4) The invited speaker Dr. Nipam Patel to the University of Puerto Rico at Rio Piedras (by Theodor Zbinden)

          Dr. Nipam Patel visited the University of Puerto Rico to be part of the Topics class of the Biology Graduated School.  Dr. Nipam Patel is a very successful evolutional developmental biology scientist and professor in the Department of Molecular and Cell Biology at the University of California, Berkeley.  He is a member of various journal editorial boards, and had research positions at the Howard Hughes Medical Institute, National Institute of Genetics in Japan, the University of Chicago and the Australian National University.  He is author and co-author of over one hundred scientific publications.

Here we will discuss just a small part of his extend research work, a scientific publication white the knock down of the Hox gene Ultrapithorax (Ubx) in the Parhyale hawaiensis to recapitulate evolutionary changes in appendage morphology.  The crustacean’s appendages are different in morphology and structure for their distinct functions.  The anterior appendages are used for feeding and the middle and posterior appendages have functions for mating, defense and locomotion.  However, some crustaceans have posterior of their head segment, appendages similar to the mouthparts named maxillipeds.  In Dr. Nipam Patel’s laboratory they found a correlation between the anterior expression of Ubx and the position and number of the maxillipeds developed in Parhyale hawaiensis.  There hypothesis was that Ubx plays a role in distinguishing between feeding appendages and the locomotory appendages.

They found with in situ hybridization technique, which detects the message RNA of the Ubx gene, that the gene is expressed in seven of the eight appendages from T2 to T8 of the Parhyale.  With the DAPI (4',6-diamidino-2-phenylindole) technique which detects DNA or RNA, they confirmed this result detecting the message RNA (see Fig. A-C).

Fig. A: Parhyale Ubx expression during embryonic development. (A) and (F) are brightfield images of PhUbx expression (purple) and (C–E and F′–H) are DAPI images (highlighting nuclei) with PhUbx expression overlaid in red. All images display a ventral view of the embryo with anterior to the top except for (B), which shows ventral views of hatchling appendages. (A) PhUbx mRNA in a stage 23 Parhyale embryo is expressed at lower levels in the second and third thoracic segments (T2 and T3) and appendages, at higher levels throughout the remaining thoracic segments and appendages (T4–T8), and is absent from the T1 segment and maxillipeds. (B) Hatchling thoracic (T1–T4) and head (MxII) appendages corresponding to the embryonic appendages shown in (A). Colors of each arrow reflect the level of PhUbx expression in that appendage. (C) Ventral view of a stage 24 Parhyale embryo stained with an antibody recognizing PhUbx protein. The PhUbx protein expression boundaries and levels are consistent with the mRNA expression in (A). (D) PhUbx mRNA expression is initially detected around stage 12 in 2 parasegment precursor rows (PSPR 5 & 6) that will give rise to parts of the second through fourth thoracic segments. (E) Higher magnification of embryo in (D). As development proceeds, PhUbx is expressed in the more posterior parasegment precursor row cells (rows 7 & 8 in F′) as well as some of the more anterior row cells that will contribute to the posterior neuroectoderm of the first thoracic segment (F and F′). (F′) Embryonic ventral midline is marked by yellow dashes, numbers indicate PSPRs and their subsequent daughter cells. By stage 18, PhUbx mRNA (H) and protein (G) are expressed in the appendages of T2–T8. In addition, low levels of PhUbx transcript are detected in the abdomen and expression is maintained in neurons of the T1 neuromere (yellow arrowhead).

The Nipal Laboratory then went on to knock down experiments, a technique in which a gene’s expression can be significantly reduced.  They used this technique to see if the gene Ubx is in fact the distinction between the maxillipeds and the remaining legs of the animal.  The knock down experiment resulted in a transformation of the T2 and T3 toward a T1 like maxilliped.  It has to be mentioned that, in general knocking down a gene in a segment results in a new phenotype adopted from the proximo anterior segment.  In our example if Ubx is knocked down in T2 and T3 they will adopt the phenotype of the anterior segment, in this case T2 from T1 and T3 from T2. Even it is a general pattern there exists exceptions in which the segment adopts the phenotype of the posterior segment.

Fig. B: Transformation of thoracic appendage identity by PhUbx knockdown. Ventral view scanning electron micrograph (SEM) of a wild-type Parhyale hatchling (A and B) and a hatchling of an embryo injected with PhUbx siRNAs (C and D). Anterior is toward the top. Higher magnifications of (A and C) are shown in (B and D) respectively. Appendage identity is indicated by color: green for maxillipeds (T1), yellow for gnathopods (T2 and T3), and magenta for walking legs (T4–T8). (A and B) In wild-type hatchlings, the first thoracic (T1) segment bears 1 pair of branched appendages, called maxillipeds (green), which function in feeding and are held against the other mouthparts of the head. The remaining thoracic appendages (T2–T8) lack these branches. (C and D) The T1 appendages (green) in PhUbx siRNA-injected animals appear unaffected (A and B). However, the second thoracic appendages of the siRNA-injected hatchlings (light green shading in C and D) possess additional branches (arrow) on the same limb segments (basis and ischium) as the maxillipeds, indicating transformation of appendage identity. The more posterior thoracic appendages in this PhUbx siRNA-injected animal retain their wild-type identity, but in more severely affected animals the third thoracic appendages are partially transformed to a more maxilliped-like identity.

The relation between evolution and development can be seen in that other species, like the malacostracan crustacean have maxillipeds like structures from T1 to T3, similar to the knock down experiment results from the Ubx gene in Parhyale hawaiensis.  There is no other species known that possess maxillipeds posterior to T3.  This could indicate an evolutionary restriction for development in which segments not expressing Ubx will form maxilliped like structures and restricted just in the possible regions from T1 to T3.

Dr. Nipam Patel (second one from left to right position) at Mojitos' Restaurant in Old San Juan, Puerto Rico [2013]

Reference:

D. M. Liubicicha, J. M. Seranob, A. Pavlopoulosc, Z. Kontarakisd, M. E. Protasb, E. Kwanb, S. Chatterjeeb, K. D. Tranb, M. Averofd, and N. H. Patel (2009).  Knockdown of Parhyale Ultrabithorax recapitulates evolutionary changes in crustacean appendage morphology. PNAS 13892–13896, vol. 106, no. 33.