Knot

{Models}

Symbol: AKA: collier (col)	Flybase ID: {Flybase_ID}
Synonyms: {Name}	{GadFly}
Function: {Short_Function}	{LocusLink}
Keywords: {Keywords}	{Interactive_Fly}

{Summary}

Kn controls the spacing of veins 3 and 4 and prevents formation of ectopic veins between them

Hedgehog
- The anterior displacement, partial loss of L3 and increased width of the L3–L4 intervein that are observed upon overexpression of hh in its own domain (UAS-hh/engrailed (en)-Gal4 driver,(Figure 1e) were suppressed by reducing col dosage (Figure 1f).(Vervoort, 1999)
Blistered/D-SRF:
- Col is expressed in bs mutant discs, but the central domain of bs expression is missing in col mutant discs, indicating that Col is needed for bs expression (Vervoort, 1999)
- The variable expressivity of the phenotype observed in the hypomorphic col/kn¹ combinations suggests that a defined threshold level of Col activity is required for activating D-SRF expression.(Vervoort, 1999)
En
- Col is not required for the hh-induced anterior en expression (Vervoort, 1999)
Ci
- Expressing a dominant-negative form of PKA in the dorsal compartment (UAS-R*/ap-Gal4) results in ectopic Ci-155 and ectopic expression of dpp, but no change in col expression (data not shown) (Vervoort, 1999). These results indicate that the presence of Ci-155 is not sufficient to activate col expression in the anterior compartment, in conditions where it activates dpp and ptc (Capdevila, 1994; Chen, 1996; Hepker, 1997; Li, 1995).
EGFR
- Double in-situ hybridization reveals that the medial region of EGFR downregulation coincides with the region of kn gene expression (Fig. 5B). In kn mutants, downregulation of EGFR expression in the medial region is almost completely eliminated (Fig. 5C), indicating that the downregulation of EGFR expression is dependent on kn. Reciprocially, EGFR expression is greatly reduced in discs in which kn is ectopically expressed under the control of the MS1096-Gal4 driver (Fig. 5D). (Mohler, 2000)
Fused
- fu phenotype is exacerbated by reducing col dosage (Vervoort, 1999)
- In fu1 mutant discs, col expression at the A/P boundary was interrupted at the dorso-ventral margin (Figure 5c), correlating with local fusion of veins L3–L4 at the margin (Figure 5a). (Vervoort, 1999) kn medial expression in the wing pouch is reduced in fu mutants (Mohler, 2000)
- In fused mutant wing discs Col and Ptc expreesion (protein and transcript) is absence approximately six rows of cells on either side of the DV boundary (Fig 1) (Glise, 2002) and the stripe is broadened due to increased range of Hh in fused mutants (Fig 1) (Glise, 2002)
PKA
- Expressing a dominant-negative form of PKA in the dorsal compartment (UAS-R*/ap-Gal4) results in ectopic Ci-155 and ectopic expression of dpp, but no change in col expression (data not shown) (Vervoort, 1999). These results indicate that the presence of Ci-155 is not sufficient to activate col expression in the anterior compartment, in conditions where it activates dpp and ptc (Capdevila, 1994; Chen, 1996; Hepker, 1997; Li, 1995).
- in Pka-C1 mutants in the anterior compartment Col, Ptc, and En are up regulated but only at some distance away from the DV boundary (Figs. 3A-3C) (Glise, 2002)

did not observe any modulation of dpp-lacZ staining along the DV axis, possible do to perdurance of B-galactosidase (Glise, 2002)
In contrast to Pka-C1 clones, clones of cells lacking Ptc activated Col in all cells within the clones except for cells along the prospective wing margin (Fig 3D) (Glise, 2002)

Notch
- Ectopically expressed N^intra (a dominat active form) produces a cell-autonomous down-regulation of both Col and Ptc (Fig 6C and 6D). By contrast, expression of En was not affected by Nintra expression in either the posterior compartment or the anterior compartment in response to Hh (Fig. 6E), consistent with the fact that en is normally activated in the prospective wing margin (Glise, 2002)

{Physical interactions}

{Regulation}

{Structure}

In third instar wind discs, Col is expressed, among other sites, in four to five rows of cells adjacent to A/P boundary (area receving highest Hh levels and overlapping ptc expression) and restricted to the wing pouch (Fig 2a) (Vervoort, 1999)
Col is expressed in the same area as the middle D-SRF stripe (Vervoort, 1999)
In situ localizes kn expression to a stripe in the middle of the wing blade and weaker expression can be detected in more posterior regions of the wing pouch and in portions of the hinge region of the disc (Fig 2A) (Mohler, 2000)
Col expression (protein and trascript) is absent in the central row of cells that correspond to the prospective wing margin (Fig. 1E). This region of refractory to Hh signaling exists along the entire DV boundary. See (Glise, 2002)
In wild-type third instar imaginal discs, rho-expressing cells flanked the col expression domain on both sides, with one to two rows of intercalary cells expressing neither gene (Fig. 4C). (Crozatier, 2002)
In wild-type discs, ara and col expression partly overlapped, with ara expression extending anteriorly by about three rows of cells into the region corresponding to presumptive L3 vein (Fig. 4I). (Crozatier, 2002)

{Modifications}

{Related to}

col mutant wings are smaller than wild type and lack L4 vein, in addition to missing the L3-L4 intervein and mis-positioning of the anterior L3 vein (Crozatier, 2002)
in col¹/kn¹ mutant wings the L3-L4 intervein was only reduced in size, with an occasional partial apposition of L3 and L4 veins (Vervoort, 1999; Crozatier, 2002: Fig 1A-C)
kn¹ is a hypomorphic viable mutation (Diaz-Benjumea and Garcia-Bellido, 1990; Nestoras et al., 1999)
in both hypomorphic and null col mutants, there is a 20% reduction in the overall wing size compared with wild type (Fig. 1D) (Crozatier, 2002)
The observation that col¹/kn¹ wings displayed both an intervein L2-L3 of normal size and a reduced L3-L4 intervein suggested that the loss of the L3-L4 intervein and the displacement of L3 vein observed in col¹ mutant were not necessarily linked. (Crozatier, 2002)
vn expression in the AP organiser is not sufficient to rescue formation of L4 vein in col mutant discs
- vn expression under the control of a dpp-Gal4 driver did not restore formation of L4 vein in col mutant wings (expressing col using the same driver did rescue L4 vein). This indicated that something else was needed to induce vein formation that was under the control of col. [other controls were done that are not mentioned here] (Crozatier, 2002)
Effect on Vein
- Flies either homozygous for a weak col/kn allele, kn¹ , or heterozygous for a strong allele of vn, vn^M2, formed a normal L4 vein. However, in kn¹, kn¹; vn^M2/+ flies, L4 vein was missing, except for its proximal- and distal-most parts, showing that vn and col cooperate to promote L4 vein formation (Fig. 5A,B). We then looked at vn expression in col1 mutant discs. In the absence of col activity, a strong downregulation of vn transcription was observed in the AP organiser, except in the cells located near the presumptive hinge. vn transcription outside the wing pouch was not affected (Fig. 5C,D and data not shown). Together, these data indicate that col acts upstream of vn transcription in the AP organiser cells. (Crozatier, 2002)
Effect on Araucan
- In col¹ mutant discs, the stripe of ara expression was both weaker and narrower. The posterior border of ara expression which is situated within the col expression domain and defined by En repression (Gomez-Skarmeta and Modolell, 1996) was not modified, but its anterior border was shifted by two to three rows of cells closer to the AP boundary (Fig. 4F,I). (Crozatier, 2002)
Effect on Notch
- In col¹ mutants, N is downregulated in the L3m provein domain (data not shown) (Crozatier, 2002) .
Effect on extramacrochaetae
- Unmodified at the A margin, emc expression was completely lost from the AP organiser cells in either col¹ or col¹/kn¹mutant discs (Fig. 3D and data not shown), showing that Col is required for emc transcription in the L3-L4 intervein primordium. (Crozatier, 2002)
Effect on Rhomboid
- In col¹ mutant discs, two types of changes are observed: first, whereas rho is only expressed in a few cells in the dorsal presumptive L4 vein, it is expressed in two to four rows of cells, rather than a single one, in the presumptive L3 vein (Fig. 4B). Second, the distance separating the L3 and residual L4 veins is reduced (Fig. 4B), suggesting a posterior shift, in addition to widening, of the presumptive L3m vein, compared with L3. (Crozatier, 2002)
- In col¹ mutant discs, the L3m rho and col expression domains partially overlapped, confirming that the position of L3m vein has been shifted posteriorwards by several cell diameters relative to wild type (Fig. 4C,D), as previously deduced from Ci labelling of 24 hours APF pupae (Fig. 2D). However, rho labelling did not abut the AP boundary, indicating that rho transcription remained repressed in the anterior cells, which express En (Blair, 1992) (Fig.4I) (Crozatier, 2002)
Effect on intervein size:
- In col¹/kn¹ wings, the size of the L3-L4 intervein was 66±6% smaller that of wild type, whereas the size of the other A interveins (anterior margin to L2 and L2-L3) was unchanged, indicating that the reduction in number of cells is specific to this intervein. (Crozatier, 2002)
- In col¹ mutant wings in which the L3-L4 intervein (and L4 vein, see below) were missing, the L2-L3 domain was 12±5% larger than in either wild-type or col¹/kn¹ wings. (Crozatier, 2002)
- In both col¹ and col¹/kn¹ wings, the L4-L5 and L5 to posterior margin intervein sectors were smaller than in wild type, by 17±5% and 27± 4%, respectively. (Crozatier, 2002)
- This data indicates that the reduced size of col¹ mutant wings was due to the reduced size (or absence) of both the L3-L4 intervein and posterior interveins (Fig. 1D). As col expression is restricted to the L3- L4 intervein (Vervoort et al., 1999), its activity on the regulation of cell proliferation in the P compartment must be cell non-autonomous. The larger size of the L2-L3 intervein in col¹ wings raised both questions of the identity of the single central thick vein observed in these wings and its position relative to the AP boundary. This vein showed the presence of campaniform organs and dorsal corrugations, which are specific for L3 vein (reviewed by Campuzano and Modolell, 1992; Milan et al., 1997). The col mutant phenotype therefore does not correspond to apposition of L3 and L4 veins, as first proposed (Vervoort et al., 1999; Mohler et al., 2000) but to loss of L4 vein and repositioning of L3 vein. (Crozatier, 2002)
- Staining for phosphorylated histone H3 (a marker for mitosis) was specifically absent from the central region of either col¹ or col¹/kn¹ mutant wings (Fig. 3B and data not shown), correlating with changes from intervein to provein fate. However, the reduced size of the medial region of the wing already detectable at this early pupal stage probably reflects an earlier proliferation defect. (Crozatier, 2002)
- The size reduction of col1 mutant wings reflects the cumulative effect of changes in Dpp signalling and decreased expression of emc and/or Nin the AP organizer. (Crozatier, 2002)
Effect on vein L3 & L4

In col¹ mutant wings, instead of L3 and L4 proveins, there was a single central stripe of cells, wider than the wild type L3, which did not express BS (Fig. 2B). A truncated stripe of BS-negative cells located near the presumptive hinge (arrow in Fig. 2B) correlated with the residual L4 vein observed in a proximal position in col1 adult wings (arrow in Fig. 1C). This indicated that L4 vein was lacking and suggested that the central vein in col¹ mutant wings was a widened L3 vein. (Crozatier, 2002)
- In order to confirm this, wings of wild-type and col¹ pupae at 28-30 hours APF were stained for Ci, which is expressed only by A cells (Blair, 1992) (Fig. 2C,D). At that stage, resolution of provein into vein has occurred and the col¹ central vein is morphologically distinguishable (arrowhead in Fig. 2D). All cells of this vein expressed Ci, indicating that it was entirely located in the anterior compartment, confirming its L3-like identity. However, although the posterior limit of Ci expression bisected the wild type L3-L4 intervein (Fig. 2C), it coincided with the posterior limit of col¹ L3m vein. This shows that the position of L3m vein is shifted towards the AP boundary, correlating with the increased size of the L2-L3m intervein of col¹ adult wings. Taken together, morphological observations and size measurements of adult wings and immunostaining of pupal discs revealed three types of defects in col1 mutant wings: a reduced number of cells, the lack of L4 vein and a posteriorwards shift in the position of a wider L3 vein.(Crozatier, 2002)

homozygous mutants (which embryonic or larval lethality was rescued by a col transgene) completelely lacked the L3-L4 intervein (Fig 1b) and L3 & L4 each maintained its own identity, as shown by the presence of campaniform sensillae on L3, and each vein was abnormally large (Vervoort, 1999)
- overall size, and both the number, ratio and position along the A/P axis of socketed and unsocketed bristles are normal (Fig 3), indicating that the domain of En activity is not modified (Vervoort, 1999)
kn and col mutations are allelic to each other (Vervoort, 1999; Mohler, 2000)
In kn1 (viable) flies vein 3 & 4 form closer and produce a corresponding shift in their primordia (as detected by rho expression) in the late third instar disc (Sturtevant, 1995)
Small kn clones (generated in early 3rd instar) have little or no effect on the position of veins 3 and 4, but are usually associated with ectopic vein material only between vein 3 & 4 (Fig. 1C). (Mohler, 2000)
- The ectopic vein material associated with these small, late clones often extends outside the clone proper (Fig. 1F); this result is consistent with the activation of vein primordia initiation with the kn clone followed by vein extension outside the clone (a process occurring later in vein differentiation that normally assures contiguity of each longitudinal vein; Garcia-Bellido, 1977; Sturtevant and Bier, 1995). The phenotypes associated with these clones indicate that kn has a role to prevent ectopic vein formation in the 3-4 intervein space, in addition to its role in positioning veins 3 and 4. (Mohler, 2000)
- ectopic activation of EGF signalling (expressing Vn and a secreted form of Spi) in the developing wing blade leads to formation of ectopic venation in the wing (Schnepp, 1998), also Vn is expressed in the medial region of the wing just anterior to the A-P compartment (Simcox, 1996); however, no alterationi of vn expression was noted either in kn/knSA1 mutant discs or in discs in which kn had been ectopically expressed by the MS1096 driver indicating that the expression of vn in this region is not controlled by kn. (Mohler, 2000)

Ectopic expression of kn elsewhere in the wing imaginal disc results in the failure to properly activate rho and Dl
Ectopic expression of kn inactivates EGFR elsewhere in the wing primordium
No detectable wing phenotype was observed upon ectopic expression of col (using UAS-col and different drivers, en-, dpp-, ap- and ptc-Gal4). (Vervoort, 1999) this suggests that Col must cooperate with another factor, probably Ci.
Ectopic expression of kn under the control of the MS1096-GAL4 driver reduces or eliminates the expression of two vein primordia markers, rho and Dl, as well as the resulting venation of the wing (lack vein 3, distal portion of 4, and the proximal portion of vein 5-due to MS1096 expressing higher in the dorsal part of the wing pouch). (Mohler, 2000)
- does not affect the expression of the vein-specific marker genes: kni-vein 2 and ara/caup-odd-numbered veins; nor does it affect dpp and sal expression, genes functioning earlier in the vein patterning hierarchy (Mohler, 2000)
In dpp-GAL4 UAS-kn flies vein 3 is often dirupted in distal portions (Fig 4A), vein 3 is shifted anteriorly (Fig 4B), and in 10-20% of the wings the campaniform sensilla are just posterior to vein 3, instead of in their normal location on vein 3. (Mohler, 2000)

{Reagents}