Table 2.

Effect of RHD mutations on protein activity in zebrafish

RNA Injecteda	RNA Concentration (pg)	Phenotypeb,c				n
		Wild-type (%)	Class I-III (%)	Class IV-V (%)
Uninjected	NA	100	—	—	—	176
zsix2.1	50	44	45	11	—	162
	100	6	57	37	—	125
zsix2.1 L43F	50	84	12	4	—	138
	100	70	23	8	—	105
Zsix2.1 Q241L	50	89	8	3	—	110
	100	91	7	2	—	111
Zsix2.1 D273N	50	96	4	—	—	124
	100	98	2	1		124

		Wild-type (Class I)	Class II	Class III	Class IV
Uninjected	NA	100	—	—	—	132
zbmp4	25	30	48	17	5	100
	50	32	16	36	16	44
	100	—	8	74	18	50
zbmp4 S84C	25	69	29	—	2	126
	50	24	30	19	27	37
	100	—	—	67	33	9
zbmp4 T109S	25	69	21	8	2	109
	50	38	13	25	25	16
	100	—	—	29	71	7
zbmp4 D144K	25	96	4	—	—	112
	50	54	19	12	15	26
	100	26	44	30	—	27

^aDifferences between six2.1 and mutated six2.1 injection results are statistically significant at both 50 pg and 100 pg using the χ ² test of independence. Differences between bmp4 and mutated bmp4 injection results are statistically significant at 25 pg only using the χ ² test of independence (see Supplemental Table 1).

^bDorsalized phenotypes resulting from six2.1 RNA injection were scored according to reference ⁵² as follows: class I-III, embryos at 24 hpf that lacked tail structures (class I and II) or had shortened twisted tails missing ventral structures (class III); class IV-V, embryos with a reduction of ventral cell types and expansion of notochord and anterior somites at 24 hpf (class IV) and bursting of embryos prior to 24 hpf due to somite expansion and constriction (class V).

^cVentralized phenotypes resulting from bmp4 RNA injection were scored according to reference ⁵³ as follows: class I, wild-type; class II, reduced head, expanded hematopoietic mesoderm; class III, loss of head and notochord; class IV, spindle-shaped embryo with no obvious dorsal/ventral polarity.

NA, not applicable.