Review of 'Serotonin is required for pharyngeal arch morphogenesis in zebrafish'

Serotonin is required for pharyngeal arch morphogenesis in zebrafish
by Saleh Bashammakh et al.

This work is about the role of serotonin during zebrafish development. First analyzing the expression patterns of Tph1A, Tph1B, and Tph2, the authors report expression of Tph2 in the raphe nuclei and of Tph1a and Tph2 in the pineal gland. Serotonin immunohistochemistry gives a pattern similar to Sox9a ISH in pharyngeal arches neural crest cells with a highly regular pattern of serotonin positive cells. Using Tph2 antisense morpholino oligonucleotides, they report induction of morphogenesis defects during pharyngeal arch development similar to Foxd3 morphants. Although the specification of neural crest cells was not modified by Tph2 morphants, they observed a reduction in the third migrating cranial neural crest cell streams in Tph2 morphant with a fusion of the first and second stream suggesting an impairment of survival and differentiation of pharyngeal arch progenitor cells. After cloning of the zebrafish 5-HT2B receptor gene, the authors identified its expression in the pharyngeal arches; they show that ritanserin (20 µM) induced defects in pharyngeal arch morphogenesis. They conclude that Tph2-derived serotonin act as a morphogenetic factor in the development of neural crest-derived tissues.

This quite straightforward study, represents a nice piece of new information for the readers. It is globally well done and sound. It extends previously published work in mice and Xenopus about the contribution of serotonin and 5-HT2B receptor in neural crest cells and pharyngeal arches morphogenesis. However, some issues should be completed in order to increase its level of interest:

-Introduction of the serotonin system is correct for the Tph genes "In the zebrafish, till now three genes of Tph, tph1a (or tphD1), tph1b (or tphD2), and tph2 (or tphR) were cloned by us (GenBank: AY616135 and AY616134) and others [5, 23]. Most likely, this increase in tph gene number is due to teleost genome duplication during evolution [24], and the postulated fourth tph gene probably got lost afterwards". However, the presentation of the receptor system is incomplete since the sentence "5-HT interacts with at least 14 different receptors grouped in 7 families, 5-HT1 to 5-HT7" is correct for most higher vertebrates but not for teleost due to their partial genome duplication. As an example, zebrafishes possess 5 different 5-HT2 receptor genes, one 5-HT2B , two 5-HT2A and two 5-HT2C-like genes (see Ensembl.org).

-The weakest part of this study is about the role of 5-HT2B receptors in neural crest-derived tissues. In particular the use of ritanserin at a single concentration of 20 µM, which was correctly labeled in the introduction as "specific 5-HT2 receptor antagonist", but not later "Ritanserin is a specific 5-HT2 receptor antagonist with specificity for the 5-HT2B receptor [10]". Reference 10 is about sea urchin and did not assess the selectivity of ritanserin, which is not selective (see Choi et al. Development, 1997 vol. 124 pp. 1745). Moreover, ignoring completely the pharmacology of the 5-HT2 receptors in zebrafish, it is difficult to conclude about the selectivity of such a dose. It would be necessary to perform a dose-response curve on the observed effects of this compound or better to use a morpholino of the 5-HT2B receptor gene, which the authors cloned, to compare the morpholino developmental effects with this compound.

-Concerning the effects of ritanserin treatment, the authors conclude "These data show that the 5-HT2B receptor plays an essential role after NC migration und (and) during their differentiation in the pharyngeal arches". However, the experimental evidences displayed in figure 4 are not really supporting this statement. Staining using a marker of neural crest cells during migration would be required and may include 5-HT, Sox9b, Dlx2a, or Foxd3 as in fig 3 (see also Reisoli et al, Development 2010 vol. 137 pp. 2927). Furthermore, previous evidence indicated apoptosis during this migration, a marker of apoptotic cells would also help. Finally, a drawing recapitulating the defects observed following the various morpholinos and ritanserin (or 5-HT2B morpholino) would help the reader in interpreting these data.

-The statement "Accordingly, defects in heart morphology which may be also due to abnormal NC development have been described for mice deficient in 5-HT2B receptors [51]" is misleading. In fact, it has been shown that neural crest cells contribute only to the mature valves and the cardiac conduction system (Nakamura, Circ res 2006 vol. 98 pp. 1547) and not to the myocardium.