Tc-smurf is involved in patterning the blastodermal fate map in

complex is maternally provided in Tribolium and anteriorly localized (Fu et al., 2012). It could be that early Wg signals play an important role already during early nuclear division cycles in Tribolium, making use of Tc-MAD. More experiments are necessary in order to completely understand the functions of Tc-Smurf during pre-blastoderm stages in Tribolium. It might also be that the early effects are due to a germ line function of Tc-smurf. Smurf function in the regulation of Dpp signaling in the germ line has been described for Drosophila (Casanueva and Ferguson, 2004;

Chang et al., 2013; Xia et al., 2010). Additionally, knock down of Tc-dpp in pupal stages causes female sterility (van der Zee et al., 2006 and data not shown) sug-gesting an important function of Dpp-signaling in the Tribolium germ line. The early defects seen before blastoderm formation might therefore be due to a general loss of egg integrity and not a result of loss of Tc-Smurf function after egg deposition.

Pupal and adult injections were performed in this study in order to knock down the function of Tc-smurf. Embryonic dsRNA injections could serve useful to elucidate whether the pre-blastodermal defects seen in some Tc-smurf RNAi embryos are due to a very early Tc-Smurf function during development.

5.2.3 Tc-smurf is involved in patterning the blastodermal fate map in Tribolium

Knock down of Tc-smurf changed the blastoderm fate map of Tribolium em-bryos. Staining for marker genes and live imaging experiments showed that the fraction of cells specified to form head tissue was clearly reduced upon Tc-smurf

knock down. This resulted in a bend in the germ serosa boundary during the dif-ferentiated blastoderm stage and eventually in headlobes that were strikingly re-duced in size during the germ rudiment stage. Interestingly, the dorsal anterior tis-sue (in the lateral midline) of the germ anlage in the blastoderm was stronger af-fected than the most anterior ventral or dorsalmost tissue. This resulted in the ob-vious bend in the germ serosa border and was most likely due to Tc-sog activity on the ventral most side of the embryo which inhibited Dpp-signaling in this region (van der Zee et al., 2006). It has previously been shown that loss of Tc-sog leads to a complete loss of the head anlage in the Tribolium blastoderm with parts of the labial segment beeing the only head part remaining, as demonstrated by staining for Hox gene expression in germ band stage embryos (van der Zee et al., 2006). If compared to the severe Tc-sog RNAi phenotype, Tc-smurf knock down has a rela-tively small impact on the overall fate map of the Tribolium differentiated blastoderm and it seems that Tc-Smurf has rather a function in fine tuning Dpp-signaling during these stages. Weak cuticle phenotypes suggest that the first re-gions to be affected by loss of Tc-smurf function are located between the clypeolabral region (CLR) and the gnathal segments. Figure 4-10 C shows a larva with missing head cuticle between the labrum and the mandibles. The labrum an-lage is located anteriorly and medial to the procephalic ocular and antennal region (Kittelmann, 2012; Posnien et al., 2010) which is missing in this larva. The loss of the ocular Tc-wg domain seen in many Tc-smurf RNAi embryos also indicates loss of at least parts of the ocular region (Figure 4-19). While Tc-sog is important to form a head per se, Tc-smurf is necessary to establish the more posterior proce-phalic head parts in concert with Tc-sog. This indicates a function of Tc-Dpp sig-naling in patterning the Tribolium head along the anterior-posterior axis. This pro-cess is most likely performed by a very tightly regulated crossplay between Tc-Sog, Tc-Tolloid (Tc-Tld) and Tc-Smurf. It has formerly been shown that Tc-Tld has an important pro-Dpp function in Tribolium (Nunes da Fonseca et al., 2010). This function is probably mediated by the cleavage of Tc-Sog molecules and concomi-tant release of bound Tc-Dpp that can then bind to its receptors as it has been de-scribed for Drosophila (Marqués et al., 1997; Srinivasan et al., 2002). My results suggest that Tc-Smurf has an essential function in fine-tuning the crossplay be-tween Tc-Tld and Tc-Sog. During differentiated blastoderm stages, Tc-dpp is ex-pressed in a fine stripe in the anterior of the germ anlage, interestingly with highest

expression levels on the ventral side. Dpp signaling is involved in defining the bor-der between embryonic cells and serosa, since interfering with this signaling path-way through Tc-sog or Tc-dpp RNAi, respectively, changes the position of the germ-serosa boundary (van der Zee et al., 2006). Tc-sog is expressed at the ven-tral most side of the blastoderm stage embryo and is necessary to transport Dpp ligands to the dorsal side of the embryo (van der Zee et al., 2006). Tc-tld is ex-pressed broadly during undifferentiated blastoderm stages, excluded only from the egg poles. During the differentiated blastoderm stage Tc-tld is expressed in a broad anterior domain within the prospective germ rudiment, uniformly along the DV-axis (Nunes da Fonseca et al., 2010). Tc-Tld is hence present in all regions of Tc-dpp expression during this stage. Tc-Smurf is expressed in all cells and is probably necessary to set a threshold level that needs to be reached before acti-vated BMP receptors result in nuclear localization of pMAD and subsequent gene regulation. According to my results, Tc-smurf prevents low level Dpp-signals which happen due to Tc-Tld mediated Tc-Sog cleavage on the ventral and lateral side of the prospective embryonic tissue. It might even shut down Dpp signals that occur due to secreted Dpp ligands which directly bind to membrane receptors before they are bound to Tc-Sog molecules. I propose a model in which Tc-Sog, Tc-Tld and Tc-Smurf act in a concerted manner to define a line that seperates extraembryonic and embryonic tissue during the differentiated blastoderm stage (Figure 5-1). According to this model, Tribolium makes use of at least four systems to define this line and control Dpp-signaling during blastoderm stages:

 Localized expression of the diffusible signaling molecule Tc-Dpp in a dor-sal-ventral stripe-like domain along the region of the prospective germ se-rosa border. After differentiated blastoderm formation, Tc-dpp expression is restricted to a thin stripe of expression along the germ serosa border, only a few cells wide.

 Localized expression of the diffusible, extracellular Dpp antagonist Tc-Sog on the ventral side of the embryo which leads to the ventral inhibition of signaling and at the same time to an amplification of the signal on the dorsal side through a shuttling function of Tc-Sog.

 Broad expression of the extracellular Tc-Sog antagonist Tc-Tld in the pre-sumptive embryonic tissue, which is necessary to reactivate Sog-bound Tc-Dpp molecules and thereby activate signaling.

 Ubiquitous expression of the intracellular Dpp signaling antagonist Tc-Smurf. Smurf is necessary to cut off ‘leaking’ Dpp-signaling due to diffus-ing Dpp molecules and untimely cleaved Sog molecules. The crossplay of all regulators results in a clear and localized border between germ and serosa tissue. In Tc-sog, Tc-dpp, and Tc-tld RNAi embryos the germ se-rosa border is shifted but still clearly defined (Nunes da Fonseca et al., 2010; van der Zee et al., 2006). This clear definition is often lost in Tc-smurf RNAi embryos. Tc-Tc-smurf is also essential to locate the germ serosa border, since Tc-smurf RNAi leads to a posterior shift especially of the lateral regions of this border. One more function of Tc-Smurf is the re-striction of peak levels of pMAD to the dorsal most region, which defines the amnion. In Tc-smurf RNAi embryos an increase of this peak level area can be detected and the amnion in many embryos is enlarged as seen in the embryo in Figure 4-23.

The extracellular Dpp agonist Tc-Twisted-gastrulation (Tc-Tsg), does not seem to play a role in defining these early Dpp-signaling regions. RNAi against Tc-tsg leads to a complete loss of Tc-Dpp function and completely ventralized embryos, identical to the phenotype observed after Tc-dpp RNAi. Tc-tsg rather seems to be necessary for Tc-dpp function per se (Nunes da Fonseca et al., 2010).

Figure 5-1 Model of Tc-smurf function during blastoderm stages of the Tribolium embryo Shown are schematic drawings of a Tribolium embryo during the differentiated blastoderm stage. (A) Simpli-fied scheme of the regulation of Dpp during late blastoderm stages in Tribolium. (B) Wild type situation, modi-fied after Nunes da Fonseca et al., (2010). (A+B) Tc-Sog binds Dpp molecules on the ventral side, resulting in a block of Dpp signaling. At the same time Sog-Dpp complexes diffuse to the dorsal side of the embryo.

Tolloid cleaves Sog. The dorsal diffusion of Sog-Dpp complexes leads to an increase of free Dpp molecules dorsally after Tolloid induced cleavage of Sog. Sog therefore indirectly enhances Dpp signaling dorsally. This allows Dpp signaling to reach peak levels in the dorsal posterior region of the embryo. Smurf inhibits Dpp sig-naling in all cells through targeting pMAD for degradation and sets a threshold level that needs to be reached before activated BMP receptors result in nuclear localization of pMAD. Dpp signal is blocked in the neuroectoderm, low in dorsal ectoderm and high in the prospective amnion. (C) Loss of Smurf function stabi-lizes pMAD in more ventral and posterior cells in the embryo. In the ventral most region Sog blocks Dpp sig-naling. More dorsally, Tld mediated cleavage of Sog allows Dpp ligands to bind to their receptors. Due to lack of Smurf threshold limits are reached more ventrally and posteriorly. Therefore more cells gain serosal fate and parts of the prospective head are lost. The germ serosa boundary is less defined. The scheme integrates results obtained during this work with previously published results (Nunes da Fonseca et al., 2010; van der Zee et al., 2006)