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Boundary cells regulate a switch in the expression of FGF3 in hindbrain rhombomeres.

Sela-Donenfeld D, Kayam G, Wilkinson DG - BMC Dev. Biol. (2009)

Bottom Line: Boundary cells display distinct molecular and cellular properties such as an elongated shape, enriched extracellular matrix components and a reduced proliferation rate compared to intra-rhombomeric cells.These findings suggest that boundary cells are required for the downregulation of segmental FGF3, presumably mediated by a soluble factor(s) that emanates from boundaries.We propose that this new function of boundary cells enables a switch in gene expression that may be required for stage-specific functions of FGF3 in the developing hindbrain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Koret School of Veterinary Medicine, The Hebrew University, The Robert H Smith Faculty of Agriculture, Food and Environment, Rehovot, Israel. seladon@agri.huji.ac.il

ABSTRACT

Background: During formation of the vertebrate central nervous system, the hindbrain is organized into segmental units, called rhombomeres (r). These cell-lineage restricted segments are separated by a subpopulation of cells known as boundary cells. Boundary cells display distinct molecular and cellular properties such as an elongated shape, enriched extracellular matrix components and a reduced proliferation rate compared to intra-rhombomeric cells. However, little is known regarding their functions and the mechanisms that regulate their formation.

Results: Hindbrain boundary cells express several signaling molecules, such as FGF3, which at earlier developmental stages is transiently expressed in specific rhombomeres. We show that chick embryos that lack boundary cells due to overexpression of truncated EphA4 receptor in the hindbrain have continued segmental expression of FGF3 at stages when it is normally restricted to hindbrain boundaries. Furthermore, surgical ablation of the boundary between r3 and r4, or blocking of the contact of r4 with boundary cells, results in sustained FGF3 expression in this segment.

Conclusion: These findings suggest that boundary cells are required for the downregulation of segmental FGF3, presumably mediated by a soluble factor(s) that emanates from boundaries. We propose that this new function of boundary cells enables a switch in gene expression that may be required for stage-specific functions of FGF3 in the developing hindbrain.

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The boundary cell marker FGF3 shows persistent segmental expression in embryos expressing truncated EphA4. (A-E): Flat-mounted hindbrains from different-staged embryos in situ hybridized with FGF3 probe. (A): 8 ss embryo showing FGF3 expression in r4-r6 and low levels in r2. (B): 16 ss embryo showing expression in r2/r4/r6. (C): 20 ss embryos showing expression in r4/r6. FGF3 expression is also becoming apparent at rhombomere borders. (D): 25 ss embryo showing FGF3 transcripts in r6 and in boundaries. (E): 30 ss embryo showing FGF3 localization to boundary cells. (F-I): Flat-mounted hindbrains from embryos electroporated unilaterally with dnEphA4 (G, I) or control (F, H) constructs at 8 ss, and left to develop for further 30 hrs. FGF3 transcripts are restricted to boundaries in control electroporations (F, H), while dnEphA4-embryos show FGF3 within even-numbered rhombomeres (G, I white arrowheads). (J, K): Views of hindbrains electroporated at 22 ss with dnEphA4 (K) or control (J) constructs and left to develop for further 18 hrs. For both, expression of FGF3 is restricted to boundary cells. Embryos in (H-K) were immunostained with anti-GFP antibody followed by Alexa-488 (H, I) or HRP (J, K) secondary antibodies to label electroporated cells. Asterisks mark the electroporated side, anterior is at the top and rhombomere numbers are indicated.
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Figure 3: The boundary cell marker FGF3 shows persistent segmental expression in embryos expressing truncated EphA4. (A-E): Flat-mounted hindbrains from different-staged embryos in situ hybridized with FGF3 probe. (A): 8 ss embryo showing FGF3 expression in r4-r6 and low levels in r2. (B): 16 ss embryo showing expression in r2/r4/r6. (C): 20 ss embryos showing expression in r4/r6. FGF3 expression is also becoming apparent at rhombomere borders. (D): 25 ss embryo showing FGF3 transcripts in r6 and in boundaries. (E): 30 ss embryo showing FGF3 localization to boundary cells. (F-I): Flat-mounted hindbrains from embryos electroporated unilaterally with dnEphA4 (G, I) or control (F, H) constructs at 8 ss, and left to develop for further 30 hrs. FGF3 transcripts are restricted to boundaries in control electroporations (F, H), while dnEphA4-embryos show FGF3 within even-numbered rhombomeres (G, I white arrowheads). (J, K): Views of hindbrains electroporated at 22 ss with dnEphA4 (K) or control (J) constructs and left to develop for further 18 hrs. For both, expression of FGF3 is restricted to boundary cells. Embryos in (H-K) were immunostained with anti-GFP antibody followed by Alexa-488 (H, I) or HRP (J, K) secondary antibodies to label electroporated cells. Asterisks mark the electroporated side, anterior is at the top and rhombomere numbers are indicated.

Mentions: FGF3 expression was previously described as occurring at early stages in specific hindbrain segments and later in hindbrain boundaries in chick and mouse embryos [22-24,26]. We carried out a more detailed analysis of the transition of FGF3 expression from a segmental pattern to boundary cells. In 8 somite stage (ss) embryos, FGF3 is expressed in the ventral half of r4-r6 and r6 (excluding the floor plate) and at lower levels in r2 (Fig. 3A). By 16 ss, expression is seen in all even-numbered segments, is downregulated in r5, and some upregulation begins at rhombomere borders (Fig. 3B). In 20 ss embryos, expression in r2 is downregulated while still present in the other even-numbered segments, and FGF3 transcripts become more apparent at r2/r3, r3/r4, r4/r5 and r5/r6 boundaries (Fig. 3C). By 25 ss, segmental expression of FGF3 is no longer detected in r4 while still present in r6, and expression in boundary cells has become even more prominent (Fig. 3D). Finally, in embryos at 30–45 ss, FGF3 transcripts are confined to the ventral part of hindbrain boundary cells and are absent from all rhombomere bodies (Fig. 3E). This analysis shows that FGF3 expression is firstly restricted to specific hindbrain segments and subsequently downregulated from these rhombomeres while expression is upregulated at rhombomere boundaries.


Boundary cells regulate a switch in the expression of FGF3 in hindbrain rhombomeres.

Sela-Donenfeld D, Kayam G, Wilkinson DG - BMC Dev. Biol. (2009)

The boundary cell marker FGF3 shows persistent segmental expression in embryos expressing truncated EphA4. (A-E): Flat-mounted hindbrains from different-staged embryos in situ hybridized with FGF3 probe. (A): 8 ss embryo showing FGF3 expression in r4-r6 and low levels in r2. (B): 16 ss embryo showing expression in r2/r4/r6. (C): 20 ss embryos showing expression in r4/r6. FGF3 expression is also becoming apparent at rhombomere borders. (D): 25 ss embryo showing FGF3 transcripts in r6 and in boundaries. (E): 30 ss embryo showing FGF3 localization to boundary cells. (F-I): Flat-mounted hindbrains from embryos electroporated unilaterally with dnEphA4 (G, I) or control (F, H) constructs at 8 ss, and left to develop for further 30 hrs. FGF3 transcripts are restricted to boundaries in control electroporations (F, H), while dnEphA4-embryos show FGF3 within even-numbered rhombomeres (G, I white arrowheads). (J, K): Views of hindbrains electroporated at 22 ss with dnEphA4 (K) or control (J) constructs and left to develop for further 18 hrs. For both, expression of FGF3 is restricted to boundary cells. Embryos in (H-K) were immunostained with anti-GFP antibody followed by Alexa-488 (H, I) or HRP (J, K) secondary antibodies to label electroporated cells. Asterisks mark the electroporated side, anterior is at the top and rhombomere numbers are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: The boundary cell marker FGF3 shows persistent segmental expression in embryos expressing truncated EphA4. (A-E): Flat-mounted hindbrains from different-staged embryos in situ hybridized with FGF3 probe. (A): 8 ss embryo showing FGF3 expression in r4-r6 and low levels in r2. (B): 16 ss embryo showing expression in r2/r4/r6. (C): 20 ss embryos showing expression in r4/r6. FGF3 expression is also becoming apparent at rhombomere borders. (D): 25 ss embryo showing FGF3 transcripts in r6 and in boundaries. (E): 30 ss embryo showing FGF3 localization to boundary cells. (F-I): Flat-mounted hindbrains from embryos electroporated unilaterally with dnEphA4 (G, I) or control (F, H) constructs at 8 ss, and left to develop for further 30 hrs. FGF3 transcripts are restricted to boundaries in control electroporations (F, H), while dnEphA4-embryos show FGF3 within even-numbered rhombomeres (G, I white arrowheads). (J, K): Views of hindbrains electroporated at 22 ss with dnEphA4 (K) or control (J) constructs and left to develop for further 18 hrs. For both, expression of FGF3 is restricted to boundary cells. Embryos in (H-K) were immunostained with anti-GFP antibody followed by Alexa-488 (H, I) or HRP (J, K) secondary antibodies to label electroporated cells. Asterisks mark the electroporated side, anterior is at the top and rhombomere numbers are indicated.
Mentions: FGF3 expression was previously described as occurring at early stages in specific hindbrain segments and later in hindbrain boundaries in chick and mouse embryos [22-24,26]. We carried out a more detailed analysis of the transition of FGF3 expression from a segmental pattern to boundary cells. In 8 somite stage (ss) embryos, FGF3 is expressed in the ventral half of r4-r6 and r6 (excluding the floor plate) and at lower levels in r2 (Fig. 3A). By 16 ss, expression is seen in all even-numbered segments, is downregulated in r5, and some upregulation begins at rhombomere borders (Fig. 3B). In 20 ss embryos, expression in r2 is downregulated while still present in the other even-numbered segments, and FGF3 transcripts become more apparent at r2/r3, r3/r4, r4/r5 and r5/r6 boundaries (Fig. 3C). By 25 ss, segmental expression of FGF3 is no longer detected in r4 while still present in r6, and expression in boundary cells has become even more prominent (Fig. 3D). Finally, in embryos at 30–45 ss, FGF3 transcripts are confined to the ventral part of hindbrain boundary cells and are absent from all rhombomere bodies (Fig. 3E). This analysis shows that FGF3 expression is firstly restricted to specific hindbrain segments and subsequently downregulated from these rhombomeres while expression is upregulated at rhombomere boundaries.

Bottom Line: Boundary cells display distinct molecular and cellular properties such as an elongated shape, enriched extracellular matrix components and a reduced proliferation rate compared to intra-rhombomeric cells.These findings suggest that boundary cells are required for the downregulation of segmental FGF3, presumably mediated by a soluble factor(s) that emanates from boundaries.We propose that this new function of boundary cells enables a switch in gene expression that may be required for stage-specific functions of FGF3 in the developing hindbrain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Koret School of Veterinary Medicine, The Hebrew University, The Robert H Smith Faculty of Agriculture, Food and Environment, Rehovot, Israel. seladon@agri.huji.ac.il

ABSTRACT

Background: During formation of the vertebrate central nervous system, the hindbrain is organized into segmental units, called rhombomeres (r). These cell-lineage restricted segments are separated by a subpopulation of cells known as boundary cells. Boundary cells display distinct molecular and cellular properties such as an elongated shape, enriched extracellular matrix components and a reduced proliferation rate compared to intra-rhombomeric cells. However, little is known regarding their functions and the mechanisms that regulate their formation.

Results: Hindbrain boundary cells express several signaling molecules, such as FGF3, which at earlier developmental stages is transiently expressed in specific rhombomeres. We show that chick embryos that lack boundary cells due to overexpression of truncated EphA4 receptor in the hindbrain have continued segmental expression of FGF3 at stages when it is normally restricted to hindbrain boundaries. Furthermore, surgical ablation of the boundary between r3 and r4, or blocking of the contact of r4 with boundary cells, results in sustained FGF3 expression in this segment.

Conclusion: These findings suggest that boundary cells are required for the downregulation of segmental FGF3, presumably mediated by a soluble factor(s) that emanates from boundaries. We propose that this new function of boundary cells enables a switch in gene expression that may be required for stage-specific functions of FGF3 in the developing hindbrain.

Show MeSH
Related in: MedlinePlus