COMMENTARY

Radar breaks the fog: Insights into dorsoventral patterning in

Thomas P. Wilm and Lilianna Solnica-Krezel* Vanderbilt University, VU Station B 351634, Nashville, TN 37235-1634

mong the first major events receptor complex. The activated BMP- during development of metazo- receptor complex phosphorylates a cyto- ans is the specification of the solic protein of the receptor-regulated anteroposterior (head͞tail or Smad family that oligomerizes with a A͞ mouth anus) and the dorsoventral co-Smad and subsequently enters the (back-belly) embryonic axes. The genetic nucleus to activate transcription of network controlling dorsoventral axis downstream targets leading to ventral specification is best understood and re- fate determination (Fig. 1). BMP antag- markably, it is evolutionarily conserved onists act by binding to the BMP ligands from fruit flies to mammals. Labeling of and prevent the formation and activa- single cells or small groups of cells and tion of the BMP receptor complexes, tracing them throughout development thus promoting dorsal fate specification. has revealed that fate specification in Misexpression of BMP2, BMP4, and and zebrafish depends on the BMP7 ligands in Xenopus and zebrafish position within the embryo during blas- cause strong reduction of dorsal struc- tula and gastrula stages, in particular tures like notochord, brain, and pharynx, along the future dorsoventral axis. The whereas ventral structures, including positional information is furnished by an blood, posterior somites, and epidermis, activity gradient of bone morphogenetic are enlarged (Fig. 2). Similar reduction proteins (BMPs) that promote ventral of dorsal structures is observed in ze- specification in a dose-dependent fash- brafish mutants in which BMP antago- ion. The key structure in the establish- nists and negative regulators are inacti- ment of the ventral to dorsal BMP activ- vated (5, 6). Conversely, functional ity gradient in vertebrate embryos is the inactivation by means of induced muta- Fig. 1. BMP signaling pathway (see text for dorsal gastrula or Spemann–Mangold tions in the zebrafish bmp2b details). organizer at the future dorsal side of the (swirl) and bmp7 (snailhouse) results in embryo (1). Embryological extirpation suppression of ventral fates and expan- experiments revealed that the formation sion of dorsal structures (7). ternal canonical Wnt pathway being the of the gastrula organizer and conse- The first key step in shaping the ven- key player during dorsal fate specifica- quently of dorsal structures is specified tral to dorsal BMP activity gradient oc- tion. In Xenopus, one possible scenario by maternally supplied determinants in curs at blastula stages and depends on includes the microtubule-dependent frog and fish. In contrast, whereas the maternally provided dorsal determi- translocation of Dishevelled protein development of ventral fates in Xenopus nants. Upon fertilization, dorsal deter- from vegetal to the future dorsal side of is associated with maternally expressed minants deposited at the vegetal pole of the embryo (9). As Dishevelled is a pos- BMP-encoding genes, in zebrafish the the egg are transported to the prospec- itive regulator of ␤-catenin, this would same set of proteins is exclusively ex- tive dorsal blastomeres. The dorsal de- ultimately cause an asymmetric distribu- pressed from the zygotic genome. This terminants promote transcriptional acti- tion of nuclear ␤-catenin accumulation poses the question about the nature of vation of the genes required to establish along the dorsoventral axis. Dorsally the upstream regulatory mechanisms the blastula organizer, which is responsi- acting ␤-catenin activates a gene cascade involved in zebrafish. In a recent issue ble for the formation of the Spemann– whose main function is to directly or of PNAS, a report by Sidi et al. (2) pro- Mangold organizer at the future dorsal indirectly eliminate dorsal expression of vided thrilling evidence that at least in side of the gastrula. As a consequence bmp genes expression in the blastula the zebrafish embryo there may be no of the translocation of the dorsal deter- (10–14). ␤ ventral default state at all. Sidi and col- minants, -catenin, a downstream com- The somewhat late activation of the ␤ laborators identified a distinct TFG- ponent of the Wnt-signaling pathway, ventralizing BMP2͞4͞7 signaling path- ligand Radar as a part of the maternal accumulates more strongly in the nuclei way during zebrafish embryogenesis dowry that activates the zygotic expres- in the dorsal region of frog and ze- does not support the simplified assump- sion of BMP genes and they start to de- brafish blastulae. Down-regulation of tion of a maternally regulated ventral ␤ lineate the pathway involved. -catenin by antisense oligonucleotides default state within the early blasto- BMP2b, BMP4, and BMP7 have been in frog embryos, interference with its derm. On the other hand, mutational implicated in dorsoventral axis forma- nuclear accumulation in the zebrafish and molecular analyses have revealed a tion in the zebrafish embryo and form a ichabod (8) mutant, or mutational inac- maternal requirement of the TGF-␤ branch of the large superfamily of trans- tivation in the mouse causes dramatic ␤ type I receptor Alk8 for the initiation of forming growth factor type proteins increase in BMP activity and complete BMP2͞4͞7 signaling, as embryos from (TGF-␤) (3, 4). Typically, BMP ligands lack of dorsal structures. Conversely, bind to the homodimeric BMP type II ventral overexpression of ␤-catenin elic- receptor (BMPR-II) that subsequently its secondary axis formation. In sum- See companion article on page 3315 in issue 6 of volume 100. recruits the homodimeric BMP type I mary, there is a wealth of experimental *To whom correspondence should be addressed. E-mail: receptor to form the active tetrameric data strongly arguing in favor of the ma- [email protected].

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0931010100 PNAS ͉ April 15, 2003 ͉ vol. 100 ͉ no. 8 ͉ 4363–4365 Downloaded by guest on September 27, 2021 Given that active TGF-␤ molecules are thought to interact with the receptor complex as either homo- or het- erodimers, overexpressing C-terminally truncated forms of Radar could act in a dominant-negative manner by forming heterodimers with endogenous ligands that are unable to signal. Overexpres- sion of such dominant-negative Radar (RadarDN) in young embryos causes stronger dorsalization than observed in radar-MO-injected embryos, providing an independent support for the idea of maternal Radar being a key player in early axis specification. How does Radar impact embryonic patterning? Induction of bmp2b and bmp4 gene expression at early blastula stages was found to be dramatically reduced in radar morphants, whereas Fig. 2. Fate specification in zebrafish embryos at the onset of gastrulation. In wild type, the BMP activity expression of bmp7 was unchanged, gradient is set up by the combination of positive autoregulation and negative regulation through leading to the conclusion that induc- BMP-antagonists like Chordin. In the absence of Chordin and Bozozok, the influence of BMP signaling tion of bmp2b and bmp4 but not bmp7 expands dorsally, and ventral tissues such as posterior somitic mesoderm and blood are enlarged. requires Radar function. Remarkably, Inactivation of bmp2b causes the opposite scenario; activity of BMP dramatically increases leading to the induction of excessive dorsal fates like neuroectoderm and anterior somitic mesoderm. the onset and expression domain of the organizer-specific BMP antagonist chordin was not affected. Similarly, females homozygous for the mutant alk8 the zygotic and maternal functions of dorsal expression of bozozok, encoding allele lost-a-fin or alk8-morphant em- Radar, Sidi et al. (2) have elegantly a negative regulator of bmp2b gene bryos are strongly dorsalized (15, 16). used an antisense RNA interference expression, and a direct downstream Interestingly, bmp2b expression is signif- technology mediated by so called mor- target of ␤-catenin was also un- icantly reduced in zebrafish gastrulae pholino (MO)-oligonucleotides (22). changed. Consistently, the functionally ͞ after RNA injection of dominant-nega- One MO-oligonucleotide was targeted redundant ventralizing genes vox vega1 ͞ tive alk8 constructs, whereas overexpres- to inhibit translation of processed tran- and vent vega2 in zebrafish, which are sion of constitutively active Alk8 causes scripts of both maternal and zygotic not dependent on BMP signaling at increase in bmp2b, without influencing origin (23). Another MO-oligonucle- early stages, are not down-regulated in bmp4 expression (17). Furthermore, em- otide was designed to interfere with bryos derived from females homozygous processing of newly, zygotically synthe- for null in the smad5 locus sized, but not with maternally provided exhibit reduced levels of the initial bmp7 RNAs (24). Injecting zebrafish em- and bmp2b expression (18, 19). These bryos with the latter MO-oligonucle- observations already predicted the exis- otide recapitulates the ob- tence of a maternal signal, likely from ␤ served in embryos carrying a genomic TGF- superfamily, inducing bmp gene deletion encompassing the radar locus. expression in zebrafish. That dorsoventral axis formation was The zebrafish gene radar represents a not affected in these embryos is consis- compelling candidate for the mysterious tent with the notion that zygotic radar maternal activator of bmp gene expres- does not regulate axis specification sion. Initial work had revealed Radar radar possessed a strong ventralizing activity (25). Strikingly, when former -MO when overexpressed in the early embryo, was used, 50% of the embryos also ex- comparable to effects seen after BMP2b hibited excessive development of dorsal and BMP4 misexpression (20), and a tissues. Three nonexclusive scenarios careful analysis of the expression profile may explain why only a fraction of em- showed that radar transcripts are already bryos was affected and to varying de- grees. Injected MO-oligonucleotides Fig. 3. Model of the maternal Radar signaling maternally contributed. pathway mediating early steps of dorsoventral axis Earlier studies had shown that zy- compete for deposited maternal radar formation. Radar acts through the TGF-␤ type I gotic expression of radar occurring at transcripts with the translation machin- receptor Alk8 and causes transcriptional activation the end of gastrulation may not be ery. In addition, active Radar protein of BMP2b and BMP4. The intracellular signal trans- needed for dorsoventral axis forma- may already be present in the oocyte ducer, believed to be a member of the Smad1͞5͞8 tion, though a function in establishing before fertilization. Functional redun- protein family is not known (indicated in gray), the integrity of the zebrafish axial vas- dancy with related factors that harbor although Smad5 may be excluded. Maternal ventralizing activity as well cannot be Smad5 may transduce the signal of a hypothesized culature may involve dorsoventral pat- distinct maternal signaling pathway acting in par- terning events as well (21). Mutational ruled out. However, another zebrafish ␣ allel to Radar. Alk8 is also the receptor for zygotic analysis of radar did not reveal any GDF6 ortholog, dynamo (gdf6 ), is not BMP2b͞7 signaling, and Smad5 transduces that participation in early dorsoventral axis involved in early dorsoventral axis for- signal, leading to the autoregulatory transcrip- formation (20). To distinguish between mation (26). tional activation of BMP2b͞4͞7.

4364 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0931010100 Wilm and Solnica-Krezel Downloaded by guest on September 27, 2021 radar morphant embryos. These obser- lated factors in the zebrafish embryo interactions between Radar and the vations clearly demonstrate that mater- that act to specify ventral fates. TGF-␤ type I receptor Alk8 suggest nally provided Radar is required to Epistatic analysis further supports that maternal Radar signals through initiate zygotic gene expression of the notion of radar acting upstream of maternal Alk8 (Fig. 3). However, the bmp2b and bmp4 specifically, and sug- bmp gene expression (3). Excess Radar dorsalization in maternal-zygotic alk8 gest that it does not interact with function in early embryo was unable to mutant embryos is much stronger than dorsal-specifying or BMP-independent rescue the mutant and morphant phe- in radar-morphant embryos. The next genes. The observation of stronger notypes of several BMP pathway com- challenge is to determine whether ad- ditional ligands cooperate with Radar dorsalization after overexpression of ponents, including bmp2b, bmp7 and ␤ in activating Alk8, whether other type RadarDN versus MO-induced down- the TGF- type I receptor alk8 (MO). I receptors are also at work in induc- regulation of Radar function can be Conversely, misexpression of bmp2b, bmp4, bmp7, and constitutively active tion of BMP gene expression, and easily explained by the stronger sup- which Smad factors are used in this pression of bmp2b, bmp4 and especially alk8 all suppressed the radar-morphant phenotype, showing that maternal pathway. Although the dominant nega- of bmp7 gene expression at blastula tive mutant allele smad5 is epistatic to and pregastrula stages. The expression function of radar is crucial for initia- tion of the ventral character in the radar as well, several newly identified domain of the BMP antagonist Chor- smad5 early zebrafish embryo. Maternal inac- null alleles, have revealed that din is expanded ventrolaterally, which in contrast to Radar positively regulat- tivation of smad5 or alk8, respectively, by itself could explain the much stron- ing bmp2b and bmp4 expression, ma- led to stronger dorsalization than inac- ger dorsalization capacity of overex- ternal Smad5 rather acts on bmp7 (ref. tivation of individual BMP ligands, pressed RadarDN. In contrast to the 19; Fig. 3). It will be important to test reminiscent of embryos with impaired radar-MO oligonucleotides that cannot whether overexpression of Radar can radar function. This raises the question suppress the strongly dorsalized pheno- interfere with putative maternally de- of whether the TGF-␤ type I receptor DN type of maternal smad5 mutant posited Radar protein, Radar may and the intracellular messenger are actually heterodimerize with maternally embryos. also involved in Radar signaling. In In contrast to the situation in Xeno- supplied wild-type Radar protein. addition, double alk8 and radar MO- ͞ ͞ Thus, overexpression of dominant neg- pus, where BMP2 4 7 are provided ma- injection studies have also indicated ternally and may create a default state, ative Radar could reflect a complete that alk8 and radar might cooperate in the delayed zygotic initiation of ubiqui- removal of Radar function. However, ventral specification. Whereas injec- tous bmp2b͞4͞7 expression in the ze- the possibility of functional interfer- tions of subinhibitory alk8 and radar brafish embryo could be interpreted as a DN ͞ ence by Radar with related BMP MO-concentrations when applied indi- ventral default setting of the prospective GDF proteins cannot be ruled out. Sidi vidually, typically produce only mildly blastoderm in a similar way. Now with and collaborators provide compelling dorsalized , coinjection of the report by Sidi et al. identifying a evidence that induction of dorsal and MO-alk8 and MO-radar at the same maternally contributed secreted factor ventral fates both seem to be con- doses caused significantly stronger dor- upstream of BMP2b͞4͞7 expression, a trolled maternally and that very likely salization, with bmp2b and bmp4 ex- revised picture of ventral fate specifica- there are additional and possibly re- pression eliminated. The synergistic tion in zebrafish is at hand.

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Wilm and Solnica-Krezel PNAS ͉ April 15, 2003 ͉ vol. 100 ͉ no. 8 ͉ 4365 Downloaded by guest on September 27, 2021