZ+ chromaffin cells compared with Gata3Z/+ littermates(Fig. 5A,C). Consistently, a smaller AM was evident in the HE sections of E18.5 Gata3Z/- adrenal glands in contrast to those of wild-type littermates (Fig. 5D,F). Phox2b immunoreactivity was diminished by 88% in the AM of drug-rescued Gata3 mutants, while Th immunoreactivity was virtually non-existent(Fig. 5G,I,J,L,S). By contrast,the adrenal medullary chromaffin population was significantly restored in Tg-rescued E18.5 Gata3 mutants(Fig. 5B,E); not surprisingly,transgene rescue resulted in similarly restored Phox2b and Th immunoreactivity(Fig. 5H,K,S). These observations suggest that Gata3 is indispensable not only for sympathetic neuronal development but also for adrenal chromaffin cell migration or differentiation. Moreover, SA system-specific Gata3 complementation restored the differentiation of adrenal chromaffin cells, as well as Phox2b and Th expression, as it did in SG.

In the AM of Tg-rescued embryos, GFP and Th immunostaining were almost completely coincident, indicating that Th immunoreactivity was restored by transgenic Gata3 complementation in a cell-autonomous manner(Fig. 5P,Q,R). Chromogranin A(ChrA), a major soluble protein occupying the core of peptide hormone and neurotransmitter secretory vesicles(Mahata et al., 2003), was markedly deficient in Gata3Z/- adrenal glands, indicating that the few remaining adrenal chromaffin cells are bereft of functional secretory vesicles (Fig. 5M,O). As an independent measure of chromaffin cell differentiation, ChrA expression was, like Phox2b and Th, largely restored in Tg-rescued Gata3 mutants(Fig. 5N).

Chromaffin cells have distinctive ultrastructural features, most notably large chromaffin granules that allow their unique identification as distinct from sympathetic neurons (Coupland and Tomlinson, 1989). Fig. 6A shows typical chromaffin cells, filled with numerous large secretory granules (core diameter >100 nm), from an E18.5 wild-type adrenal gland. By contrast, the adrenal gland of an E18.5 Gata3-/-embryo harbors few cells that can be readily identified as chromaffin cells,based on the presence of similar granules(Fig. 6A,C). As anticipated, SA tissue-specific transgenic Gata3 rescue restored chromaffin cell ultrastructural characteristics (Fig. 6B). Taken together, these observations suggest that Gata3 is indispensable for conferring the functional properties attributed to fully differentiated chromaffin cells. Despite the fact that Gata3 mutant embryos lack a compact AM, the size of the adrenal cortex (as measured by examining Sf1 expression, a transcription factor exclusively expressed in steroidogenic cells) (Luo et al.,1994), is unaltered in the Gata3 mutants(Fig. 6F). Sf1-negative chromaffin cells were abundant in wild-type and Tg-rescued Gata3mutants, whereas a clearly defined AM was not apparent in drug-rescued Gata3 mutant embryos (Fig. 6D-F).

Reduced number of adrenal chromaffin progenitors in Gata3mutant mice

We next asked whether Gata3-deficient chromaffin cells are able to home to the adrenal gland anlagen during early embryogenesis. This was addressed by following lacZ expression from the knock-in mutant allele as a marker for the SA lineage cells. At E13.5, fewerβ-gal+ cells were detected in the adrenal gland primordium of homozygous mutant embryos in comparison with heterozygotes(Fig. 7A,C).β-Gal+ cells clustered in the center of the AM in Gata3 heterozygous mutants by E15.5, while Gata3-deficient chromaffin cell number was clearly diminished and failed to aggregate normally in the medullary region (Fig. 7D,F). Quantification of β-gal+ cells in serial sections showed that the chromaffin cell population in homozygous mutants amounted to only 30% of heterozygous controls(Fig. 7G). In Tg-rescued Gata3 mutants, the chromaffin cell number was partially restored at E13.5 and E15.5 of embryogenesis (Fig. 7B,E,G).

To determine whether an increase in programmed cell death might contribute to the observed reduction in chromaffin cell number in Gata3-null mutants, TUNEL assays were performed. A marked increase (4- to 5-fold over wild-type) in the number of TUNEL-positive cells was observed in the adrenal glands of pharmacologically rescued E13.5 and E15.5 Gata3 mutants(Fig. 8C,F,G), while TUNEL-positive nuclei were rarely detected in the organs of wild-type mice(Fig. 8A,D,G). Although TUNEL labeling was observed primarily in Th-negative cells, those apoptotic structures were almost exclusively localized in the medulla. In Tg-rescued Gata3 mutants, the TUNEL-positive cell number was markedly diminished(Fig. 8B,E,G). Taken together,these data suggest that a progressive reduction of chromaffin cells in the AM of Gata3 mutants can be attributed to apoptotic cell death, which is largely reversed by SA tissue-specific Gata3 rescue.

Quantitative analysis supports a co-regulatory relationship among SA lineage control genes

To address the consequences of Gata3 deficiency on other SA cell-specific transcription factors, we enriched lacZ+ E18.5 chromaffin cells by flow cytometry using the Gata3 knock-in allele as a chromaffin cell-specific marker. Hematopoietic or dead cells were eliminated by gating out CD45+ and/or propidium-iodide (PI)+ cells. The lacZ+CD45- cells constituted 4.06±1.14%(Gata3Z/-; n=5), 5.43±0.005%(Gata3Z/-:TgDBH-G3; n=5) or 8.02±1.48% (Gata3Z/+; n=5) of the E18.5 adrenal gland single cell suspension (Fig. 9A). The percent changes of fractionally recovered lacZ+ cells is consistent with their fractional representation observed in the X-gal-stained histological analyses of embryos of the same genotypes (Fig. 7).

The purity of the flow-sorted chromaffin cells was assessed by monitoring the expression of Sf1 mRNA, encoding an adrenal cortical cell-specific nuclear receptor (Luo et al., 1994). The lacZ+CD45- fraction was largely devoid of Sf1, whereas the lacZ-CD45-population contained abundant Sf1+ cells, indicating that adrenal chromaffin cells were highly enriched in the lacZ+CD45- fraction(Fig. 9B).

The expression of chromaffin cell-restricted genes, including Gata3,Th, Dbh, Hand2, Phox2b and Mash1, was assessed by real-time quantitative RT-PCR using total RNA prepared from purified lacZ+CD45- chromaffin cell populations. As anticipated,Th and Dbh mRNAs were markedly suppressed in the chromaffin cells of pharmacologically rescued Gata3Z/- mutants(Fig. 9C). Remarkably, we discovered that the expression of Phox2b, a putative upstream regulator of Gata3, was suppressed by 50% in Gata3-deficient chromaffin cells,suggesting that reduced Phox2b immunoreactivity in the Gata3 mutant adrenal gland could be due to both the loss of chromaffin cells through apoptosis and suppressed Phox2b expression in the remaining viable chromaffin cell population. To further investigate this possibility, we examined the expression of other genes that are proposed to act both before and after Phox2b in the cascade. We found that the bHLH transcription factor Hand2,which is purportedly activated downstream of Phox2b, but before Th and Dbh(Goridis and Rohrer, 2002), is more depleted than Phox2b, demonstrating an even stronger influence of Gata3 on Hand2 expression in Gata3-deficient chromaffin cells. Unexpectedly, we found that Mash1 expression is elevated (approximately twofold) in purified Gata3-deficient chromaffin cells, demonstrating that Gata3 (either directly or indirectly) negatively regulates Mash1, the SA regulator that is proposed to act earliest in this cascade.

If the nascent hypothesis that Gata3 (directly or indirectly) acts in a feedback loop to either enforce or repress the expression of other SA system-restricted regulatory genes, SA cells-specific rescue of Gata3 should restore their expression to nearly wild-type levels. In purified TghDBH-G3-rescued mutant chromaffin cells, transgenic Gata3 was expressed at ∼50% of haploid levels(Fig. 9C), which might generate either a hypomorphic or complete response if Gata3 indeed regulates those other transcription factor genes. As anticipated, restoring even 25% of wild-type Gata3 by breeding the TghDBH-G3 into the mutant background created a strong hypomorphic allele, and significantly restored Th,Dbh, Hand2 and Phox2b expression in those purified chromaffin cells, while Mash1 levels reverted to almost the same level as in Gata3Z/+ animals (Fig. 9C). These data clearly demonstrate that Th and Dbh expression as well as the expression of putative regulators of Gata3 (Mash1, Phox2b and Hand2) are under pronounced (direct or indirect) influence of Gata3 in chromaffin cells. Importantly, Gata3 complementation in the mutant neuroendocrine cells largely restores those transcription factor expression levels. Taken together, these quantitative analyses are inconsistent with a simple linear hierarchical model in which Mash1 activates Phox2a and Hand2, while Phox2b activates Phox2a, Hand2 and Gata3, which in concert activate Th and Dbh, but instead indicate that Gata3 levels significantly affect the expression of those `upstream' genes(Goridis and Rohrer, 2002).

Of the six Gata factor family members, only Gata2 and Gata3 are reportedly expressed in the nervous system (Nardelli et al., 1999; Pata et al.,1999). Gata3 has previously been shown to be involved in the development of serotonergic neurons in the caudal raphe nuclei(Pattyn et al., 2004; van Doorninck et al., 1999),in sensory otic neurons (Karis et al.,2001; van der Wees et al.,2004) and in the expression of the noradrenergic genes Th and Dbh(Lim et al., 2000; Tsarovina et al., 2004). Here,we show that Gata3 is indispensable for the differentiation of adrenal chromaffin cells as well as sympathetic neurons during murine embryogenesis. Moreover, SA lineage-specific Gata3-expressing transgenic lines were able to rescue developmental deficiencies of the sympathoadrenal system that are attributable to Gata3 loss-of-function mutation.

Gata3 is thought to act as a `downstream' regulator in the molecular pathway leading to SA differentiation(Goridis and Rohrer, 2002). In Gata3 mutants, sympathetic ganglia are formed and express Phox2 mRNA and protein, but they exhibit dramatically reduced levels of Th and Dbh(Lim et al., 2000). Conversely, Phox2b-dependent expression of Gata3 has previously been demonstrated in the sympathetic chain of Phox2b-null mutant mice(Tsarovina et al., 2004). Furthermore, chicken Gata2, which appears to be the functional counterpart of murine Gata3 in the primary sympathetic chain, is expressed later than Mash1,Phox2b, Hand2 and Phox2a during chick embryogenesis(Tsarovina et al., 2004).

In contrast to our expectations, the present observations revealed that Gata3 plays a more complex role in SA differentiation than previous studies had indicated. The Gata3 homozygous mutants developed significantly smaller embryonic SG and AM throughout embryogenesis (E13.5 to E18.5),demonstrating that SA development was not simply delayed but permanently altered. There was increased apoptotic activity in Gata3 homozygous mutant adrenal glands from E13.5 onwards. This temporally and spatially restricted progressive cell death in SA tissues resulted in the accumulation of substantially fewer chromaffin cells by E18.5, demonstrating that Gata3 is essential for survival of adrenal chromaffin cells.

As previously reported, Th immunoreactivity was almost completely abolished in Gata3-/- sympathetic ganglia and adrenal glands. Meanwhile, only a fraction of viable sympathetic neurons and chromaffin cells expressed (weak) Phox2b immunoreactivity in E18.5 Gata3-/-mutants, whereas Phox2b is detected intensely in all sympathetic neurons and adrenal chromaffin cells of control animals. This qualitative observation was verified by Q-PCR as Gata3-/- chromaffin cells exhibited modestly lower Phox2b (halved) and significantly diminished Hand2, Th and Dbh mRNA accumulation. Consistent with these observations, previous studies reported suppressed Hand2 expression in E10.5 Gata3 mutant sympathetic ganglia (Tsarovina et al.,2004), suggesting that the same pathways regulate noradrenalin synthesis in all SA cells. Regulation of Hand2 by Gata factors has previously been reported in various tissues. For example, Hand2 is prominently expressed in the first branchial arch, which gives rise to lower jaw structures, and is significantly reduced in the branchial arches of Gata3 mutants(Ruest et al., 2004). In the developing cardiac primordium, Hand2 is regulated by Gata4(McFadden et al., 2000),implying a potential regulatory function for Gata factors on Hand2 expression during cardiogenesis.

These experiments additionally revealed that Mash1 expression was induced in Gata3-/- chromaffin cells but reverted to almost normal levels in transgene-rescued mutants, suggesting a possible negative regulatory role for Gata3 on Mash1 expression. Mash1 transcription is gradually downregulated from E14.5 onwards in SA lineage cells with progressive differentiation (Huber et al.,2002; Tsarovina et al.,2004), and the observed de-repression of Mash1transcription could be the causal event leading to differentiation failure in Gata3-deficient chromaffin cells. The results presented here demonstrate that Mash1, Phox2b and Hand2, all of which are putative upstream regulators of Gata3 in early SA development, are under the control of Gata3 regulatory influence in chromaffin cells.

Previous reports documented the transient expression of Th and Dbh in the ventrolateral neural tube (NT) of mid-gestational embryos (E10.5-E13.5),indicative of the transient noradrenergic cell lineage in this region(Son et al., 1996; Teitelman et al., 1981). It was also reported that an hDBH promoter transgene faithfully recapitulated the transient NT expression at this stage(Kapur et al., 1991). As anticipated, hDBH promoter-driven transgenic eGFP expression was observed transiently in the NT of E10.5 and E12.5 transgenic rescued embryos. Of potential significance here, Gata3 is also transiently expressed in the ventral NT between E10.5 and E13, including expression in a subpopulation of V2 interneurons (Smith et al.,2002). Consistently, our observations reflected a significant suppression of Th expression both in the SG and NT of E10.5 Gata3mutant embryos and the restoration in both regions in the Tg-rescued Gata3 mutants, suggesting that Gata3 directly or indirectly regulates transient Th expression in the ventrolateral neural tube.

Several lines of evidence support the notion that sympathetic ganglia and adrenal chromaffin cells share a common developmental origin(Anderson, 1993; Anderson et al., 1991; Unsicker, 1993). Adrenal chromaffin cells arise from a population of neural crest cells that initially localize in the primary sympathetic chain of the embryo. When the primary sympathoblasts re-migrate dorsolaterally to form the definitive sympathetic ganglia, other progenitors migrate ventrally and then penetrate the medial cranial end of the developing adrenal gland to become adrenal chromaffin cells(Yamamoto et al., 2004). When these neural crest derivatives colonize the adrenal anlagen, they become associated with mesodermal cells that are destined to form the adrenal cortex. In the environment of the adrenal gland, the ultimate development of these precursor cells is thought to benefit from growth factors provided by the developing cortex (e.g. IGF-1, FGF and glucocorticoids), and then be converted into mature adrenalin-producing chromaffin cells(Unsicker, 1993). The experiments reported here show that the expression of Phox2b, Th and Dbh genes and the sympathoadrenal development in the Gata3 mutants is progressively suppressed until around the time of birth (E18.5). These observations, when taken together with the observation that Gata3 is expressed throughout SA cell development, suggest that Gata3 may play some role in the initial specification of the common sympathoadrenal progenitor cell in the primary sympathetic chain, but unquestionably demonstrate that Gata3 is indispensable for successful execution of sympathetic neuronal and adrenal chromaffin cell differentiation.

Previous reports have shown that Phox2b mutant animals are also severely compromised in the number of adrenal chromaffin cells(Huber et al., 2005; Pattyn et al., 1999). This observation suggests a common requirement for Phox2b and Gata3 in the survival of sympathoadrenal progenitors in the adrenal medulla. Reduced Phox2b expression in Gata3 mutant SA cells and the reported observation of Phox2b-dependent Gata3 expression in Phox2b mutant raises the possibility that there exists a reciprocal and mutually reinforcing crossregulation by these transcription factors. Explicitly, these data are inconsistent with the hypothesis that a simple genetic hierarchy exists between these genes in the cascade of sympathoadrenal developmental events(Goridis and Rohrer, 2002).

Unfortunately, the current literature sheds no additional light on whether Gata3 directly or indirectly regulates the Hand2, Mash1 or Phox2b genes, as SA lineage-specific transcriptional regulation of these loci has not yet been reported. A neuroepithelial-specific proximal promoter that regulates Phox2b transcription has been characterized,but without specific reference to SA-restricted activity(Samad et al., 2004). Similarly, two in vitro studies have characterized the transcriptional behavior of the Phox2a promoter in tissue culture cells(Flora et al., 2001; Hong et al., 2001) without evidence that the regulation is SA lineage specific. Thus, the transcriptional data characterizing the Phox2a and Phox2b (to date)expression provide an inadequate foundation on which to base further regulatory analysis.

Real-time quantitative RT-PCR demonstrated that transgenic Gata3 mRNA was expressed at lower levels than the endogenous Gata3 mRNA in E10.5 embryo caudal halves and in the E18.5 mutant adrenal gland; both transgenic lines expressed comparable levels of transgene-derived Gata3 mRNA (data not shown). Presumably, high level Gata3 expression in the SA system could result in embryonic lethality in wild-type embryos as a consequence of overstimulation of catecholamine synthesis, possibly preventing the establishment of highly expressing transgenic lines. Alternatively, the nature of the hDBHgene promoter may be intrinsically limited in its ability to express foreign DNAs. The human Dbh gene is postulated to lie downstream of Gata3 in the hierarchy of SA development as the onset of hDBH-directed transgene expression occurs later than endogenous Gata3 expression(George et al., 1994) and is suppressed in the Gata3-null background (T.M., data not shown). A SA system-specific enhancer of the Gata3 gene, which would completely recapitulate endogenous Gata3 spatiotemporal expression, could be a more appropriate element for directing full genetic complementation.

Taken together, the observations reported here show that Gata3 is essential for the development and survival of both sympathetic ganglia and adrenal chromaffin cells. These experiments are incompatible with a simple hierarchical regulatory cascade, but rather are more consistent with the hypothesis that Mash1, Phox2a/b, Hand2 proteins and Gata3 are individually required for mutually reinforcing activation or repression of one another. Whether these cell-autonomous, mutually reinforcing activities are direct or are mediated by intermediate effectors will be resolved only when the cis elements controlling the SA system-specific activity for each of these regulatory genes is defined. Finally, the hDBH-rescued Gata3mutants should prove to be a useful tool to investigate currently cryptic Gata3 functions in many tissues (e.g. otic neurons, the thymus, parathyroid gland and kidneys) that display dysmorphic/amorphic phenotypes in the late gestational hDBH-Gata3-rescued animals.

The authors thank R. Palmiter, C. Gordis and G. Hammer for sharing valuable reagents. The excellent technical assistance of S. Meshinchi, D. McPhee and X. Jiang is gratefully acknowledged. We also thank the staff in the Microscopy and Imaging Lab at the University of Michigan for assistance with electron microscopy. This work was supported by an NIH grant (GM28896; to J.D.E.), by grants from the National Kidney Foundation (to K.-C.L.) and by the Genome Network Project from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to S.T.).

Anderson, D. J. (
1993
). Molecular control of cell fate in the neural crest: the sympathoadrenal lineage.
Annu. Rev. Neurosci.
16
,
129
-158.
Anderson, D. J., Carnahan, J. F., Michelsohn, A. and Patterson,P. H. (
1991
). Antibody markers identify a common progenitor to sympathetic neurons and chromaffin cells in vivo and reveal the timing of commitment to neuronal differentiation in the sympathoadrenal lineage.
J. Neurosci.
11
,
3507
-3519.
Coupland, R. E. and Tomlinson, A. (
1989
). The development and maturation of adrenal medullary chromaffin cells of the rat in vivo: a descriptive and quantitative study.
Int. J. Dev. Neurosci.
7
,
419
-438.
Flora, A., Lucchetti, H., Benfante, R., Goridis, C., Clementi,F. and Fornasari, D. (
2001
). Sp proteins and Phox2b regulate the expression of the human Phox2a gene.
J. Neurosci.
21
,
7037
-7045.
George, K. M., Leonard, M. W., Roth, M. E., Lieuw, K. H.,Kioussis, D., Grosveld, F. and Engel, J. D. (
1994
). Embryonic expression and cloning of the murine GATA-3 gene.
Development
120
,
2673
-2686.
Goridis, C. and Rohrer, H. (
2002
). Specification of catecholaminergic and serotonergic neurons.
Nat. Rev. Neurosci.
3
,
531
-541.
Hendriks, R. W., Nawijn, M. C., Engel, J. D., van Doorninck, H.,Grosveld, F. and Karis, A. (
1999
). Expression of the transcription factor GATA-3 is required for the development of the earliest T-cell progenitors and correlates with stages of cellular proliferation in the thymus.
Eur. J. Immunol.
29
,
1912
-1918.
Hong, S. J., Kim, C. H. and Kim, K. S. (
2001
). Structural and functional characterization of the 5′ upstream promoter of the human Phox2a gene: possible direct transactivation by transcription factor Phox2b.
J. Neurochem.
79
,
1225
-1236.
Howard, M. J. (
2005
). Mechanisms and perspectives on differentiation of autonomic neurons.
Dev. Biol.
277
,
271
-286.
Huber, K., Bruhl, B., Guillemot, F., Olson, E. N., Ernsberger,U. and Unsicker, K. (
2002
). Development of chromaffin cells depends on MASH1 function.
Development
129
,
4729
-4738.
Huber, K., Karch, N., Ernsberger, U., Goridis, C. and Unsicker,K. (
2005
). The role of Phox2B in chromaffin cell development.
Dev. Biol.
279
,
501
-508.
Kapur, R. P., Hoyle, G. W., Mercer, E. H., Brinster, R. L. and Palmiter, R. D. (
1991
). Some neuronal cell populations express human dopamine betahydroxylase-lacZ transgenes transiently during embryonic development.
Neuron
7
,
717
-727.
Karis, A., Pata, I., van Doorninck, J. H., Grosveld, F., de Zeeuw, C. I., de Caprona, D. and Fritzsch, B. (
2001
). Transcription factor GATA-3 alters pathway selection of olivocochlear neurons and affects morphogenesis of the ear.
J. Comp. Neurol.
429
,
615
-630.
Ko, L. J. and Engel, J. D. (
1993
). DNA-binding specificities of the GATA transcription factor family.
Mol. Cell. Biol.
13
,
4011
-4022.
Ko, L. J., Yamamoto, M., Leonard, M. W., George, K. M., Ting, P. and Engel, J. D. (
1991
). Murine and human T-lymphocyte GATA-3 factors mediate transcription through a cis-regulatory element within the human T-cell receptor delta gene enhancer.
Mol. Cell. Biol.
11
,
2778
-2784.
Lakshmanan, G., Lieuw, K. H., Lim, K. C., Gu, Y., Grosveld, F.,Engel, J. D. and Karis, A. (
1999
). Localization of distant urogenital system-, central nervous system-, and endocardium-specific transcriptional regulatory elements in the GATA-3 locus.
Mol. Cell. Biol.
19
,
1558
-1568.
Lim, K. C., Lakshmanan, G., Crawford, S. E., Gu, Y., Grosveld,F. and Engel, J. D. (
2000
). Gata3 loss leads to embryonic lethality due to noradrenaline deficiency of the sympathetic nervous system.
Nat. Genet.
25
,
209
-212.
Luo, X., Ikeda, Y. and Parker, K. L. (
1994
). A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation.
Cell
77
,
481
-490.
Mahata, S. K., Mahapatra, N. R., Mahata, M., Wang, T. C.,Kennedy, B. P., Ziegler, M. G. and O'Connor, D. T. (
2003
). Catecholamine secretory vesicle stimulus-transcription coupling in vivo. Demonstration by a novel transgenic promoter/photoprotein reporter and inhibition of secretion and transcription by the chromogranin A fragment catestatin.
J. Biol. Chem.
278
,
32058
-32067.
McFadden, D. G., Charite, J., Richardson, J. A., Srivastava, D.,Firulli, A. B. and Olson, E. N. (
2000
). A GATA-dependent right ventricular enhancer controls dHAND transcription in the developing heart.
Development
127
,
5331
-5341.
Mercer, E. H., Hoyle, G. W., Kapur, R. P., Brinster, R. L. and Palmiter, R. D. (
1991
). The dopamine beta-hydroxylase gene promoter directs expression of E. coli lacZ to sympathetic and other neurons in adult transgenic mice.
Neuron
7
,
703
-716.
Nardelli, J., Thiesson, D., Fujiwara, Y., Tsai, F. Y. and Orkin,S. H. (
1999
). Expression and genetic interaction of transcription factors GATA-2 and GATA-3 during development of the mouse central nervous system.
Dev. Biol.
210
,
305
-321.
Pandolfi, P. P., Roth, M. E., Karis, A., Leonard, M. W.,Dzierzak, E., Grosveld, F. G., Engel, J. D. and Lindenbaum, M. H.(
1995
). Targeted disruption of the GATA3 gene causes severe abnormalities in the nervous system and in fetal liver haematopoiesis.
Nat. Genet.
11
,
40
-44.
Pata, I., Studer, M., van Doorninck, J. H., Briscoe, J., Kuuse,S., Engel, J. D., Grosveld, F. and Karis, A. (
1999
). The transcription factor GATA3 is a downstream effector of Hoxb1 specification in rhombomere 4.
Development
126
,
5523
-5531.
Patient, R. K. and McGhee, J. D. (
2002
). The GATA family (vertebrates and invertebrates).
Curr. Opin. Genet. Dev.
12
,
416
-422.
Pattyn, A., Morin, X., Cremer, H., Goridis, C. and Brunet, J. F. (
1999
). The homeobox gene Phox2b is essential for the development of autonomic neural crest derivatives.
Nature
399
,
366
-370.
Pattyn, A., Simplicio, N., van Doorninck, J. H., Goridis, C.,Guillemot, F. and Brunet, J. F. (
2004
). Ascl1/Mash1 is required for the development of central serotonergic neurons.
Nat. Neurosci.
7
,
589
-595.
Ruest, L. B., Xiang, X., Lim, K. C., Levi, G. and Clouthier, D. E. (
2004
). Endothelin-A receptor-dependent and -independent signaling pathways in establishing mandibular identity.
Development
131
,
4413
-4423.
Samad, O. A., Geisen, M. J., Caronia, G., Varlet, I.,Zappavigna, V., Ericson, J., Goridis, C. and Rijli, F. M.(
2004
). Integration of anteroposterior and dorsoventral regulation of Phox2b transcription in cranial motoneuron progenitors by homeodomain proteins.
Development
131
,
4071
-4083.
Smith, E., Hargrave, M., Yamada, T., Begley, C. G. and Little,M. H. (
2002
). Coexpression of SCL and GATA3 in the V2 interneurons of the developing mouse spinal cord.
Dev. Dyn.
224
,
231
-237.
Son, J. H., Min, N. and Joh, T. H. (
1996
). Early ontogeny of catecholaminergic cell lineage in brain and peripheral neurons monitored by tyrosine hydroxylase-lacZ transgene.
Brain Res. Mol. Brain Res.
36
,
300
-308.
Teitelman, G., Gershon, M. D., Rothman, T. P., Joh, T. H. and Reis, D. J. (
1981
). Proliferation and distribution of cells that transiently express a catecholaminergic phenotype during development in mice and rats.
Dev. Biol.
86
,
348
-355.
Thomas, S. A., Matsumoto, A. M. and Palmiter, R. D.(
1995
). Noradrenaline is essential for mouse fetal development.
Nature
374
,
643
-646.
Tsarovina, K., Pattyn, A., Stubbusch, J., Muller, F., van der Wees, J., Schneider, C., Brunet, J. F. and Rohrer, H. (
2004
). Essential role of Gata transcription factors in sympathetic neuron development.
Development
131
,
4775
-4786.
Unsicker, K. (
1993
). The chromaffin cell:paradigm in cell, developmental and growth factor biology.
J. Anat.
183
,
207
-221.
van der Wees, J., van Looij, M. A., de Ruiter, M. M., Elias, H.,van der Burg, H., Liem, S. S., Kurek, D., Engel, J. D., Karis, A., van Zanten,B. G. et al. (
2004
). Hearing loss following Gata3 haploinsufficiency is caused by cochlear disorder.
Neurobiol. Dis.
16
,
169
-178.
van Doorninck, J. H., van Der Wees, J., Karis, A., Goedknegt,E., Engel, J. D., Coesmans, M., Rutteman, M., Grosveld, F. and De Zeeuw, C. I. (
1999
). GATA-3 is involved in the development of serotonergic neurons in the caudal raphe nuclei.
J. Neurosci.
19
,
RC12
.
Van Esch, H., Groenen, P., Nesbit, M. A., Schuffenhauer, S.,Lichtner, P., Vanderlinden, G., Harding, B., Beetz, R., Bilous, R. W.,Holdaway, I. et al. (
2000
). GATA3 haplo-insufficiency causes human HDR syndrome.
Nature
406
,
419
-422.
Yamamoto, M., Ko, L. J., Leonard, M. W., Beug, H., Orkin, S. H. and Engel, J. D. (
1990
). Activity and tissue-specific expression of the transcription factor NF-E1 [GATA] multigene family.
Genes Dev.
4
,
1650
-1662.
Yamamoto, M., Yanai, R. and Arishima, K.(
2004
). Study of migration of neural crest cells to adrenal medulla by three-dimensional reconstruction.
J. Vet. Med. Sci.
66
,
635
-641.
Zhou, Q. Y. and Palmiter, R. D. (
1995
). Dopamine-deficient mice are severely hypoactive, adipsic, and aphagic.
Cell
83
,
1197
-1209.