Slit molecules prevent entrance of trunk neural crest cells in developing gut

Abstract

Neural crest cells emerge from the dorsal neural tube early in development and give rise to sensory and sympathetic ganglia, adrenal cells, teeth, melanocytes and especially enteric nervous system. Several inhibitory molecules have been shown to play important roles in neural crest migration, among them are the chemorepulsive Slit1-3. It was known that Slits chemorepellants are expressed at the entry to the gut, and thus could play a role in the differential ability of vagal but not trunk neural crest cells to invade the gut and form enteric ganglia. Especially since trunk neural crest cells express Robo receptor while vagal do not. Thus, although we know that Robo mediates migration along the dorsal pathway in neural crest cells, we do not know if it is responsible in preventing their entry into the gut. The goal of this study was to further corroborate a role for Slit molecules in keeping trunk neural crest cells away from the gut. We observed that when we silenced Robo receptor in trunk neural crest, the sympathoadrenal (somites 18-24) were capable of invading gut mesenchyme in larger proportion than more rostral counterparts. The more rostral trunk neural crest tended not to migrate beyond the ventral aorta, suggesting that there are other repulsive molecules keeping them away from the gut. Interestingly, we also found that when we silenced Robo in sacral neural crest they did not wait for the arrival of vagal crest but entered the gut and migrated rostrally, suggesting that Slit molecules are the ones responsible for keeping them waiting at the hindgut mesenchyme. These combined results confirm that Slit molecules are responsible for keeping the timeliness of colonization of the gut by neural crest cells.

Keywords: Cell migration; Enteric nervous system; Neural crest; Robo.

Figure 1. Slit and Robo expression in chicken embryos

Whole mount and sections of in situ hybridization for Slit and Robo in HH16–20 in chicken embryos. A Slit2 is expressed in dorsal neural tube (arrow), mesonephros (arrowhead) and not in vagal region (red arrow) in HH18 embryo. B Robo2 receptor is expressed in migrating trunk neural crest (arrowhead) but not in vagal (arrow). C Slit2 is not expressed in the ventral region at vagal level (arrow), although it is expressed in gut region posterior to the heart (arrowhead) at HH18. D Slit2 is expressed in dorsal neural tube (arrow) in HH10 with lines for sections shown in H and I. E Slit1 embryo at HH11 with lines for sections shown in L and M arrow points to pharynx. F sections in hindbrain region showing Slit1 expression in gut (arrow) of a HH20 embryo. G whole mount for Slit1 showing expression in dorsal neural tube (arrowhead) and absence where the gut is beginning to develop (arrow and line point to primordial gut mesenchyme). H, I sections of Slit2 embryo in D showing lack of Slit2 expression in pharynx region. J section at sacral level showing Robo2 expression of cells in gut (arrow) at HH20. K ventral side of HH19 embryo showing expression of Slit2 along gut mesenchyme (arrows) L, M sections of Slit1 embryo in E showing lack of Slit1 expression in pharynx region (arrows) Arrowhead corresponds to section on M at higher magnification.

Figure 2. Whole mount and rostro-to-caudal sections of HH14 embryos

Sections and whole mounts pictures for GFP and DAPI (blue) of HH14 electroporated embryos incubated for 24hrs. A–H shows a range of sections through control GFP embryo shown in G, forelimb (C) and hindlimb (F) sections are marked to highlight axial levels. G: shows the composite image of the embryo. H–L shows a range of sections through RD2 embryo shown in M, forelimb (I) and hindlimb (L) sections are marked to highlight axial levels. Arrows in J–L point to RD2 cells that migrated past trunk regions beneath the dorsal aorta. M: composite image of RD2 embryo showing posterior region of the embryo.

Figure 3. Whole mount and rostro-to-caudal sections of HH16 embryos

Sections and whole mounts pictures for GFP and DAPI (blue) of HH16 electroporated embryos incubated for 24hrs. A–G shows a range of sections through control GFP embryo shown in H, forelimb (C) and hindlimb (E) sections are marked to highlight axial levels. H: shows the composite image of the embryo with labels for sacral somite region and hindlimb somites (ss26–33). I–N shows a range of sections through RD2 embryo shown in O, forelimb (I) and hindlimb (L) sections are marked to highlight axial levels. Arrows in L–N point to RD2 cells that migrated past trunk regions into gut mesenchyme. O: composite image of RD2 embryo showing posterior region of the embryo with labels for sacral a hindlimb somites (ss26–33).

Figure 4. Sacral neural crest cells enter gut in Robo LOF embryos

Sections and whole mounts pictures for GFP and DAPI (blue) of HH17 electroporated embryos incubated for 24hrs. A–D correspond to control GFP shown in I. E, F correspond to RD2 embryo shown in J while G, H correspond to embryo shown in K. AD Control GFP embryo does not have GFP cells in the gut folds. E, F RD2 embryos show GFP cells in the gut folds rostral to hindlimb (arrows). G, H RD2 embryos show GFP cells in the gut folds at hindlimb level (arrows). Lines and letters in I–K indicate corresponding section region in A–H.

Figure 5. Slit mutant mice have altered enteric neural crest cell migration

Sections through whole mount in situ hybridization for Sox10 in E12.5 Slit1 mutant (A) and mice mutant for Slit1,2,3 (B, C) showed that neural crest cells were in larger number at the gut mesenchyme compared with no detectable Sox10 cells in Slit1 mutant embryo (arrows in A–C). All mice were Slit1 −/− background. D–G: Sections at more rostral level (lung region) show the presence of many Sox10 cells in Slit triple knockout mouse compared with Slit1 mutant (arrows in D–G). F, G corresponds to higher magnification of gut region by lung from D, E, notice that in control mouse there is no clear plexus forming (F), while it is quite advanced in the triple mutant (G).

Figure 6. Graphical abstract

The graphical abstract summarizes what we know about Slit/Robo interaction in neural crest migration into the gut: at vagal levels neural crest cells enter the gut by migrating through area without Slit expression. Trunk neural crest cells before somite 18 are repelled by Slits as well as another yet unknown factor(s). The trunk neural crest between somite 17–24 (sympathoadrenal) is kept away from the gut by Slit molecules as well as sacral crest is prevented from populating the gut before vagal crest arrives by Slit expression along the gut mesenchyme.