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PMID:21073867

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Citation

Choudhry, P, Joshi, D, Funke, B and Trede, N (2011) Alcama mediates Edn1 signaling during zebrafish cartilage morphogenesis. Dev. Biol. 349:483-93

Abstract

The zebrafish pharyngeal cartilage is derived from the pharyngeal apparatus, a vertebrate-specific structure derived from all three germ layers. Developmental aberrations of the pharyngeal apparatus lead to birth defects such as Treacher-Collins and DiGeorge syndromes. While interactions between endoderm and neural crest (NC) are known to be important for cartilage formation, the full complement of molecular players involved and their roles remain to be elucidated. Activated leukocyte cell adhesion molecule a (alcama), a member of the immunoglobulin (Ig) superfamily, is among the prominent markers of pharyngeal pouch endoderm, but to date no role has been assigned to this adhesion molecule in the development of the pharyngeal apparatus. Here we show that alcama plays a crucial, non-autonomous role in pharyngeal endoderm during zebrafish cartilage morphogenesis. alcama knockdown leads to defects in NC differentiation, without affecting NC specification or migration. These defects are reminiscent of the phenotypes observed when Endothelin 1 (Edn1) signaling, a key regulator of cartilage development is disrupted. Using gene expression analysis and rescue experiments we show that Alcama functions downstream of Edn1 signaling to regulate NC differentiation and cartilage morphogenesis. In addition, we also identify a role for neural adhesion molecule 1.1 (nadl1.1), a known interacting partner of Alcama expressed in neural crest, in NC differentiation. Our data shows that nadl1.1 is required for alcama rescue of NC differentiation in edn1(-/-) mutants and that Alcama interacts with Nadl1.1 during chondrogenesis. Collectively our results support a model by which Alcama on the endoderm interacts with Nadl1.1 on NC to mediate Edn1 signaling and NC differentiation during chondrogenesis.

Links

PubMed PMC3036164 Online version:10.1016/j.ydbio.2010.11.006

Keywords

Activated-Leukocyte Cell Adhesion Molecule/metabolism; Animals; Branchial Region/cytology; Branchial Region/metabolism; Cell Differentiation/physiology; Chondrogenesis/physiology; Cloning, Molecular; DNA Primers/genetics; Endothelin-1/genetics; Endothelin-1/metabolism; Gene Expression Profiling; Gene Knockout Techniques; Immunohistochemistry; In Situ Hybridization, Fluorescence; Leupeptins; Models, Biological; Neural Cell Adhesion Molecules/metabolism; Neural Crest/cytology; Neural Crest/physiology; Signal Transduction/physiology; Zebrafish/embryology; Zebrafish Proteins/genetics; Zebrafish Proteins/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

DANRE:A0A2R8QME4

involved_in

GO:0048703: embryonic viscerocranium morphogenesis

ECO:0000315: mutant phenotype evidence used in manual assertion

ZFIN:ZDB-GENO-980202-1487

P

Seeded From UniProt

complete

DANRE:A0A2R8QU31

involved_in

GO:0048703: embryonic viscerocranium morphogenesis

ECO:0000315: mutant phenotype evidence used in manual assertion

ZFIN:ZDB-GENO-980202-1487

P

Seeded From UniProt

complete

DANRE:A5WW41

involved_in

GO:0014033: neural crest cell differentiation

ECO:0000315: mutant phenotype evidence used in manual assertion

ZFIN:ZDB-GENO-980202-1068

P

Seeded From UniProt

complete

DANRE:A5WW41

involved_in

GO:0048703: embryonic viscerocranium morphogenesis

ECO:0000315: mutant phenotype evidence used in manual assertion

ZFIN:ZDB-GENO-980202-1068

P

Seeded From UniProt

complete

DANRE:B0S6Y3

involved_in

GO:0048703: embryonic viscerocranium morphogenesis

ECO:0000315: mutant phenotype evidence used in manual assertion

ZFIN:ZDB-GENO-980202-1487

P

Seeded From UniProt

complete

DANRE:F1QQ43

involved_in

GO:0048703: embryonic viscerocranium morphogenesis

ECO:0000315: mutant phenotype evidence used in manual assertion

ZFIN:ZDB-GENO-980202-1487

P

Seeded From UniProt

complete

DANRE:C166A

involved_in

GO:0060536: cartilage morphogenesis

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

DANRE:C166A

involved_in

GO:0010770: positive regulation of cell morphogenesis involved in differentiation

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

DANRE:C166A

involved_in

GO:0014033: neural crest cell differentiation

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

DANRE:C166A

involved_in

GO:0014033: neural crest cell differentiation

ECO:0000315: mutant phenotype evidence used in manual assertion

ZFIN:ZDB-MRPHLNO-110125-5

P

Seeded From UniProt

complete

DANRE:C166A

involved_in

GO:0048703: embryonic viscerocranium morphogenesis

ECO:0000315: mutant phenotype evidence used in manual assertion

ZFIN:ZDB-MRPHLNO-110125-5

P

Seeded From UniProt

complete

DANRE:C166A

involved_in

GO:0010628: positive regulation of gene expression

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

DANRE:C166A

GO:0060536: cartilage morphogenesis

ECO:0000315:

P

5 dpf alcama morphants are characterized by a protruding lower jaw, cardiac edema and absence of swim bladder (Fig. 1G and I). Alcian blue staining of 5 dpf larvae revealed that the percentage of individuals with affected cartilage was equivalent in wild-type (WT) and the p53−/− mutant lines. alcama RNA can rescue the ventral cartilage defect caused by alcama knockdown (Fig. S1). Mc and ch are fused to their respective dorsal elements, palatoquadrate (pq) and hyosymplectic (hm) (Fig. C).

complete
CACAO 1999

DANRE:C166A

GO:0010628: positive regulation of gene expression

ECO:0000315:

P

In alcama morphants, the expression of hand2, dlx3b, dlx5a and dlx6a is strongly reduced in the first and the posterior arches, and mildly reduced in the second arch at 30 hpf (Fig. 4B, E, H, K).

complete
CACAO 2068

DANRE:C166A

GO:0014033: neural crest cell differentiation

ECO:0000315:

P

Co-injection of alcama RNA with alcama MO decreases the number of affected individuals (absent dlx5a staining) to WT levels. The percentage of affected larvae is similar in WT and p53−/− mutants (Fig. S6D)

complete
CACAO 2076

DANRE:C166A

GO:0010770: positive regulation of cell morphogenesis involved in differentiation

ECO:0000315:

P

5 dpf alcama morphants are characterized by a protruding lower jaw, cardiac edema and absence of swim bladder (Fig. 1G and I). Alcian blue staining of 5 dpf larvae revealed that the percentage of individuals with affected cartilage was equivalent in wild-type (WT) and the p53−/− mutant lines. alcama RNA can rescue the ventral cartilage defect caused by alcama knockdown (Fig. S1).

complete
CACAO 2078

DANRE:Q1KHF9

involved_in

GO:0060536: cartilage morphogenesis

ECO:0000316: genetic interaction evidence used in manual assertion

UniProtKB:Q90460

P

Seeded From UniProt

complete

DANRE:Q1KHF9

GO:0060536: cartilage morphogenesis

ECO:0000316:

UniProtKB:Q90460


P

Particularly striking is the similarity of alcama morphants to furinA−/− mutants (Fig. 2 E).

complete
CACAO 2069

DANRE:Q1KHF9

involved_in

GO:0048703: embryonic viscerocranium morphogenesis

ECO:0000315: mutant phenotype evidence used in manual assertion

ZFIN:ZDB-GENO-980202-1487

P

Seeded From UniProt

complete

DANRE:Q9I8G3

GO:0060536: cartilage morphogenesis

ECO:0000315:

P

The three characteristic phenotypes of the ventral cartilages in alcama morphants; shortening, change of orientation and fusion to dorsal cartilages, are typical of the edn1-class of mutants (Fig. 3 G)

complete
CACAO 2070

DANRE:Q9I8G3

GO:0050714: positive regulation of protein secretion

ECO:0000315:

P

edn1 RNA injection results in more than 50% increase in Alcama protein in WT and edn1−/− mutants (Fig. 3G)

complete
CACAO 2071

DANRE:Q9I8G3

GO:0032435: negative regulation of proteasomal ubiquitin-dependent protein catabolic process

ECO:0000315:

P

To test whether Edn1 stabilizes Alcama protein by interfering with its degradation by the proteasome, we treated edn1−/− larvae with the proteasome inhibitor MG132 and measured Alcama levels. Alcama levels in the pharyngeal pouches increased by more than 300% upon treatment with MG-132 (Fig. 3H)

complete
CACAO 2072

DANRE:Q9I8G3

GO:0045597: positive regulation of cell differentiation

ECO:0000315:

P

edn1 over-expression……resulting in a 300% increase in the number of edn1−/− mutants with dlx5a staining (sum of strong and partial) (Fig. 5D).

complete
CACAO 2073

DANRE:Q9I8G3

involved_in

GO:0045597: positive regulation of cell differentiation

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

DANRE:Q9I8G3

involved_in

GO:0032435: negative regulation of proteasomal ubiquitin-dependent protein catabolic process

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

DANRE:Q9I8G3

involved_in

GO:0050714: positive regulation of protein secretion

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

DANRE:Q9I8G3

involved_in

GO:0048703: embryonic viscerocranium morphogenesis

ECO:0000315: mutant phenotype evidence used in manual assertion

ZFIN:ZDB-GENO-980202-1068

P

Seeded From UniProt

complete

DANRE:Q9I8G3

involved_in

GO:0014033: neural crest cell differentiation

ECO:0000315: mutant phenotype evidence used in manual assertion

ZFIN:ZDB-GENO-980202-1068

P

Seeded From UniProt

complete

DANRE:L1CA1

involved_in

GO:0010628: positive regulation of gene expression

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

DANRE:L1CA1

involved_in

GO:0060536: cartilage morphogenesis

ECO:0000316: genetic interaction evidence used in manual assertion

UniProtKB:Q9I8G3

P

Seeded From UniProt

complete

DANRE:L1CA1

GO:0060536: cartilage morphogenesis

ECO:0000316:

UniProtKB:Q9I8G3


P

Phenotypically, nadl1.1 morphants are similar to alcama morphants: by 5 dpf they have a protruding lower jaw, cardiac edema and absent swim bladder (data not shown). Alcian blue staining reveals cartilage defects reminiscent of alcama morphants: mc and ch are changed in orientation and are fused to pq and hm, respectively (Fig. 6G–H'). Injection of nadl1.1 MO into p53−/− mutants yields similar percentages of larvae with dlx5a down-regulation and jaw abnormalities (Fig. 6F, I), indicating that the observed effect is specific to nadl1.1 knockdown.

complete
CACAO 2074

DANRE:L1CA1

GO:0010628: positive regulation of gene expression

ECO:0000315:

P

Similar to alcama MO, nadl1.1MOaffects edn1-dependent gene expression: hand2 and dlx5a expression is strongly reduced in the arches with a more pronounced effect on the posterior arches (Fig. 6B–E).

complete
CACAO 2075


See also

References

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