Our analysis has made three contributions towards understanding the circuitry that governs cell fate specification in the C. elegans foregut. First, we identified tbx-2, which is required for ABa-derived precursors to adopt a pharyngeal muscle fate (). This is the first locus identified that is essential to produce a particular cell type within the pharynx. Second, tbx-2 is repressed by Notch signaling and the REF family of transcription factors. This regulation confines TBX-2 to ABa descendants, which may be important for limiting the number of pharyngeal muscles. Third, TBX-2 and PHA-4 are mutually dependent on each other to maintain expression, suggesting a direct or indirect positive regulatory loop. We propose that this regulatory loop is essential for commitment to pharyngeal muscle fate since pharyngeal muscles are lost in either pha-4 or tbx-2 mutant embryos.
A model of pharyngeal muscle specification in the ABa lineage
PHA-4 expression normally initiates at the 2-4E stage whereas TBX-2 initiates at the 8E stage. These time-points correspond precisely to the period when embryonic blastomeres lose their developmental plasticity (Fukushige and Krause, 2005
; Gilleard and McGhee, 2001
; Horner et al., 1998
; Zhu et al., 1998
). After this stage, blastomeres are not able to transform to a different cell-type identity (reviewed in Labouesse and Mango, 1999
). Conversely, the loss of pha-4
eliminates pharynx or epidermis, respectively, demonstrating that these factors are necessary for early patterning (Mango et al., 1994a
; Page et al., 1997
). An intriguing notion is that the positive regulatory loop between PHA-4 and TBX-2 contributes to the cell-fate restriction that occurs in ABa descendants by the 8E stage. This model could explain the lack of a transformation in MS-derived cells that express TBX-2. TBX-2 expression initiates after the 8E stage in MS descendants, which may be too late to contribute to cell fate commitment.
directly regulates most pharyngeally-expressed genes via a TRTTKRY consensus sequence (Gaudet and Mango, 2002
). The C. elegans tbx-2
5.2 kb promoter includes 19 PHA-4 consensus sites, six of which are located within the 4.7 – 5.2 kb upstream sequence. While not conserved in position, PHA-4 binding sites are also found in tbx-2
from C. ramanei
(13) and C. briggsae
(8). Thus, PHA-4 may activate tbx-2
transcription directly during the maintenance phase. We note that the large number of consensus sites and the lack of direct alignment of sites complicate the analysis of direct vs. indirect regulation.
Previous studies of the Tbx2/3/4/5 subfamily suggested a role in anteroposterior patterning. For example, misexpression of Tbx2 in the chick limb can convert digit III into digit IV, and Tbx3 can convert digit II to digit III (Suzuki et al., 2004
). Similarly, Tbx4 is required for hindlimb development whereas Tbx5 is required for the forelimb bud (Rodriguez-Esteban et al., 1999
). These activities, in addition to the contribution of C. elegans tbx-2
for anterior pharyngeal muscles, suggest that an ancient role for the Tbx2/3/4/5 subfamily involves anterior/posterior patterning. However, the phenotypes associated with these genes are not entirely clear-cut. For example, inactivation of Tbx5 in mouse leads to loss of FGF expression and forelimb outgrowth, but patterning of the limb field appears normal (Takeuchi et al., 2003
). Therefore, the effects of Tbx5 on anterior cells may reflect proliferation rather than anterior vs. posterior fate. In C. elegans
, we do not observe alterations in cell division of ABa descendants in tbx-2
mutants. Moreover, FGF, which is an important Tbx target for growth and morphogenesis in vertebrates, is probably not downstream of tbx-2
in C. elegans
. FGF ligands and receptors have no described phenotypes in the anterior pharynx (Birnbaum et al., 2005
). Rather, our phenotypes are best explained as alterations of cell fate progression.
signaling is abrogated (or its downstream effectors lag-1/su(H)
and the REF proteins), we observe extra cells expressing TBX-2::GFP. These data suggest that Notch signaling represses tbx-2
and ensures only some cells progress towards anterior muscle fate. Studies with Drosophila
have suggested that one role for Notch is to block the progression of development (Cagan and Ready, 1989
). Inhibition of tbx-2
by Notch could serve this purpose, by repressing a gene required for developmental progression.
How does TBX-2 function? In other organisms, members of the Tbx2/3/4/5 subfamily of T-box factors can be either activators (e.g. Tbx4, Tbx5) (Takeuchi et al., 2003
; Zaragoza et al., 2004
) or repressors (e.g. Tbx2, Tbx3) (Carreira et al., 1998
; He et al., 1999
). Examination of C. elegans tbx-2
reveals that it has sequences similar to the amino terminal repression domain of vertebrate Tbx2 and Tbx3 () (He et al., 1999
). On the other hand, the carboxyl terminal sequence of TBX-2 is rich in serine, proline and glutamate, suggesting it could function as an activation domain, similar to some other transcription factors (Matsuzaki et al., 1995
; Nagy et al., 2002
; Stepchenko and Nirenberg, 2004
). Thus, a speculative possibility is that in C. elegans
, the sole Tbx2 family member functions as both an activator and a repressor, whereas in vertebrates, with four Tbx2 members, activation and repression activities are segregated into different proteins.
TBX-2 is both nuclear and cytoplasmic
We observe TBX-2 in the cytoplasm beginning at the 1.5 fold stage. This location agrees with previous studies where endogenous TBX-2 was observed in the cytoplasm of post-embryonic neurons (Miyahara et al., 2004
). Tbx factors from other organisms also accumulate in the cytoplasm, suggesting this may be a conserved mechanism to regulate Tbx proteins (Bruce et al., 2003
; Fuchikami et al., 2002
; Krause et al., 2004
). Our cytoplasmic expression appears filamentous, as though TBX-2 were associated with the cytoskeleton. The pattern resembles that of microtubules, although this idea remains to be tested. The cytoplasmic expression pattern was detected in 12/12 independent transgenic lines, and the cytoplasmic transition was consistent even in the weakest expressing lines; arguing that this is not an over expression phenotype. Recent studies with chicken Tbx5 suggested this protein could bind to LMP-4 in vitro
, and was targeted to actin filaments in COS cells (Krause et al., 2004
). Thus, members of the Tbx2 subfamily may be regulated by cytoskeletal association.